The insulin analogue with activity reduction of glucose level in the blood

 

(57) Abstract:

Use: in clinical practice to reduce the level of glucose in the blood. The essence of the invention: insulin analogues modified in position 29 of his chain and optionally other provisions are modified physico-chemical and pharmokinetics properties. 6 C.p. f-crystals, 5 tab., 31 Il.

The invention relates to insulin analogues modified in position 29 amino acids of the B-chain of natural human insulin and optionally in other positions. These insulin analogues are less prone to dimerization or spontaneous associations with more high molecular weight forms, so that they more quickly increase their activity, while retaining the biological activity of natural human insulin.

The invention encompasses the insulin analogs of the formula (I) shown in Fig. 1, in which A21 represents alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, threonine, or serine; B1 represents phenylalanine or aspartic acid, or is absent; B2 represents a valine or may not be present in the absence of B1; B3 represents asparagine of blending or aspartic acid; B28 is any amino acid; B29 is an L-Proline, D-Proline, D-hydroxyproline, L-hydroxyproline, L-(N-methyllysine), D-lines, L-(N-methylarginine) or D-arginine; B30 is an alanine, threonine, or is absent; Z represents-OH, -NH2, -SON3or-CH2CH3; X represents Arg, Arg-Arg, Lys, Lys-Lys, Arg-Lys, Lys-Arg, or absent; Y may be present only when there is X, and if present, represents Glu or an amino acid sequence, which contains all or part of the sequence-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Ary, which begins at the N-terminal Glu this sequence.

Described and claimed is also a method of treating hyperglycemia by entering into the patient in need of such treatment, an effective amount of an insulin analog of the formula (I). In addition, described and claimed pharmaceutical compositions containing an effective amount of an analog of formula (I) in combination with one or more pharmaceutically suitable excipients.

In Fig. 2 depicts a restrictive point and functional map of plasmid pKC283; Fig. 3 - restrictive point and functional fold increases point and functional map of plasmid pKC283-LB; in Fig. 6 - restrictive point and functional map of plasmid pKC283-PRS; Fig. 7 - restrictive point and functional map of plasmid pL32; Fig. 8 - restrictive point and functional map of plasmid pNM789; Fig. 9 schematically shows the construction of plasmid 120 of Fig. 10 depicts a restrictive point and functional map of plasmid pL47; Fig. 11 - restrictive point and functional map of plasmid pPR12, Fig. 12 - restrictive point and functional map of plasmid pPR12AR1; Fig. 13 - restrictive point and functional map of plasmid pL110 in Fig. 14 and 15 schematically shows the construction of plasmids pL110C; Fig. 16 depicts a restrictive point and functional map of plasmid pCZR126S; Fig. 17 shows the nucleotide sequence of the gene synthesized human proinsulin; Fig. 18 depicts a restrictive point and functional map of plasmid pPB145; Fig. 19 - restrictive point and functional map of plasmid pRB164A; Fig. 20 - restrictive point and functional map of plasmid pRB172; Fig.21 - restrictive point and functional map of plasmid pRB173; Fig. 22 - restrictive point and functional map of plasmid pRB175.

Formula (I) vosproizvodyaschaya. The abbreviations for amino acids can be decoded as follows.

Abbreviated Amino acid

Aba - Aminobutyric acid

Ala - Alanine

Arg - Arginine

Asn - Asparagine

Asp - Aspartic acid

Cys - Cysteine acid

Cys - Cysteine

Gln - Glutamine

Glu - Glutamic acid

Gly is Glycine

His - Histidine

Il - Isoleucine

Leu - Leucine

Lys - Lysine

Met - Methionine

Nle - Norleucine

Nva - Norvaline

Orn is Ornithine

Phe - Phenylalanine

Pro - Proline

Ser - serine

Thr - Threonine

Trp - Tryptophan

Tyr - Tyrosine

Val - Valine

B28 can be any amino acid, natural or non-natural origin. This amino acid is preferably aspartic acid, valine, leucine, isoleucine, norleucine, Proline, arginine, histidine, citrulline, ornithine, lysine, phenylalanine, alanine, or glycine. Of the above amino acids are particularly preferred subgroup is aspartic acid, valine, leucine, isoleucine, norleucine, Proline, arginine, histidin, ornithine or lysine. Lysine is the most preferred amino acid for B28. B29 is a before the ACLs to the invention is such, in which B28 represents the B29 lysine and represents a Proline, i.e. the inversion of the amino acid sequence of natural human insulin in regulations 28 and 29 of the B-chain.

According to the following features of the invention, as mentioned above, when B28 is an L-Proline, the position B29 is a preferably L-(N-methyllysine), L-lysine, L-(N-methylarginine) or L-arginine. The preferred analog according to this particular is one in which B28 is an L-Proline and B29 is a L-(N-methyllysine) or D-lysine.

Below are also other modifications of the insulin analogues according to the invention, i.e. modification of insulin analogues in positions other than position B28 and B29. In particular, it may be desirable to replace groups of Asn at position 21 of the A chain (i.e. carboxyl end groups) groups of Ala, Asp, Gln, Glu,Gly, Thr or Ser, and when the replacement is the preferred substitute group is Ala. Likewise, it may be desirable to replace the group in position 3 circuit B aspartic acid (Asp). Such a possible replacement to increase the stability of the analogues at extreme pH values, because Asn is particularly sensitive to the reaction of the Asti should be clear also, what glutamic group insulin analogues can be equally sensitive to the reactions of deliciouse and regrouping. Thus, their substitution of glutamic acid is also covered by the scope of the present invention. Other modifications are available insulin analogues according to the invention include (in any combination) replacement of his-tag group in position B10 aspartic amino acid substitution of phenylalanine group in position A1 aspartic acid substitution of ser / thr group in position B30-alanine, the replacement of the serine group at position B9 aspartic acid, a deletion of amino acids at position B1 (des-B1) either as such or in combination with deletion at position B2 (des-B2) and a deletion of threonine from position B-30 (des-B30).

Insulin analogues according to the invention can also be modified in limit positions B30 by entering any of the following amino acids or dipeptides: Arg, Arg-Arg, Lys, Lys-Lys, Arg-Lys or Lys-Arg. In case of their presence, they are indicated in the formula (I) as X. Preferred of them is Arg-Arg.

In addition, if these added links in position B30 similar can be further modified by adding to the formed B30-untinen sequence-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala - Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-, beginning at its N-terminal Glu. This amino acid or sequence in case of its presence is indicated in the formula (I) by the group of Y. When this sequence is only part of the above, it can be any of those parts, which start at the N-Terminus of the sequence, i.e. the group of glutamic acid (Glu).

X or a combination of X and Y defined above represents the whole or part of the connecting peptide found in human proinsulin, the molecule that is the biological source of the natural human insulin.

Preferred sequences for Y are

-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu - Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala - Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-;

-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu - Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala - Leu-Glu-Gly-Ser-Leu-Gln-Les-;

Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu - Leu-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Glu-Pro-Leu-Ala - Leu-Glu-Gly-Ser-Leu-Gln-;

-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu - Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala - Leu-Glu-;

-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu - Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-;

-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-.

In addition, regardless of whether there is or there is no X individually or present or H2CH3. The preferred value of Z is-OH.

As mentioned above, the invention encompasses pharmaceutically suitable salts of insulin analogues. Such preferred salts are salts of zinc, sodium, potassium, magnesium and calcium.

Insulin analogs corresponding to this invention, can be obtained from one of the known methods of peptide synthesis, including classical (solution) methods in solid state systems, methods poluentes known and recently the methods of recombinant DNA.

In the methods using solid-phase systems amino acid sequence is a concatenation of the original insoluble C-terminal amino acid with a resin carrier. These solid-phase methods described T. Stewart and other Solid-phase synthesis of peptides. Freeman and Co., San Francisco, 1969

Usually when performing solid-phase method, the amino acid corresponding to the C-terminal amino acid group of the desired peptide is attached to Smolyan media and then a peptide chain, which starts coated with resin C-terminal amino acids. Individual amino acids are introduced sequentially until then, until Poluchenie fragments, which can be introduced into the peptide chain in the desired order. Peptide chain remains bonded with the resin during the synthesis process, and upon completion of the build chain this peptide is cleaved from the resin.

The peptide chain is attached to the polystyrene resin by ester bonds formed between the carboxyl group of the C-terminal part and the specific methylene group present in the resin matrix, which is the point of this bond.

Amino acids are linked conjugate connections well known in the field methods of formation of peptide bonds.

One such technique is the conversion of the amino acid derivative, which gives a carboxyl group are more sensitive to reaction with the free N-terminal amino group of a peptide of the enzyme. For example, the amino acid can be converted into a mixed anhydride by reaction of the protected amino acids with etelcharge.com, phenylcarbamates, verbatimliteraltwo or isobutylparaben. As a possible variant of the amino acid can be converted into an active ester, such as 1,4,5-trichloranisole ester, pentachlorphenol ester, para-the ash.

Another technique of pairing involves the use of a suitable agent such as N,N'-dichlorochlordene (BCA) or N,N'-diisopropylcarbodiimide (DIC). Other suitable interfacing agents are compounds that are already known to specialists in this field (see Schroder and Lubke, "the Peptides", Academic Press, 1965, Chapter III).

It should be borne in mind that the amino group of each amino acid used in the peptide synthesis must be protected during the reaction mates to prevent side reactions in which it participates-amine functional group. It should also be borne in mind that some amino acids containing side chains, which are the functional groups (for example, sulfhydril, -amino, - and - carboxyl, imidazole, guanidino and hydroxy), and that such functional groups must also be protected in the initial and subsequent stages of the reaction pair. Suitable protective groups are known in the art (see , for example, "Protective groups in organic chemistry". M. McOmie. Ed. Plenum Press, N 4, 1973, and U.S. patent N 4617149).

When choosing the type of the protective group must comply with certain conditions. Protective for the amino group should give functionally is eacli mates in such conditions, where not removed the protective groups of the side chain, should not change the structure of the peptide fragment and should eliminate the possibility of racemization upon activation immediately prior to reaction pair. The protective group of the side chain should give this a functional side-chain inert under the conditions used for the reaction pair, must be stable under the conditions occurring when removing protective for the amino group, and should be easily removed upon completion of construction of the desired amino acid sequence under such reaction conditions that do not alter the structure of the peptide chain.

For specialists in this area should be clear that the protective group, which is already known as the group used for synthesis of peptides will be on different active agents used to remove them. For example, some protective group, such as triphenylmethyl and 2-(para-biphenyl)isopropoxycarbonyl, is very unstable and can be broken down in mildly acidic conditions. Other protective groups such as tert-butyloxycarbonyl, tert-aminostyrene, dematiaceae and para-methoxybenzeneboronic, less stable and require moderately removal. Some protective group, such as benzyloxycarbonyl, glaivemaster, para-nitrobenzenesulfonyl, cycloalkylcarbonyl and isopropoxycarbonyl, even less sustainable and for their removal requires the use of stronger acids, such as hydrofluoric acid, Hydrobromic acid or triptorelin boron in triperoxonane acid.

Upon completion of the desired construction sequence of the peptide is protected peptide should be useplan from resin media and all protecting groups must be removed. The reaction of the cleavage and removal of protective groups can be carried out simultaneously or in stages. When the resin media is klimatisierung polystyrene resin, the link that binds the peptide from the resin is an ester bond formed between the free carboxyl group of the C-terminal link and one of the many chloromethylene groups present in the resin matrix.

It should be borne in mind that the clamping connection can be cleaved by reagents which are known to be able to break the ester bond and to penetrate into the resin matrix. Especially easy way , and remove all protective group. Therefore, the use of this reagent can provide completely devoid of the protection of the peptide. When it is desirable removal of the peptide without removal of the protective groups, the protected peptide - resin can be subjected to methanolysis, resulting protected peptide in which the C - terminal carboxyl group is subjected to methylation. This methyl ester can then be hydrolyzed in a weakly alkaline conditions, and the result is a free C-terminal carboxyl. The protective group of the peptide chain can be removed by treatment with a strong acid, such as liquid hydrofluoric acid. Especially interesting methanolysis method described in the work of G. Moore and other Peptides. Proceedings of the 5th American Symposium on peptides. M. Goodman and J. Meinhofer, editor John Wiley, N 9., 1977, S. 518-521, where the substituted peptide-resin is treated with methanol and potassium cyanide in the presence of a simple crown-ether.

Following cleavage of the protected peptide from the resin and is linolite or treatment with hydrazine. If desired, formed by the C-terminal amide or hydrazide can be hydrolyzed to the free C-terminal carboxyl, and the protective group can be removed common is kinogruppy, removed preferably either before or simultaneously with the cleavage of the protected peptide from the resin media.

A - and B-chain insulin analogues corresponding to this invention, can also be produced by recombinant DNA. The resulting nucleotide sequence encoding the desired peptide A - or B - chain, using conventional technology for such synthesis. These methods generally involve obtaining oligonucleotides encoding as fragments of the desired coding sequence, and the full sequence. Oligonucleotides provide overlap one fragment of the coding sequences of the two fragments complementary sequence and Vice versa. These oligonucleotides kongugiruut and associated with the formation of the ultimately desired gene sequence.

This sequence is inserted in a cloning vector at a point which allows expression of the peptide product that it encodes. Suitable cloning vector comprises at least a portion of the control sequence of the gene expression.

Chains A and B insulin analogues according to the invention can also be perceived by the other Peptides: Synthesis - Structure - Function. Proceedings of the 7th American Symposium on peptides. Ed. D. Rich and E. Gross (1981).

However, the step of combining separate receive chains A and B can be carried out by a method described in the work of Chance, etc., Peptides: Synthesis, structure and functions. Proceedings of the 7th American Symposium on peptides, 1981

For illustration of this invention are the following examples, which, however, are not intended to limit the scope of the invention.

Example 1. Human insulin, Pro (B29), Lys (B28).

A. Obtaining educated from the recombinant A-chain.

A-chain of human insulin produced by recombinant DNA by chemical synthesis of the gene encoding A-chain and its expression in E. Coli. The gene for the A-chain is synthesized from various trinucleotide, synthetic fragments that make up the length from deck to pentadecanolide, through the method of obtaining the block phosphotriester. This gene has a single cohesive end to restrictive endonucleases Eco RI and Bam HI. Insert it in the suitable pronounced vector containing-galactose gene (-gae), resulting in chimeric plasmid containing A-chain-related gene-gae through meinenemy codon. To access the e promoter, not-gae-gene. The chimeric plasmid is transformed into E. Coli, resulting in the expression of the original protein (protein precursor-gae-met A-chain (or trp LE'-met A-circuit, when the system is used promoter trp LE'). The processing of the original protein cyanogenmod leads to the splitting of methioninol connection with obtaining after clearing the A-chain of human insulin. Oxidative sulfites leads to the formation of S-from sulphonated A-chain, which is used for combination with the B-chain S-sulfonate), as described below.

A detailed description of the chemical synthesis of genes for A-human insulin is given in the publication Crea and other Proc. Natl., Acad. Sci., USA, 75, 57655769, 1978. For complete information about the expression in E. Coli of a chemically synthesized gene for the A-chain of human insulin, see publication Goeddel, etc. Proc. Natl. Acad. Sci., USA, 76, 101-110, 1979.

B. Obtaining similar B-chain [Lys (B28), Pro (B29)].

To obtain the raw peptidases resin is used, the peptide synthesizer Applied Biosystems 430A (including modification A4). Use 0.5 mmol original solid-phase resin (t-BOC-Thr (BZl) OCH2Pam resin) (0,76 mmol/g,658 g). All amino acids are protected with BOC, and, except for glutamic acid and histidine, all amino acids used in the second amino acid, supplied by the company Applied Biosystems Inc). Glutamic acid and histidine Peptides are delivered by the company International Corporation, and they are in such packages that each contain approximately 2 mmol of the desired protected amino acids. After drying, the crude peptidases resin in vacuum at room temperature over night) is determined by its weight and is mapped to the initial weight to guarantee the desired gain. A small portion of the sample is subjected to amino acid analysis to ensure that the desired amino acids are inserted in a precisely defined quantities.

The peptide is cleaved from peptidases resin, and the protection of the side chain is removed in the mixing for approximately 1 h at 0oC in a solution of 10 parts (about./weight.) HF (containing 5% vol./about. ethyl mercaptan and 5% vol./about. metacresol) with 1 part of peptidases resin. After vacuum distillation, the greater part of the HF, the peptide is precipitated in a simple ethyl ether. After a few simple leaching with ethyl ether, followed by vacuum filtration, the peptide is dissolved in about 200 ml of 7 M deionized water containing 0.1 mol of Tris, 0.1 mol Na2SO3and 0.01 mol of Na2S4O6. The pH of the solution is brought to 8.5 by 5 N. NaOH, and the solution II peptide solution is introduced into the column size 5 215 cm filled with Sephadex G-25 (fine) at room temperature. The sample is eluted with 50 mm ammonium bicarbonate with a flow rate of 20 ml/min at room temperature. The output stream is analyzed (at 276 nm). Fractions of 20 ml are collected, and receive the desired fractions, which is then purified additionally by liquid chromatography high resolution (HPLC), as described below.

Combined desired fractions pumps in a column 2.5 to 30 cm with Iu Pont C-8, 9-12 μm (column HPLC), and elution is carried out using linear ingredient with increasing concentration of acetonitrile in 100 ml of ammonium bicarbonate at room temperature (2,6 ml/min). Emerging from the column flow is analyzed (280 nm). Fractions of 25 ml are collected. Selected HPLC fractions are analyzed to determine what fraction remain. The desired fractions are collected, lyophilizers and used in further combination with a chain, obtained as described above.

C. Obtaining human stroke Pro (29 B), Lys (28 B).

The combination of chain A and B is carried out according to the procedure described in the publication of Chance and others, see above. 700 mg of S-sulfonate A-chain formed from recombinant DNA, and 140 mg sinteticheskoe 0.1 M buffer solution of glycine and the pH value of each is brought to 10.5 by 4 N. NaOH, then they are cooled to 5oC. Prepared 6 ml of dithiothreitol (DTT) concentration of 10.5 mg/ml in 0.1 M glycine buffer solution at room temperature, the pH value of this solution is brought to 10.5 by 5 N. NaOH, then the solution is cooled to 5oC.

These solutions A-chain and B-chain are mixed with each other, after which this mixture is quickly introduced to 5.21 ml DTT (SH/SSO3= 0,90). This reaction solution is stirred at a temperature of 5oC in open flasks 200 ml test tubes for centrifuges for 2.5 h at 5oC. is Injected 45 ml of glacial acetic acid, and the solution to stand at a temperature of 5oC during the night.

The resulting mixture was then treated with centrifuged for 20 min with a speed of 2000 rpm at a temperature of 5oC. the Surface layer is mixed with 1 M acetic acid, washed from the solid particles, is introduced into column 5 200 cm) with Sephadex G-50 (superfine) in 1 M acetic acid in 5oC and eluted at acceleration of gravity. Twenty minute elution fractions collected within 3 days. These fractions are analyzed (at 276 nm), and some samples are analyzed using analytical methods HPLC. The fractions containing PEFC is the sample additionally purified by liquid chromatography high resolution (HPLC) with a reversible phase (using column 2,12 25 cm with Iu Pont C-8 with elution at room temperature with a rate of 2.6 ml/min using a linear gradient with increasing concentration of acetonitrile in 0.1 M NaH2PO4pH of 2.2). The output stream is analyzed (at 276 nm). Selected fractions are analyzed by analytical HPLC method. The desired fractions are collected and optionally purified using pH 7 HPLC as described below.

Fused product from HPLC low pH is diluted with about twice in an ice bath 0.1 M (NH4)2HPO4. The pH is brought to 7 by cold 3 N. NaOH in an ice bath. This sample is entered in the same column and are eluted from it in the same conditions as in the preparation of the sample with low pH, with the difference that an eluting buffer solution in this case is 0.1 M (pH 7) (NH4)2HPO4/acetonitrile.

Fused product from the HPLC with a pH of 7 is rapidly cooled in an ice bath and two times diluted with a 0.1% aqueous solution triperoxonane acid (TFA). Enter 1 N. HCl (cold sample in an ice bath) to reduce the pH to 3. The sample is introduced into the column Vydae C4 or, as an option in column Iu Pont C-8 HPLV (2,12 25 cm), and elution is carried out with a linear gradient of increasing concentration of acetonitrile in 0.1% TFA aqueous solution. Emerging from the column, the product is analyzed (at 214 or 276 nm). The desired fractions are merged and liofilizirovannoe phase.

Example 2. Human insulin, Pro (B29), Lys (B28).

The second method of obtaining human insulin, Pro (B29), Lys (B28) is to use an enzyme prosentase (reversible proteolysis) for combining the des - octapeptide insulin (A1-21-B1-22with synthetic oktapeptidom. des-Octapeptide insulin is obtained tripticase digestion natural porcine or human insulin as described in Bromer and Chance "preparation and properties les-oktapeptid-insulin", Biochem. Biophys Acta 133, 219-223, (1967). Synthetic oktapeptid Gly-Phe-Phe-Tyr-Tyr-Lys-Pro-Thr is obtained by the method of automatic solid-phase synthesis as described above.

les-octapeptide insulin (435 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Lys-Pro-Thr (465 mg) are mixed in 15 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part of a 0.25 M buffer solution of Tris-acetate, pH 7.3. Peptides are completely dissolved by heating the solution on a hot plate. Then the solution is incubated at 37oC and added 90 mg of porcine trypsin. The solution from time to time stirred for 90 minutes at a temperature of 37oC. the Reaction is terminated by mixing the solution with 135 ml of 0.05 N. HCl.

Okolona HPLC sizes 2.5 to 25 cm, filled with C-8 Bond, and elution with a linear gradient of increasing concentration of acetonitrile in the buffer solution of 0.1 M monobasic phosphate, pH of 2.2. Facing speakers product is analyzed (at 276 nm). The desired fractions merge two times diluted with water and injected into HPLC column sizes 1 25 cm C-8 Ultrasphere. This analog eluted by a linear gradient with increasing concentration of acetonitrile in 0.5% TFA aqueous solution. Emerging from the column, the product is analyzed (at 276 nm). The desired fractions meet again and lyophilizers, yielding 125 mg of the pure analog. This structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5809,2 (theoretically: 5808,7).

This analysis is carried out by the method of fast atomic bombardment - mass spectroscopy using a mass spectrometer with double focusing VG-AB-25E, with a resolution of approximately 1500. Analogues of human insulin are dissolved in a mixture of glycerin and triglycerine with the content of oxalic acid. For calibration of the measuring device, which performs magnetic scan range from m/Z 5300 to m/Z 6500, use of cesium iodide. The obtained data are presented as mass-average value of +1.

oC. is Injected porcine trypsin (75 g). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this point of time the reaction is terminated as a result of the entry of this mixture in 137 ml of 0.05 N. HCl. This entire solution is introduced into the column sizes 21 250 mm C-8 Bond, and products suiryudan at small acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

The appropriate fractions, which were determined by analytical HPLC, merge, twice diluted with water and injected into the column 25 to 300 mm C-18 Vydae. The desired protein is eluted from the column combined acetonitrile gradient in 0.05% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers with obtaining yield 59 mg of this structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5765,7 (theoretically: 5765,6).

Example 4. Human insulin Pro (29 B), Ala (B28).

Pork des-octapeptide insulin (290 mg) and systematic oktapeptid Gly-Phe-Phe-Tyr-Thr-Ala-Pro-Thr (310 mg) are mixed in 10 ml rastvoriaetsia porcine trypsin (60 mg). The solution is thoroughly mixed and from time to time stirred for 60 min at 37oC. during this time the reaction is terminated due to introduction of the mixture into 90 ml of 0.05 N. HCl. The entire solution is introduced into the column sizes 21 250 mm C-8 Bond, and products suiryudan in small combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

The appropriate fractions, which were determined by analytical HPLC, merge, twice diluted with water and injected into the column sizes 10250 mm C-8 Ultrasphere. The desired protein is eluted from the column using the combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers with obtaining the outputs 43 mg. this structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5752,3 (theoretically: 5751,6).

Example 5. Human insulin, Pro (B29), Arg (B28).

Pork des-octapeptide insulin (290 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Arg-Pro-Thr (310 ng) is mixed in 10 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. is Injected porcine insulin (60 mg). The solution Spremeni reaction is terminated as a result of the entry of this mixture in 90 ml of 0.05 N. HCl. The entire solution is introduced into the column sizes 21250 mm C-8, and products suiryudan in small combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

The appropriate fractions, which are defined by analytical HPLC, merge, twice diluted with water and injected into the column 10250 mm C-8 Ultrasphere. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, and obtained 103 mg of the product. This structure is confirmed by amino acid analysis (table. 1) and mass spectrometric analysis (MS). MS: 5836,1 (theoretically: 5836,7).

Example 6. Human insulin, Pro (B29), Asn (B28).

Pork des-octapeptide insulin (409 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Asn-Pro-Thr (398 mg) are mixed in 14 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7,9, at 37oC. Enter porcine trypsin (81 mg). The solution is mixed and from time to time thoroughly stirred for 120 min at 37oC. during this time the reaction is terminated due to introduction of the mixture in 136 ml of 0.5 N. HCl. The entire solution was poured into kolorowego phosphate, pH 2.

The appropriate fractions, as detected by analytical HPLC, merge, twice diluted with water and injected into the column sizes 25300 μm C-18 Vydae. The desired protein is eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is 56 mg of product. This structure is confirmed by amino acid analogue (table. 1) and mass spectroscopy (MS). MS: 5794,7 (theoretically: 5794,6).

Example 7. Human insulin, Pro (B29), Asp (B28).

Pork des-octapeptide insulin (400 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Asp-Pro-Thr (388 mg) are mixed in 13 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3 at 37oC. is Injected porcine trypsin (78 mg). The solution is thoroughly mixed and from time to time stirred for 120 minutes at 37oC.

The reaction is stopped at this time by entering this mixture in 137 ml of 0.05 N. HCl. The entire solution is introduced into the column 21250 mm C-8 Bond, and products suiryudan small combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate rnu times with water and introduced into the column sizes 25300 mm C-8 Vydae. The desired protein is eluted from the column combined acetonitrile gradient in 0.1% triperoxonane acid. The fractions containing the purified insulin analogue, drained and lyophilizers, the result is 85 mg of product. This structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5795,7 (theoretically: 5795,6).

Example 8. Human insulin, Pro (B29), Asp (B10), Lys (B28).

A. obtaining the B-chain of human insulin, Pro (B29), Asp (B10), Lys (B28).

To obtain the crude peptide resin is used peptide synthesizer, Applied Biosystems 430 A (including modification of 1.4). Use 0.5 mmol original solid-phase resin (t-BOC-Thr (BZI) OCH2Pam resin) (0,72 mmol/g 0,705 g). All the amino acids are protected with BOC, with the exception of glutamic acid, aspartic acid and histidine, all amino acids are used in the form as they are received (i.e. in the packaging supplied by the company Applied Biosystems Inc.; each pack contains approximately 2 mmol of the protected amino acids). Glutamic acid, aspartic acid and histidine are received by the industrial acids, they are packing so that each package contains approximately 2 mmol sulaimania night and its weight is matched with the weight of the original resin to guarantee the desired gain. A small portion of the sample is subjected to amino acid analysis to ensure that amino acids are inserted in the correct proportions.

The peptide is cleaved from peptidases resin, and protection of the side chain is removed in the mixing for about 1 h at 0oC in a solution of 10 parts (about. /weight. ) HF (containing 5% vol./about. para-thiocresol and 5% vol./about. metacresol) with 1 part of peptidases resin. After removing the largest part of the HF under vacuum distillation of the peptide precipitates simple ethyl ether. After a few simple leaching with ethyl ether, followed by vacuum filtration, the peptide is dissolved in about 120 ml of 8 M guanidine HCl, pH 11, containing 0.1 mol of Tris, 35 mg/ml of Na2SO3and 25 mg/ml of Na2S4O9. The pH of the solution is brought to 8.8 by 5 N. NaOH, and the solution rapidly stirred for 3 h at room temperature.

Received S-peptide from sulphonated solution is introduced into the column 5215 cm Sephadex G-25 at room temperature. The sample is eluted with a speed of 21 ml/min at room temperature using 50 mmol ammonium bicarbonate. Emerging from the column, the product is analyzed (at 276 nm). Fractions of 25 ml are collected and merged, and the right (HPLC), as is described below.

Fused product comprising the desired fractions is introduced into the chromatographic column 2,530 cm with Iu Pont C-8, 9-12 µm, and elution is carried out using a linear gradient with increasing concentration of acetonitrile in 100 mm ammonium bicarbonate at room temperature (2,6 ml/min). Emerging from the column, the product is analyzed at 280 nm. Fractions (25 ml) are collected and merged. Selected fractions are analyzed by the method of analytical HPLC to determine which fractions are preserved. The desired fractions are merged lyophilizers and used in the following combinations with the A-chain.

B. combination of the B-chain of human insulin, Pro (B29), Lys (B28), Asp (B10) with the A-chain of human insulin.

The combination of A - and B-chains is carried out according to the procedure described in the work of Chance, and others, see above.

Two grams of S-sulfonate A-chain formed from recombinant DNA, and 400 mg of synthetic S-sulfonate B-chain Pro (B29), Asp (B10), Lys (B28) each dissolved in 200 ml and 40 ml, respectively 0.1 M buffer solution of glycerol at room temperature, the pH value of each is brought to 10.5 by 5 N. NaOH and then each is cooled to 5oC. Preparing the re, the pH is brought to 10.5 by 5 N. NaOH, and then the solution is cooled to 5oC.

Solutions A-chain and B-chain are mixed, and then this mixture is quickly introduced to 15.9 ml DTT (su/SSO-3=1) . The reaction solution is stirred at 4oC in open 200-ml glass test tubes for centrifugation for 19.6 hours at 4oC. is Introduced glacial acetic acid (129 ml), and the solution to stand at 4oC for 1 h

The resulting mixture with loose sediment centrifuged for 30 min with a speed of 2000 rpm at 4oC. the Surface layer is mixed with 292 ml Milli-Q-water and 73 ml of acetonitrile, and the mixture is additionally purified by HPLC with a reversible phase (using a column of 2.5 to 30 cm with Vydae C18 with elution at room temperature with a rate of 2.6 ml/min using a linear ingredient with increasing concentration of acetonitrile in 0.1 M NaH2PO4pH 2,1). Emerging from the column, the product is analyzed at 280 nm. Selected fractions are analyzed by analytical method HPLC and the desired fractions are merged and diluted two times with 0.1% aqueous solution triperoxonane acid (TFA), and then merged product is introduced into chromatographica the Oia acetonitrile in 0.1% TFA aqueous solution. Emerging from the column, the product is analyzed at 280 nm. Selected fractions are analyzed by analytical method HPLC and the desired fractions are merged and re-purified using the same column and under the same conditions as described above, when a slightly different gradient of acetonitrile. The appropriate fractions are merged and lyophilizers, the result is 65 mg insulin analogue with a degree of purity of more than 93% as a result of HPLC with the reversible phase. This structure is confirmed by amino acid analysis (table. 1) and a method of mass spectroscopy (MS). MS: 5786,1 (theoretically: 5786,7).

Example 9. Human inulin Pro (B29), Cya (B28).

Is the oxidation of naturaline acid in order to convert cysteine of octapeptide in the form of cysteine acid. Synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Lys-Pro-Thr (363 mg) was dissolved in 36 ml of the freshly prepared naturalyou acid in ice flask, and the solution is gently stirred for 1 h Oxidized material is diluted ten times with water and liabilitiesa. In this prosentase used this lyophilisate.

Pork des-octapeptide insulin (222 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Cya-Pro-Thr (225 mg) are mixed in 18 ml races, the ri 37oC. is Injected porcine trypsin (45 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this point of time the reaction is terminated as a result of the entry of this mixture in 242 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 cm C-8 Bond, and products suiryudan weak combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

The appropriate fractions, which are determined by the analytical method HLC, merge, diluted twice with water and introduced into the column 25 to 300 mm C-18 Vydae. Desalted protein eluted from the column using the combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is a yield 16 mg of the Specified product structure is confirmed by amino acid analysis (table. 1) and a method of mass spectroscopy (MS). MS: 5831,5 (theoretically: 5831,7).

Example 10. Human insulin, Pro (B29), Gln (B28).

Pork des-octapeptide insulin (290 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Gln-Pro-Thr (310 mg) are mixed in 10 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer is from time to time stirred for 60 min at 37oC.

At this point of time the reaction is terminated as a result of introduction of the mixture into 90 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Zordax, and products suiryudan using weak combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

The appropriate fractions, which are defined by analytical HPLC, merge, diluted twice with water, inserted in the column with the dimensions of 10 to 250 mm C-8 Ultraphase. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, turns out to yield 87 mg of this structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5809,4 (theoretically: 5808,6).

Example 11. Human insulin, Pro (B29), Glu (B28).

Pork des-octapeptide insulin (402 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Glu-Pro-Thr (398 mg) are mixed in 14 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, at 37oC. is Injected porcine trypsin (80 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37o

The appropriate fractions, which are defined by analytical HPLC, merge, diluted four times with water and introduced into the column 25 to 300 mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is a yield 59 mg of This structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5809,6 (theoretically: 5809,6).

Example 12. Human insulin, Pro (B29), Gly (B28).

Pork des-octapeptide insulin (412 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Gly-Pro-Thr (376 mg) are mixed in 13 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3. Injected pork trypsin (79 mg). The solution is thoroughly mixed and from time to time stirred for 180 min at 37oC.

At this point of time the reaction is terminated by introduction of the mixture in 147 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Bond, and products suiryudan weak acetone is, is the quiet, the analytical HPLC, merge, diluted four times with water and introduced into the column 25 to 300 mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.1% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is 11 mg of product. This product structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5737,2 (theoretically: 6537,6).

Example 13. Human insulin, Pro (B29), His (B28).

Pork des-octapeptide insulin (400 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-His-Pro-Thr (398 mg) are mixed in 13 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. is Injected porcine trypsin (79 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this point of time the reaction is terminated by introduction of the mixture in 237 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Zordax, and products suiryudan weak in the combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

The appropriate fractions, which defined Anelik eluted from the column combined acetonitrile gradient in 0.1% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is a yield 79 mg of This structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5816,9 (theoretically: 5817,7).

Example 14. Human insulin, Pro (B29), Ile (B28).

Pork des-octapeptide insulin (409 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Ile-Pro-The (398 mg) are mixed in 13 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. Enter porcine trypsin (81 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this point of time the reaction is terminated by introduction of the mixture in 136 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Bond, and products suiryudan weak in the combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

Appropriate fractions defined by analytical HPLC, merge, diluted twice with water and introduced into the column 25 to 300 mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing purified insulin is ridesa amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5793,7 (theoretically: 5793,7).

Example 15. Human insulin, Pro (B29), Leu(B28).

Pork des-octapeptide insulin (418 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Leu-Pro-Thr (410 mg) are mixed in 14 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. is Injected porcine trypsin (83 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this point, the reaction is terminated by entering this mixture in 136 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Bond, and products suiryudan weak in the combined acetonitrile gradient in 0.1 M buffer solution of monobasic sodium sulfate, pH 2.

The appropriate fractions defined by analytical HPLC, merge, diluted four times with water and introduced into the column 25 to 300 mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is a yield 74 mg of the Structure of this product is confirmed by amino acid analysis (table. 1) and mass spectroscopically insulin (290 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Nle-Pro-Thr (310 mg) are mixed in 10 ml of solution, containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. Enter porcine trypsin (60 mg). The solution is thoroughly mixed and from time to time stirred for 60 min at 37oC.

At this point, the reaction is terminated by typing a mixture of 90 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Bond, and products suiryudan weak in the combined acetonitrile gradient in 0.1 M buffer solution of monobasic sodium phosphate, pH 2. The appropriate fractions, which are defined by analytical HPLC, merge, diluted twice with water and introduced into column 10 259 mm C-8 Ultraphere. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing insulin analogue, merge and lyophilizers, the result is a yield 54 mg of this structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5794,6 (theoretically: 5793,7).

Example 17. Human insulin D-Lys (B29).

Pork des-octapeptide insulin (392 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Pro-D-Lys-Thr (387 mg) are mixed 23.5 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butandiol Suaeda and from time to time stirred for 120 min at 37oC.

At this point, the reaction is terminated by entering mixture to 136.5 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Bond, and products suiryudan weak in the combined acetonitrile gradient in 0.1 M buffer solution of monobasic sodium sulfate, pH 2.

Appropriate fractions defined by analytical HPLC, merge, diluted four times with water and introduced into the column 25 to 300 mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is 94 kg of product. This structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5809,0 (theoretically: 5808,7).

Example 18. Human insulin, Pro (B29), Met (B28).

Pork des-octapeptide insulin (350 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Met-Pro-Thr (366 mg) are mixed in 12 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7, at 37oC. is Injected porcine trypsin (71 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this time the reaction is Wirayuda weak in the combined acetonitrile gradient in 0.1 M buffer solution of monobasic sodium phosphate, pH 2.

Appropriate fractions defined by analytical HPLC, merge, diluted four times with water and introduced into the column 25 to 300 mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.1% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is 72 mg of product. This structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5811,0 (theoretically: 5811,7).

Example 19. Human insulin, Pro (B29), Orn (B28).

Pork des-octapeptide insulin (290 mg) and synthetic oktapeptid Gly-Phe-Phe-Thr-Orn-Pro-Thr (310 mg) are mixed in 10 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. is Injected porcine trypsin (60 mg). The solution is thoroughly mixed and from time to time stirred for 90 min at 37oC.

At this point of time the reaction is terminated by entering this mixture in 90 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Bond, and products suiryudan weak in the combined acetonitrile gradient in 0.1 M buffer solution of monobasic sodium phosphate, pH 2.

Suitable fracc the Ultraphase. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing purified insulin analogue, merge and lyophilizers, the result is 89 mg of the product. Product structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5795,2 (theoretically: 5794,7).

Example 20. Human insulin, Pro (B29), Phe (B28).

Pork des-octapeptide insulin (290 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Phe-Pro-Thr (310 mg) are mixed in 10 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. is Injected porcine trypsin (60 mg). The solution is thoroughly mixed and from time to time stirred for 80 min at 37oC.

At this point of time the reaction is terminated by typing a mixture of 90 ml of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Bond, and products suiryudan weak in the combined acetonitrile gradient in 0.1 M buffer solution of monobasic sodium phosphate, pH 2.

Appropriate fractions defined by analytical HPLC, merge, diluted four times with water and introduced into the column 25 to 300 mm C-18 Vydae. Desalted protein eluted from Collina, merge and lyophilizers, the result is 17 mg of product. Product structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5827,9 (theoretically: 5827,7)

Example 21. Human insulin, Pro (B29).

Pork des-octapeptide insulin (339 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Pro-Pro-Thr (363 mg) are mixed in 9 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3 at 37oC. is Injected porcine trypsin (70 mg). The solution is thoroughly mixed and from time to time stirred for 80 min at 37oC.

This reaction at this time is terminated by introduction of the mixture in 108 ml of 0.05 N. HCl. The entire solution is introduced into the column 10 250 mm C-8 Bond, and products suiryudan weak combined acetonitrile gradient in 0.1 M buffer solution odnoosnownogo phosphate, pH 2.

The appropriate fractions, which are defined by analytical HPLC, merge, diluted two times with water and introduced into column 10 250 mm C-8 Ultrasphere. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, as a result . S: 5778,6 (theoretically: 5777,6).

Example 22. Human insulin, Pro (B29), Ser (B28).

Pork des-octapeptide insulin (412 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Ser-Pro-Thr (390 mg) are mixed in 13 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. is Injected porcine trypsin (80 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this time the reaction is terminated by typing a mixture of 137 mg of 0.05 N. HCl. The entire solution is introduced into the column 21 250 mm C-8 Bond, and products suiryudan weak in the combined acetonitrile gradient in 0.1 M buffer solution of monobasic sodium phosphate, pH 2.

Appropriate fractions defined by analytical HPLC, merge, diluted twice with water and introduced into the column h mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, and going lyophilizers with obtaining product yield 37 mg of the product Structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5768,1 (theoretically: 5767,6).

Note the peptide Gly-Phe-Phe-Tyr-Thr-Thr-Pro-Thr (420 mg) are mixed in 14.5 ml, containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. Enter porcine trypsin (86 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this point of time the reaction is terminated by introduction of the mixture into 135,5 ml of 0.05 N. HCl. The entire solution is introduced into the column h mm C-8 Lorbax, and products suiryudan small combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

Appropriate fractions defined by analytical HPLC, merge, twice diluted with water and injected into the column h mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, turns out to yield 78 mg of This structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5781,9 (theoretically: 5781,6).

Example 24. Human insulin, Pro (B29), Trp (B28).

Pork des-octapeptide insulin (310 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Thr-Trp-Pro-Thr (325 mg) are mixed 10.5 ml of a solution containing 1 part of dimethyl sulfoxide, 2 chaser thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this point of time the reaction is terminated by typing a mixture of 140 ml of 0.05 N. HCl. The entire solution is introduced into the column h mm C-8 Lorbax, and products suiryudan weak combined acetonitrile gradient in 0.1 M buffer solution of monobasic sodium phosphate, pH 2.

The appropriate fractions, which are defined by analytical HPLC, merge, twice diluted with water and injected into the column sizes h mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin, merge and lyophilizers, the result is a yield 47 mg of This structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5866,2 (theoretically 5866,7).

Example 25. Human insulin, Pro (B29), Tyr (B28).

Pork des-octapeptide insulin (391 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Thr-Tyr-Pro-Thr (400 mg) are mixed in 13 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. is Injected porcine trypsin (79 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

In this mo is h mm C-8 Bond, and products suiryudan weak combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

The appropriate fractions, which are defined by analytical HPLC, merge, diluted twice with water and introduced into the column sizes h mm C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is a yield of 30 mg of This structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5843,7 (theoretically 5843,7).

Example 26. Human insulin, Pro (B29), Tyr (B28).

Pork des-octapeptide insulin (400 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Tyr-Pro-Thr (383 mg) are mixed in 12 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. is Injected porcine trypsin (78 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this point of time the reaction is terminated by introduction of the mixture in 238 ml of 0.05 N. HCl. The entire solution is introduced into the column h mm C-8 Bond, and products suiryudan adamie faction, defined by analytical HPLC, merge, diluted four times with water and introduced into the column sizes h with C-18 Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.1% triperoxonane acid. The fractions containing the purified insulin analogue, merge and lyophilizers, the result is a yield 74 mg of the product Structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5780,0 (theoretically: 5799,6).

Example 27. Human insulin, Pro (B29), Nva (B28).

Pork des-octapeptide insulin (292 mg) and synthetic oktapeptid Gly-Phe-Phe-Tyr-Thr-Nva-Pro-Thr (279 mg) are mixed in 10 ml of a solution containing 1 part of dimethyl sulfoxide, 2 parts of 1,4-butanediol and 1 part buffer 0.25 M Tris, pH 7.3, at 37oC. is Injected porcine trypsin (57 mg). The solution is thoroughly mixed and from time to time stirred for 120 min at 37oC.

At this point of time the reaction is stopped by adding a mixture of 240 ml of 0.05 N. HCl. The entire solution is introduced into the column h mm C-8 Bond, and products suiryudan weak combined acetonitrile gradient in buffer solution of 0.1 M monobasic phosphate, pH 2.

Appropriate fractions that ODA is Vydae. Desalted protein eluted from the column combined acetonitrile gradient in 0.5% triperoxonane acid. The fractions containing purified insulin analogue, merge and lyophilizers, the result is the product yield 51 mg of the product Structure is confirmed by amino acid analysis (table. 1) and mass spectroscopy (MS). MS: 5870,0 (theoretically: 5779,6).

Example 28. Carrying out the procedure as described in this application, receive the following additional insulin analogs:

(a) Human insulin, Asp(B1), Lys(B28), Pro(B29);

(b) Human insulin, des(Phe-B1), Lys(B28), Pro(29);

(c) Human insulin, des(Phe-B1), Asp(B10), Lys(B28), Pro(B29);

(d) Human insulin, des(Phe-B1, Val-B2), Lys(B28), Pro(B29);

(e) Human insulin, des(Phe-B1, Val-B2), Asp(B10), Lys(B28), Pro(B29);

(f) Human insulin Gly(A21), Asp(B10), Lys(B28), Pro(B29);

(g) Human insulin Ala(A21), Asp(B10), Lys(B28), Pro(B29);

(h) Human insulin, des(Thr-B30), Lys(B28), Pro(B29);

(i) Human insulin Asp(B10), Arg(B28), Pro(B29);

(j) Human insulin Ala(A21), Arg(B28), Pro(B29);

(k) Human insulin, Asp(B1), Arg(B28), Pro(B29).

Example 29. Human insulin, Pro (B29), Lys (B28).

A. Construction of plasmids pCP126.

1. The selection plasmid pKC283.

Lively E. Coli K12 BE1201/pKC283 by the social number NRRL B-15830. Lively decantered in tubes containing 10 ml LB medium (10 g Bacto-tryptone, 5 g Bacto-yeast extract and 10 g NaCl per liter; pH is brought to 7.5), and then incubated for 2 h at 32oC, during this time the culture is brought to a concentration of 50 μg/ml ampicillin and then are incubated with the 32oC during the night. Cells of E. Coli K12 BE1201 / pKC283 growing in the 32oC because they contain temperaturesalinity inhibitory gene, integrated into the cellular DNA. When used in selection procedures plasmids (as described in the following examples) cells, which contain the inhibitory gene lambda p wild type or do not contain promoter lambda p, temperature of incubation is 37oC.

A small part of the grown overnight culture is placed in a Cup with LB-agar (LB medium with 15 g/l Bacto-agar) containing 50 μg/ml of ampicillin, to get a single isolate the columns of E. Coli K12 BE1201/pKC283. This only received a colony inoculated in 10 ml of LB medium containing 50 μg/ml ampicillin, and incubated overnight at 32oC with vigorous shaking. 10 ml overnight culture inoculated in 500 ml of LB medium containing 50 μg/ml ampicillin, and incubated at 32oSukegawa procedure, described by Maniatis and others, 1982, Moulular Cloning (Cold Spring Harbor Laboratory).

Cells are collected by centrifugation acceleration 4000 g for 10 min at 4oC, and the surface layer is removed. The cellular residue is washed with 100 ml ice STE buffer solution (0.1 mol NaCl, 10 mmol Tris-HCl, pH 7.8, and 1 mmol EDTA). After washing, the cell sediment is re-suspended in 10 ml of solution 1 (50 mm glucose, 25 mmol Tris-HCl, pH 8, and 10 mmol EDTA) containing 5 mg/ml lysozyme, and left at room temperature for 10 minutes Then secrete lysozyme-treated cells injected 20 ml of solution 2 (2 N. NaOH and 1% SDS), and the solution is subjected to careful inversion mixing. The mixture is incubated on ice for 10 minutes 15 ml ice 5 M potassium acetate, pH of 4.8, are lysed cell mixture, and the solution is subjected to inversion mixing. Prepared 5 M potassium acetate solution by entering 11.5 ml glacial acetic acid 28.5 ml of water and 60 ml of 5 M potassium acetate, the resulting solution is 3 M with respect to the concentration of potassium and 5 M with respect to the concentration of acetate.

Lysed cell mixture is centrifuged in a centrifuge Beckman SW 27 (or its equivalent) with a speed na bottom of the tube. Retrieved approximately 36 ml of the surface layer and injected with 0.6 volume of isopropanol, is stirring, and the resulting solution is aged at room temperature for 15 minutes Plasmid DNA recovered by centrifugation with acceleration 12000 g for 3 min at room temperature. The surface layer is decanted, and the precipitated DNA is washed with 70% ethanol at room temperature. Then ethanol rinsing decanted, and the precipitate is dried in a desiccator. The residue is again suspendered in 8 ml of TE buffer solution (10 ml of Tris-HCl, pH 8, and 1 ml of ethylenediaminetetraacetate (EDTA).

The DNA solution is injected 8 g CaCl2enter approximately 0.8 ml of a solution of italianbased in the water for every 10 ml CaCl2-DNA. The final density of this solution is about 1.55 g/ml, and the concentration of italianbased approximately 600 µg/ml of This solution is fed into the centrifuge tube type Beckmann 50, is filled from the top with paraffin oil, sealed and centrifuged at the rate of 45,000 rpm for 24 h at 20oC. After centrifugation in normal light there are two layers of DNA. After removal of the cap from the tube bottom layer DNA is removed through the Omid is removed by several extraction of l-butanol, water-saturated. CaCl2is removed by dialysis with TE-buffer. After extraction buffer solution of phenol and then with chloroform, the DNA is precipitated, washed with 70% ethanol and dried. It is about 1 mg of plasmid pKC283, and it is kept at 4oC in buffer solution, THE concentration of approximately 1 μg/μl. Restrictive point and functional map of plasmid pKC283 shown in Fig. 2.

2. Construction of plasmids pKC283PX.

About 10 μl of DNA plasmid pKC283, prepared as described in example 1, is mixed with 20 ál of 10X srednesetevogo restrictive buffer (500 mm NaCl, 100 mmol Tris-HCl, pH 7.5, 100 mmol MgCl2and 10 mmol DTT), 20 μl of BSA (1 mg/ml), 5 ál of restrictive enzyme PVU11 (50 units, as defined in the laboratory Bethesda Research Laboratories (BEL), from which all these restrictive enzymes obtained), and 145 μl of water, and this reaction mixture is incubated at 37oC for 2 h the Reaction restrictive enzyme described in this application is completed in the usual manner by extraction with phenol and then chloroform, after which the precipitation of the DNA, which is washed with ethanol and again suspended in buffer solution. After evaporation with Pvu, as described above, vivares> Approximately 500 pmol connecting links XhoI (5'-CCTCGAGG-3') kinesiologist in a mixture containing 10 ál of 5X kinase buffer (300 mmol Tris-HCl, pH 7.8, 50 mmol MgCl2and 25 mmol DTT), 5 μl of 5 M ATP, 24 μl of H2O, 0,5 µl polynucleotides T4(about 2.5 units as defined by P-L Biochemicals), 5 μl of 1 mg/ml BSA and 5 μl of 10 mm spermidine, by incubating the mixture at 37oC for 30 minutes

Approximately 12.5 ál kinesiology links are introduced in 5 ál extracted with PvuII DNA plasmid pKC283, and then 2 μl of 10X buffer ligase (300 mmol Tris-HCl, pH 7,6, 100 mmol MgCl2and 50 mmol DTT), and 2.5 μl BSA concentration of 1 mg/ml, 7 μl of 5 mm ATP, and 2.5 μl of ligase T4(DNA is about 2.5 units as defined by P-L Biochemicals), and 2.5 μl of 10 mm spermidine and 3 μl of water are introduced into a specified DNA. The resulting reaction mixture knitting incubated at 4oC during the night. After the crosslinking reaction, the reaction mixture is controlled so as to achieve a high salt buffer solution (0.1 mol NaCl, 0.05 mol Tris-HCl, pH 7.5, 10.0 mmol MgCl2and 1 mmol DTT). The mixture is introduced about 10 μl (100 units) restrictive enzyme XhoI, and the resulting reaction mixture is incubated at 37oC for 2 h

The reaction is finished, and which connecting links XhoI not introduced into the mixture of crosslinking. Crosslinked DNA consists of the desired plasmid pKC283PX. Restrictive point and functional map of plasmid pKC283PX shown in Fig. 3.

3. Construction of E. Coli K12 MO (+)/pKC283PX.

E. Coli K12 MO (+) can be obtained from the Northern Regional Research Laboratories in lyophilized form under the official number NRRL B-15993. E. Coli K12 MO includes suppressing gene of the lambda PL of this type, so that transcription from the hybrid promoter pL-Ipp, corresponding to this invention, does not occur in cells of E. Coli K12 MO (+). Is the selection of lifelow and rebuilt single colonies MO (+), and is prepared 10 ml overnight culture of cells MO (+) generally according to the procedure described in example 29 A1, with the difference that the temperature of incubation is 37oC and in the growth medium is not used any of ampicillin.

50 µl of the overnight culture is used to inculati 5 ml of LB medium, which also contains 2 mmol MgSO4and 10 mmol MgCl2. This culture is incubated at 37oC overnight with vigorous shaking. The next morning the culture was diluted to 200 ml LB medium containing 10 mmol MgSO4and 10 mmol MgCl2. The diluted culture is incubated at 35, that shows cell density of about 110 cells/ml of This culture is cooled for 10 min in an ice water bath, and then the cells are removed by centrifugation at acceleration 4000 g for 10 min at 4oC. Cellular precipitate on the bottom of the tube again suspended in 100 ml of cold 10 mm MgSO4and then immediately re-granulated by centrifugation. Cellular precipitate again suspended in 100 ml of 30 mm CaCl2and incubated in ice for 20 minutes

These cells are again collected by centrifugation and again suspensions in 10 ml of 30 mm CaCl2. An aliquot of these cells, a component of gender-ml, is introduced into the crosslinked DNA, prepared as in example 29 A2, prepared DNA concentration of 30 mmol in CaCl2. The mixture of cells - DNA incubated in ice for 1 h, heated at 42oC for 90 s and then cooled on ice for 2 min. the Mixture of cells DNA is diluted in 10 ml of LB medium, and 125-ml flasks and incubated at 37oC for 1 h Aliquots of 100 μl are planted in cups with LB-agar containing ampicillin, and then incubated at 37oC up until not appear colony.

These colonies are individually cultivated, and DNA p is NC plasmids is carried out in small quantities according to the procedure of example 29 A1, but step CsCl gradient is not up until indefinida E. Coli K12 MO (+) pKC283PX transformants. Restrictive point and functional map of plasmid pKC283PX shown in Fig. 3.

4. Construction of E. Coli K12 MO (+) /pKC283-l

10 μg of DNA plasmid pKC283PX obtained according to the procedure of example 29 A1, dissolved in 20 μl of 10X high salt buffer, 20 μl 1 mg/ml BSA, 5 μl (50 units) restrictive enzyme BgeII, 5 μl (50 units) restrictive enzyme XhoI, and 150 μl of water, and the resulting reaction mixture is incubated at 37oC for 2 h the Reaction is terminated and after deposition boiled with BgeII - XhoI DNA, this DNA is again suspended in 5 μl of TE buffer solution.

Synthesized and gynasiums connecting link DNA with single-strand-DNA ends, characteristic splitting restrictive enzyme BgeII and XhoI. This link gynasiums according to the procedure of example 29 A2. Glue the DNA has the structure shown in Fig. 23.

The above link is synthesized from a single line of deoxyoligonucleotide according to the already well-known in the field procedure. These single deoxyoligonucleotide can be synthesized through in the 1850's Lincoln Centre Irive, Foster City (A 94404), which is used in the chemistry of phosphoramidites. There are also many other treatments of DNA synthesis. Conventional modified method of phosphocreatine for the synthesis of single-strand DNA is described in the work of Itakura and others, 1977, Science, 198 : 1056 and Crea and others, 1978, Proc. Nat. Acad. Sci. USA, 75 : 5765. In addition, especially preferred method of DNA synthesis is described in the work of the Hsiung and others, 1983, Nucleic Acid Resarch, 11, 3227; and in the work Narang and others, 1980, Methods in Enzymology, 68 : 90.

It is a link and boiled with BgeII - XhoI plasmid pKC283PX bound mainly according to the procedure of example 29 A2. Crosslinked DNA constitutes the desired plasmid pKC283-L. Restrictive point and functional map of plasmid pKC283-L shown in Fig. 4. DNA plasmid pKC283-L is used to transform E. Coli K12 MO (+), and the resulting transformants of E. Coli E12 MO (+)/pKC283-L are identified mainly according to the procedure of example 29 A3.

5. Construction of E. Coli K12 MO (+) / pKC283-LB.

Approximately 10 μg of DNA plasmid pKC283-L, obtained according to the procedure of example 29 A1, dissolved in 20 μl of 10X high salt buffer, 20 μl 1 mg/ml BSA, 5 μl (50 units) restrictive enzyme XhoI, and 155 μl of H2O, and the reaction mixture is incubated at 37oC for 2 h, Extracted with XhoI DNA p is Tata sodium, incubation in a bath of dry ice - ethanol for 5 min and centrifugation. The precipitate DNA is washed with 70% ethanol, dried and re-suspended in 2 μl of 10X Nick-translation buffer (0.5 mol Tris-HCl, pH to 7.2, 0.1 mol MgSO4and 1 mmol DTT), 1 μl of a solution 2 mm in each of deoxynucleotidase, 15 μl of H2O, 1 μl (6 units as defined by P-L Biochemical) fragment maple, which is a large fragment 1 DNA polymerase, E. Coli, and 1 μl of 1 mg/ml BSA. The reaction mixture is incubated at 25oC for 30 min, the reaction is terminated as a result of the incubation solution at 70oC for 5 minutes

Connecting links of Bam HI (5'- CCGGATCCCG-/3') kinesiologist and fastened with boiled with XhoI, treated with fragment maple DNA plasmid pKC283-L mainly according to the procedure of example 29 A2. After the reaction of the crosslinking DNA vivariums approximately 100 units of Bam AI for 2 h at 37oC in high salt buffer solution. After digestion with Bam AI DNA is prepared for joining according to the procedure of example 29 A2.

Restrictive fragment of 5.9 KB Bam HI is closed in a ring by joining and transformed into E. Coli K12 MO (+) according to the procedure of example 29 A2 and 29 A3. Transformants K12 MO (+

6. Construction of E. Coli K12 MO (+) / pL32.

About 10 µg of plasmid pKC283PX vivariums with restrictive SalI fragment in high salt buffer solution, processed by the fragment maple and stitched the binding units of Eco RI (5'-GAGGAATTCCTC-3') generally according to the procedure of example 29 A5, with the difference that it uses the original plasmid, restrictive fragments and connecting links. After digestion with restrictive fragment Eco RI, which forms in the fragment of 2.1 Lb from DNA, restrictive fragment of 4 KB Eco RI closes the ring by stitching with obtaining plasmids pKC283pPS. This crosslinked DNA is used to transform E. Coli K12 MO (+) generally according to the procedure of example 29 A3. After identification of transformant E. Coli K12 MO (+) / pKC283pPS DNA plasmids pKC283PRS processed according to the procedure of example 29 A1. Restrictive point and functional map of plasmid pKC283PRS shown in Fig. 6.

About 10 μl of the plasmid pKC283PRS vivariums in 200 μl of high salt buffer solution with about 50 units each of restrictive fragments > PST and SphI. After incubating the reaction mixture at 37oC for about 2 h, the reaction mixture was subjected to electrophoresis on 0.6% hours at 130 B and 75 mA in Tris-acetate buffer solution.

This gel is painted in diluted solution italianbased, and a layer of DNA, comprising restrictive fragment of 0.85 KB > PST - SphI, which is visually detectable in the far-ultraviolet rays, cut off from the gel in a small segment. The volume of this segment is determined by weight and density, and the tube containing this segment introduces an equal volume of 10 mmol Tris-HCl, pH to 7.6. Then this segment is melted by incubation at 72oC. is approximately 1 µg restrictive fragment of 0.85 KB > PST - SphI plasmids pKC283PRS and volume of approximately 100 μl. Similarly, plasmid pKC283-LB vivariums with restrictive enzymes > PST and SphI obtained restrictive fragment of 3 KB is isolated by electrophoresis on agarose gel and processed for preparation for stitching.

Restrictive fragment of 0.85 KB > PST - SphI plasmids pKC283PRS stitched with restrictive fragment of 0.3 KB > PST - SphI plasmid pKC283-LB according to the procedure of example 29 A2. Crosslinked DNA constitutes the desired plasmid pL32. Restrictive point and functional map of plasmid pL32 shown in Fig. 7. Plasmid pL32 transformed into cells of E. Coli K12 MO (+) generally according to the procedure of example 29 A3. DNA plasmid pL32 prigotovlu pL32 demonstrated more than one link Eco RI is attached to the processed fragment maple ends SalI plasmids pKC283PX. The presence of more than one link Eco RI does not affect the usefulness of plasmid pL32 or derivatives of plasmid pL32 and can be identified by the presence of restrictive point XhoI, which is formed regardless of when filed both a liaison Eco RI together with each other. As a possible option, plasmid pL32 can be constructed by excision of the fragment SalI-Eco RI and stitching under the first section of this example with the description of plasmid pKC283-LB.

7. Construction of E. Coli K12 MO (+) / pL47.

E. Coli K12 RV308/pNM789 can be obtained from the Northern regional research laboratory in lyophilized form under the number NRRL B-18216. Restrictive point and functional map pNM789 shown in Fig. 8. Plasmid DNA extracted from the culture as described in example 1, with the difference that the temperature of incubation is 37oC. 10 μg pNM789 suspensions in 200 μl PvuII buffer solution (50 mmol Tris-HCl, pH 7.5, 60 mm NaCl and 6 mmol MgCl2). Enter 1 unit of PvuII, and the reaction mixture incubated for 5 min at 37oC. This enzyme inactiveusers 8), 1 mol NaCl and 70 mmol MgCl2), 70 μl of H2O and 10 units of BamHI, and the reaction mixture was incubated for 1 h at 37oC. then injected with 5 units of Alp and incubation for 1 h at 65oC. DNA Fragments were separated on 1% agarose gel, and purification of the DNA fragment (Fig. 9) the size of a single slice segment.

Connecting link DNA with cut off end and a Bam HI-end is synthesized mainly according to the procedure of example 29 A4. This connecting link (shown in Fig. 9) has the structure shown in Fig. 24.

This connecting link gynasiums and sewn in boiled Bam HI - PvuII plasmid pNM789 mainly according to the procedure of example 29 A2. This mixture stitching as transformation of cells of E. Coli K12 RV308, and isolation of plasmids is carried out using the transformant according to the description of example 29 A3. Select some plasmids that contain the PvuII fragment (494 hp) suitable size and fragment XbaI - Bam HI (628 hp). The sequence of at least two of them is determined from a sequence of point-Bam HI to a unique point SmaI, and selects one clone with the desired sequence. This intermediate plasmid, oboznachaet is and plasmids 120 shown in Fig. 9.

To highlight coding EK-BGH DNA about 10 µg of plasmid 120 vivariums in 200 μl of high salt buffer containing about 50 units each of restrictive enzymes XbaI and Bam HI. The digestion products are separated by electrophoresis on agarose gel, and the restrictive enzyme of 0.6 KB XbaI-Bam HI, which encodes the EK-BGH, stands out and disassembled the crosslinking according to the procedure described in example 29 A6.

Plasmid pL32 also vivariums with restrictive enzymes XbaI and Bam HI, and restrictive fragment of 3.9 KB extracted and prepared for joining. Restrictive fragment of 3.9 KB XbaI-Bam HI plasmid pL32 stitched with restrictive fragment of 0.6 KB XbaI-Bam HI plasmids 120 mainly according to the procedure of example 29 A2, resulting in plasmid pL47. Restrictive point and functional map of plasmid pL47 shown in Fig. 10. Plasmid pL47 transformed into E. Coli K12 MO (+) generally according to the procedure of example 29 A3, and identified transformants of E. Coli K12 MO (+) / pL47. DNA plasmid pL47 data obtained from the transformant mainly according to the procedures of example 29 A1.

8. Construction of E. Coli K12 P308 /pPR12AR1.

Plasmid pPR12 embodies chuvstvitelnaja pPR12 described and claimed in U.S. patent N 4436815, released March 13, 1984. Restrictive point and functional map of plasmid pPR12 shown in Fig. 11.

About 10 µg of plasmid pPR12 vyvalivaetsya approximately 50 restrictive enzyme Eco RI in 200 μl of high salt buffer solution at 37oC for 2 h, Extracted with Eco RI DNA plasmid pPR12 deposited and processed by the fragment maple according to the procedure approximate 29 A5. When the reaction maple boiled Eco RI-treated fragment maple DNA plasmid pPR12, again closes in the ring by stitching according to the procedure of example 29 A2. This custom made DNA, which constitutes the desired plasmid pPR12AR1, is used to transform E. Coli K12 RV308 mainly according to the procedure of example 29 A3, with the difference that this choice is based on resistance to tetracycline (5 μg/ml), and not to ampicillin. E. Coli K12 RV308 has an official number NRRL B-15624. After identification of transformant E. Coli K12 RV308/pPR12R1 from the transformant obtained DNA plasmids pPR12AR1 according to the procedure of example 29 A11.

About 10 µg of plasmid pPR12AR1 vyvalivaetsya approximately 50 restrictive enzyme AvaI 200 ál srednesetevogo buffer solution at 37oC for 2 h

Boiled with AvaI DNA plasmids pPR12AR1 sagduyusuna with AvaI, processed fragment maple DNA plasmids pPR12AR1, sewn with a bridge Eco RI (5'-GAGGAATTCCTG-3') generally according to the procedure of example 29 A2.

After stitching connecting link DNA is deposited and then re-suspended in about 200 μl of high salt buffer containing about 50 units of restrictive enzyme Eco RI. The reaction mixture is incubated at 37oC for about 2 hours After digestion with Eco RI, the reaction mixture is placed on an agarose gel, and restrictive fragment of 5.1 KB Eco RI is cleared mainly according to the procedure of example 29 A6. Restrictive fragment of 5.1 KB Eco RI again closed in the ring by stitching according to the procedure of example 29 A2. This crosslinked DNA constitutes the desired plasmid pPR12AR1. This DNA plasmids pPR12AR1 transformed into E. Coli K12 RV308 according to the procedure of example 29 A3, with the difference that the choice is based on resistance to tetracycline, and not to ampicillin.

After identification of transformant E. Coli K12 RV308/pPR12AR1 obtained DNA plasmids pPR12AR1 mainly according to the procedure of example 29 A1. Restrictive point and functional map of plasmid pPR12AR1 shown in Fig. 12.

9. Construction of E. Coli K12 RV308/pL110.

Approximately 10 mg of DNA PL is the Treaty of restrictive enzymes > PST and Eco RI, and the reaction mixture digestion incubated at 37oC for about 2 hours Then the reaction mixture is placed on an agarose gel, and restrictive fragment of 2.9 KB > PST - Eco RI plasmids pPR12AR1 is allocated and prepared for joining mainly according to the procedure of example 29 A6.

About 10 ug of plasmid pL47 vyvalivaetsya with restrictive enzymes > PST and Bam HI in 200 μl of high salt buffer solution at 37oC for 2 h, Extracted with > PST - Bam HI DNA is placed on the agarose gel, and is restrictive fragment of 2.7 KB > PST -Bam HI, which is the source of the replication part resistant to the ampicillin gene and which is prepared for crosslinking according to the procedure of example 29 A6. In the reaction, separation of about 10 μg DNA plasmid pL47 vyvalivaetsya with restrictive fragments Eco RI and Bam HI in 200 μl of high salt buffer solution at 37oC for 2 h, and is restrictive fragment of 1.03 KB > PST - Bam HI, which encompasses new transcriptional and translational activating sequence and encoding EK-BGH DNA, and this fragment is prepared for joining according to the procedure of example 29 A6. About 2 μg of the obtained restrictive fragment of 1.03 KB Eco RI - Bam HI uses the restrictive fragment of 2.9 KB > PST - Eco RI plasmids pPR12AR1 for construction of plasmid pL110, and cross-linked DNA is used to transform E. Coli K12 RV308 mainly according to the procedure of examples 29 A2 and 29 A3, with the difference that the basis for selection of transformant is resistance to tetracycline, and not to ampicillin. In the coding area of the EK-BGH are two probability point of the bounding > PST fragment, which does not affect the bounding point and functional maps in the figures of this application. Restrictive point and functional map of plasmid pL110 shown in Fig. 13.

10. Construction of E. Coli K12 RV308 /pL110C.

A. Construction of E. Coli K12 RV308 /pL110A.

Approximately 1 μg DNA plasmid pL110 vivariums with restrictive enzyme NdeI in 20 µl total volume containing 2 μl of 10X high salt buffer solution (1 mol NaCl, 0.50 mol Tris-HCl, pH 7.5, 0.10 mol MgCl2and 10 mmol of dithiothreitol) and 3 units of enzyme NdeI, for 1 h at 37oC. the Reaction mixture is extracted with phenol /chloroform and the DNA precipitated with ethanol. Boiled with NdeI DNA plasmid pL110 dissolved in 50 ál of 1X buffer solution maple (40 mmol K2HPO4, pH 7.5, 6.6 mmol MgCl2, 1 mmol 2-mercaptoethanol, 33 µm dATP, 33 µmol dGTP and 33 µmol TTP). 2 μl ( 10 units , New England Biolabs) kr is the DNA and the reaction mixture is incubated at 16oC for 1 h the Reaction is terminated by extractable phenol, and the DNA is purified by usual methods. Boiled with NdeI, treated with fragment maple DNA are then sewn with T4DNA ligase at 4oC for 16 hours, DNA is used for normal transformation of E. Coli K12 RV308 (NRRL B-15624). Transformants were selected on plates with L-agar containing 100 μg/ml ampicillin, and the plasmid extracted from resistant colonies by the rapid alkaline extraction, as described by Birnboim and Doly. Selected plasmid (pL110A in Fig. 14) no point NdeI.

b. Construction of phage pL110 B through a point-specific mutagenesis.

The procedure of removal point Bam HI gene, indicating resistance to tetracycline by point-specific mutagenesis, shown on the right side of Fig. 14.

b (i) Construction of phage m13 Tc3.

Plasmid pL110 serves as a source that makes them resistant to tetracycline gene. About 50 μg of plasmid pL110 in 50 μl of buffer solution THOSE entered in 25 µl buffer 10X Hind III and 170 μl of H2O. About 5 μl ( 50 units ) restrictive enzyme Hind III are introduced into a solution of DNA plasmid pL110, and units) restrictive enzyme Eco RI are entered in boiled with HindIII DNA plasmid pL110, and the reaction mixture is incubated for more than 2 h at 37oC. the Reaction is terminated as a result of extraction of the reaction mixture with a saturated TE-phenol, phenol is removed by extraction with chloroform. Boiled with Eco RI-HindIII DNA plasmid pL110 then recovered by precipitation and centrifugation, placed on 1% agarose gel, and a major limiting enzyme of 4.3 KB Eco RI-HindIII allocated and cleared.

Approximately 5 μg of phage m13 mp18 (New England Biolabs) dissolved in 50 μl TE buffer solution, and then evaporated HindIII and Eco RI, as described above.

Cut Hindlll - Eco RI DNA of phage m 13 mp18 purified as described for pL110, with the difference that carried out the isolation and purification of restrictive fragment of 7.25 KB.

Approximately 100 ng of the fragment of 4.3 KB HindIII - Eco RI presidi pL110 is mixed with about 100 ng of the fragment of 7.25 KB HindIII - Eco RI phage m13 mp18, 2 ál of 10X buffer ligase, 1 μl ( 100 units) T4DNA ligase and 14 μl of H2O. the Reaction mixture knitting incubated at 15oC for 1.5 h, crosslinked DNA is desired DNA phage m13 Tc3. Restrictive point and functional map of the phage m13 Tc3 shown in Fig. 14.

1 ml of overnight culture of E. Coli K12 JM109 E. Coli K12 JM101 obtained from New England Biolabs, can use the tour incubated at 37oC with aeration until until OD660(optical density) will not be from 0.3 to 0.4. Cells re-suspensions 25 ml and 10 mmol of NaCl, and then incubated on ice for 10 min and extracted by centrifugation. These cells are placed in 1.25 ml of 0.75 mm CaCl2, 200 μl of an aliquot of these cells is removed, is introduced into 10 ál of crosslinked DNA obtained as described above, and incubated on ice for 40 minutes and Then a mixture of cells - DNA incubated at 92oC for 2 min, and different aliquots (1,10 and 100 μl) are removed and introduced into 3 ml of top agar (L-broth with 0.5% agar supported in the molten state at 45oC), which also contains 50 μl of 2% X-Gal, 50 μl of 100 mm IPTG and 200 μl of E. Coli K12 JM109 in the phase of logarithmic growth. Then the upper cell portion of the agar mixture is planted in cups with L-agar containing 40 μg/ml X-Gal (5-bromo-4-chloro-3-indolyl-D-thiogalactoside) and 0.1 mm IPTG (isopropyl- -D-thiogalactoside), and cups are incubated overnight at 37oC.

The next morning, somewhat transparent, but not blue colonies are used for insulinopenia 2 ml L-Buelna, and the resulting cultures are incubated at 37oC with aeration for 2 hours No blue staining pokazushnogo layer are introduced into 10 ml cultures (OD550= 0,5) E. Coli K12 JM109 grown at 37oC with aeration. These cultures are incubated for 30 min at 37oC, then these cells precipitate in the centrifuge and are used to obtain replicating forms of recombinant phage, which they contain. Of these cells is highlighted line replicatory form ragovoy DNA using procedures (in smaller quantities), described in example 1. Transformant containing phage m13 Tc3 DNA are identified by analysis of restrictive fragment their ragovoy DNA.

b (ii) Obtaining single DNA phage m13 Tc3.

1.5 ml overnight culture Coli K12 JM109/m13 Tc3 centrifugeuse, and 100 µl of the upper layer, containing the phage m13 Tc3, are used for inoculation of 25 ml culture Coli K12 JM109 with an optical density OD660about 0.4 to 0.5. This culture is incubated for approximately 6 h at 37oC with aeration, and then centrifuged culture, formed the top layer of about 20 ml is transferred into a new tube. Approximately 2 ml of a solution containing 20% polyethylene glycol (PEC) 6000 and 14.6% NaCl, are introduced into the surface layer, which is then incubated on ice for 20 min.

The surface layer of centrifuged for 25 EUETS in 500 μl of TE buffer solution. The DNA solution is extracted twice with saturated TE-phenol and twice - chloroform. Then single-strand DNA is precipitated using NaOAc and ethanol and centrifuged. The precipitate is washed with 70% ethanol, dried and then dissolved in 60 μl of H2O.

b (iii) Mutagenesis.

A single fragment of DNA used in the process of mutagenesis, is synthesized in an automatic DNA synthesizer. This fragment has the sequence 5'-CCCGTCCTCTGGATACTCTACGCCGA-3' and is the homologue of the area surrounding the point Bam HI (5'-GGATCC-3'), which imparts resistance to tetracycline gene from plasmid pBR322, with the difference that the A-group, the second from the 5'-end (or third from the 3'-end) is C in plasmid pBR322. This modification does not change the amino acid composition of the protein that makes them resistant to tetracycline, but excludes the point Bam HI.

Approximately 10 pmol mutagenic seed and universal priming M 13 (Bethesda Research Laboratories (BRL) P. O. Box 6009, Galesburg, MD 20760) are processed separately 10 units (BRL) polynucleotides T4in 20 μl of buffer solution of 1X kinase (60 mmol Tris-HCl, pH 7.8, 15 mm 2-mercaptoethanol, 10 mmol MgCl20.41 µmol ATP) for 30 min at 37oC. Processed kinase DNA used in the described Rog with another 300 ng (1,2 µl) per single phage m13 Tc3, 1 pmol (2 ál) of the universal seed, 1 pmol (2 ál) mutagenic seed, 2 ál 10X renaturalised buffer solution (100 mmol Tris-HCl, pH 7.5, 1 mmol EDTA and 500 mm NaCl) and 12.8 μl of H2O. the Reaction mixture was incubated at 80oC for 2 min, at a temperature of 50oC for 5 min, and then cooled to room temperature.

The reaction of chain elongation by typing 5 ál 10X buffer solution stretching (or elongation) (500 mmol Tris-HCl, pH 8, 1 mm EDTA and 120 mmol MgCl2), 5 μl of 2 mm dATP, 1 μl of a solution of 6 mm each of dGTP, TTP, and dCTP, 1 μl ( 2 units, Pharmacia P-L Biochemicals, 800 Centennial Avenue Piseataway, NJ 08854) enzyme maple, 1 μl (100 units) T4DNA ligase and 17 μl of H2O in the mixture denaturirovannah DNA. The reaction mixture of chain elongation incubated at room temperature for 1 h, then at 37oC for 2.5 h and then overnight at 4oC.

The reaction is terminated by implementing two extraction saturated TE-phenol, after which there are two extraction with CHCl3. DNA is precipitated with ethanol and sodium acetate (NaOAc). DNA is extracted by centrifugation and again suspended in 50 μl of H2O, and then 6 μl of 10X buffer solution SI are introduced into the DNA solution.

The amount of precipitation the DNA re-suspensions in 20 μl of H2O, and used 10 µl of the resulting solution to transform E. Coli K12 JM109 E. Coli K12 JM101 can also be used) according to the procedure carried out for construction of phage m13 TS, with the difference that the cups do not enter either IPTG or X-Gal. Double-strand replicatory form DNA from approximately 48 colonies stands out as described above, and selected for the presence of restrictive point Bam HI. Isolates without point Bam HI optionally selected from obtaining single DNA as described above.

The amino acid sequence of the single strand DNA is determined ispolzuyetsa as pL110B (Fig. 14).

C. Construction of plasmids pL110C.

About 50 μg replicating form of phage pL110B DNA vyvalivaetsya 250 mcg 1X buffer solution NheI (50 mmol NaCl, 6 mmol Tris-HCl, pH 7.5, 6 mm MgCl2and 6 mmol-mercaptoethanol) containing 50 units restrictive enzyme NheI, at 37oC for 2 h with 5 ál of 5 M NaCl are then entered in boiled with NheI phage pL110B DNA with subsequent input 5 μl ( 50 units) restrictive enzyme Sal. The digestion is continued for 2 h at 37oC. Then the desired fragment 422 BP NheI-SalI containing the mutated area of the gene that makes them resistant to tetracycline, stands out from acrylamide gel according to standard procedures. DNA plasmids pL110A vivariums with NheI and SalI in identical conditions, with the difference that instead of plasmids pL110B used plasmid pL110A. Restrictive fragment of 6.1 KB NheI-SalI plasmids pL110A purified from agarose.

The construction of the desired plasmid pL110C is carried out by merging with each other at 100 ng each of the NheI-SalI fragments pL110A ( 6,1 KB) and pL110B ( 422 BP) by well-known procedures. Restrictive point and functional map of plasmid pL110C shown in Fig. 15. The desired plasmid pL110C reported resistance to tetracycline 10 μg/ml of tetracycline what s pCZRIII.

Plasmid pL110C contains the limiting point ClaI, which is removed during the course of the following reactions. Approximately 1 µg of plasmid pL110C vivariums with ClaI according to the procedure of example 29 A2, with the difference that use restrictive enzyme ClaI and 10X buffer solution ClaI (500 mmol NaCl, 100 mmol Tris-HCl, pH 7,9, and 100 mmol MgCl2). Boiled with ClaI DNA is then processed by the fragment maple as described in example 29 A5, with the difference that you enter only dCTP, and not all four dNT.

Then the DNA is precipitated and re-suspended in 50 µl of buffer solution bean nuclease Mung (50 mmol sodium acetate, pH 5, 30 mmol NaCl and 1 mmol ZnSO4). 1 unit of bean nuclease Mung (industrial issued New England Biolabs) is introduced into this suspension and the reaction mixture is incubated at 30oC for 30 minutes and Then the tube is placed in the ice and NaCl is introduced at a concentration of 0.2 mol, after which the mixture is extracted with phenol (chloroforme), precipitated with ethanol and re-suspended in 10 mmol Tris-HCl (pH 8). Then DNA is sumissive and transformed into E. Coli cells according to examples 29 A3 and 29 A4. The resulting plasmid is a boiled plasmid pCZR111.

12. Construction of plasmids pC which consists of 600 mmol Tris-HCl, 100 mmol MgCl21 mol NaCl and 10 mmol 2-mercaptoethanol, pH 7.5 (at 37oC), 50 µl buffer 10X XbaI, 15 μl XbaI (10 u/ál) and 185 μl of H2O introduced in 250 μl of water containing approximately 25 µl of plasmid pL110. The digestion is carried out at 37oC for 1 h pL110, boiled with XbaI, and then extracted in phenol, introduced 1/10 volume of ethanol, the mixture is incubated in a bath of dry ice-ethanol for 5 min and then centrifuged. Precipitated DNA is re-suspended in 50 μl of H2O. Boiled with XbaI plasmid pCZR111, vivariums in Bam HI in the following way. 0.2 µl Bam HI (10 u/μl), 10 μl of buffer solution Bam HI (100 mmol Tris-HCl, 50 mmol MgCl21 mol NaCl and 10 mmol 2-mercaptoethanol, pH 8, at 37oC) and 90 μl of H2O are introduced into 50 ál pL110, boiled with XbaI, obtained as described above. The digestion is carried out for 5 min at 37oC. Boiled pCZR111 extracted in phenol, introduced 1/10 volume of CH3COONa, then entered 3 volumes of ethanol. Precipitated DNA again suspended in 50 ál 10 mm Tris, 1 mmol EDTA, pH 8, buffer solution.

pCZR111, boiled with XbaI and Bam HI, is then placed on the agarose gel, and a layer of DNA approximately 5.8 KB. Plasmid pCZR126S obtained by crosslinking fragment of about 5.8 KB pCZR111 with svateho 5'-end and the point of the Bam HI at its 3'-end. The XbaI sequence to NdeI obtained using the method of constructing standard oligonucleotide sequence, and it comprises the sequence shown in Fig. 25.

This sequence is constructed by chemical synthesis of both strands, followed by mixing with the aim of hybridization. Encoding EK-bGH gene is constructed of 16 chemically synthesized segments of single-strand DNA containing from 71 to 83 nucleotides, which together constitute the complementary strands of the entire gene. The synthesis is carried out in the device Applied Biosystems (ABS) and includes the sequence shown in Fig. 26 and 27.

Construction of plasmids pCZR126S by blending the following components: 0,28 μg of the fragment of about 5.8 KB was obtained from the plasmid pL110 after complete digestion with XbaI and partial digestion c Bam HI in a total volume of 2 μl, of 0.18 µg synthetic gene encoding the derived factor bullish growth, which has a 5' end corresponding to the point XbaI, and the end of the 3' corresponding to the point of Bam HI in a total volume of 2.5 μl, 8,75 pmol chemically synthesized XbaI - NdeI link 1 mm. These components plasmids are introduced in 8 ál of 5X buffer solution knitting: 250 mmol TeV>O. This mixture knitting incubated over night at 16oC. Then closed in a ring plasmid pCZR126S is used to transform cells of E. Coli RV308 according to the method of example 29 A3. Restrictive point and functional map of plasmid pCZR126S shown in Fig.16.

B. Building expressed plasmids human proinsulin pRB172

1. Construction of plasmids pRB145.

Gene human proinsulin first synthesized in the usual manner and is cloned into the plasmid PuC18 (industrial manufactured by BRL). This gene is synthesized by standard techniques and comprises the sequence shown in Fig. 28.

One of the clones with the correct sequence is selected for producing purified on cesium chloride DNA. This plasmid is extracted by standard procedures (see, for example, the publication "Molecular cloning. Laboratory manual, 1982, ed. Maniatis, T.; Fritsh, E. F. u Sambrook, J., Cold Spring Heorbour Laboratory Publucations, new York). About 6 μl (29 mcg) of this DNA plasmids are introduced into 20 ál of 10X NdeI buffer solution (150 mmol NaCl, 10 mmol Tris-HCl, pH 7.8, 6 mm MgCl2, 6 mmol 2-mercaptoethanol, 100 μg/ml BSA), 5 μl of restrictive enzyme Nde I ( 40 units ) and 169 μl of H2O. After stirring implement the PEX volumes of ethanol, stirring, cooling to -70oC and centrifugation. Precipitated DNA is washed with 70% ethanol (1 ml), dried and dissolved in 20 μl of 10X Bam HI buffer (150 mmol NaCl, 6 mmol Tris-HCl, pH 7,9, 6 mmol MgCl2, 100 μg/ml BSA), 2 μl of restrictive enzyme Bam HI (40%) and 178 μl of H2O. weak After stirring the reaction mixture incubated at 37oC for 2 h DNA again precipitated with three volumes of ethanol, as defined above, and subjected to electrophoresis on 1% molten agarose gel (FMC, sea agarose). The desired DNA fragment corresponding to approximately 270 BP, cut from the gel, and then the DNA recovered by melting the agarose and pass through the column Elutip-d (Schlicher and Schuell, Kune N. H) according to the procedure recommended by the seller. After precipitation and drying, the DNA is aged in 30 μl of 10 mm Tris-HCl, pH 8.

Approximately 15 μg of plasmid pCZR126S (from example 29 A12) suspensions in 20 μl of 10X NdeI buffer solution, 5 μl of restrictive enzyme NdeI (40%) and H2O (175 ml), poorly mixed, and the mixture is incubated at 37oC for 2 h After incubation, the DNA is precipitated with three volumes of ethanol, as above, dried and then re-suspended in 20 μl of 10X buffer solution Bam HI, 2 μl of OGRA 37oC for 2 h DNA again precipitated with three volumes of ethanol, and electrophoresis on 1% low melting agarose gel. The largest fragment corresponding to the vector DNA cut from the gel and the DNA recovered by the procedure in column Elutip-d, as described above. After precipitation and drying, the vector DNA is maintained in 35 μl of 10 mm Tris-HCl, pH 8.

Approximately 2.5 ál vector DNA are mixed with 12 µl gene fragment purified human insulin, 4 μl 10 mm ATP, and 0.5 µl and 1 mol of dithiothreitol, 5 ál 10X buffer ligase (50 mmol Tris-HCl, pH 7,6, and 100 mmol MgCl2), 26 μl water and 0.5 μl of T4DNA ligase (Pharmacia, a 3.5%). This reaction mixture is incubated at 4oC for 16 hours Stitched mixture is diluted with 50 ál of 10 mm Tris-HCl (pH 7.6) and 3 μl of 1 M CaCl2and then transformed into E. Coli K12 RV308 according to the description of example 29 A3. Cells inoculated on THE cups containing 5 μg/ml tetracycline, and then are incubated overnight at 32oC.

Plasmids from 3 ml cultures stand out from resistant to tetracycline colonies by the rapid alkaline extraction, as described in the publication "Molecular cloning". Laboratory manual, 1982, as amended Moniatis T., Frits is determined by procedures miniature using electrophoresis on polyacrylamide gel for analysis, extracted with XbaI/Bam HI fragment. Plasmids with the correct size inserts (approximately 316 BP) were selected for amplification and purification. A plasmid containing the gene for human proinsulin, is a pRB145. Restrictive point and functional map of plasmid pRB145 shown in Fig. 18.

2. Construction of plasmids pRB164A.

About 30 μg of plasmid pRB145 suspensions in 20 μl of 10X NdeI buffer solution, 5 μl of restrictive enzyme NdeI (New England Biolbs, 40%) and 175 μl of H2O with careful stirring, and the mixture is incubated at 37oC for 1 h Then the reaction mixture is injected 2 μl of restrictive enzyme Bam HI (New England Biolabs, 40%), and incubation at 37oC for a further 2 hours the DNA is precipitated with three volumes of ethanol and 0.3 mol NaOAc and subjected to electrophoresis on 1% low melting agarose gel. Smaller restrictive fragment (approximately 270 BP) NdeI/Bam HI gene encoding human proinsulin, cut from the gel, and the DNA is extracted by passing through a column Elutip-d, as described above. After precipitation and drying, the DNA is aged in 30 μl 10 mm Tris, pH 8.

Then this DNA (30 μl) is added to 20 ál of 10X buffer solution AvaII (50 mmol NaCl, 6 mmol Tris-HCl, pH 8, 10 mm MgCl2, 6 mmol 2-meringo stirring this reaction mixture is incubated at 37oC for 2 h DNA precipitated with three volumes of ethanol and 3 mmol NaOAc (20 ml) and then subjected to electrophoresis on a 1.2% low-melting agarose gel. Larger bounding AvaII fragment/Bam HI (approximately 156 BP) cut from the gel, and then the DNA is extracted by passing through a column Elutip-d, as described above. After precipitation and drying, the DNA is aged in 30 μl 10 mm Tris, pH 8.

DNA ( 115 BP), corresponding restriction fragment NdeI/AvaII gene of human proinsulin, obtained by synthesis. The first step involves the synthesis of the four-lane of deoxyribooligonucleotides through DNA synthesizer Applied Biosystems model 380B. The nucleotide sequences of the four oligonucleotides shown in Fig. 29.

After cleaning each of the oligonucleotide by electrophoresis on polyacrylamide gel oligonucleotides two and three fosfauriliruyutza as described publications Brown E. L. R. Belagaje Kyan, M. J., and Khorana, H. G. (1979), Methods in Enzymology. Ed. Wu R., Academic Precc, New York, 68, 109-151. Phosphorylated oligonucleotides two and three (715 pmol each) are then mixed with one and two oligonucleotides ( 860 pmol), denaturiruty and fastened in a buffer solution (200 μl) containing 50 mmol Tris-HCl (pH 7,6), 10 masuki purified by 15% polyacrylamide gel. DNA extracted from the gel layer by electrophoresis with subsequent desalting column Sephadex C-50. The output of the desired cross-linked product is 485 pmol.

Approximately 100 pmol of DNA fosfauriliruetsa in buffer solution (50 μl) containing 50 mmol Tris (pH 7,6), 10 mmol MgCl2, 10 mmol DTT and ATP, as described in the publication of E. L. Brown and others, 1979, Methods in Enzymology, 68, 109-151. After filtration through a column of Sephadex C-50 DNA is aged in 50 ál 10 mm Tris, pH 8.

Approximately 2.5 ál vector DNA (boiled in NdeI-Bam HI pZR126) are mixed with 18 μl of restrictive fragment AvaIII/Bam HI from plasmids pPRB145 and 10 μl (10 pmol) synthetic link NdeI/AvaII in buffer solution (50 μl) containing 50 mmol Tris (pH 7,6), 10 mmol MgCl2, 10 mmol DTT, 800 μl of ATP and 3.5 units of T4DNA ligase. The reaction mixture was incubated at 4oC overnight and then transformed into E. Coli K12 RV308 according to the procedure of example 29 A3. Desired of transformant E. Coli K12 RV308/pRB164A identified by analysis of their plasmid DNA. The desired transformant grown at 30oC in the environment of T4containing 5 µg/ml tetracycline until D550approximately 0.2 (early logarithmic growth phase) and then at the 42oC for 3-3,5 hours of dbuffer (0.125 mol Tris-HCl, pH of 6.8, 2% SDS, 30% glycerol, 1 mol of 2-mercaptoethanol, 6 mol of urea). Samples heated to 97oC for 2 minutes before placing them on acrylamide gel. Layers easily visually examined by staining dye Kumasi Diamond blue. For the quantitative determination of the percentage of cell protein used gel scanning densitometer. Restrictive point and functional map of plasmid pPR16 4A shown in Fig. 19.

3. Construction of plasmids pRB172.

Approximately 25 μg of plasmid pRB145 suspensions in 15 ál 10X buffer solution DraIII (200 mmol NaCl, 50 mmol Tris-HCl, pH 7.5, 1 mmol DTT, 10 μg/ml BSA), 6 μl of restrictive enzyme DraIII (Bochringer Mnnheim, 27%) and 129 μl of H2O, gently mixed and then incubated at 37oC for 6 hours After incubation, the DNA is precipitated, dried and re-suspended in 10 µl of 10X buffer XbaI (150 mmol NaCl, 6 mmol Tris-HCl, pH 7,9, 6 mmol MgCl2, 7 mmol 2-mercaptoethanol, 100 μg/ml BSA), 3 μl of restrictive enzyme XbaI (Bochringer Mannheim, 36%) and 77 μl of H2O. the Reaction mixture was gently mixed and incubated at 37oC for 4 h DNA again precipitated with three volumes of ethanol and sodium acetate, subjected to electrophoresis on 1% of nescopeck is one proinsulin, cut from the gel and the DNA recovered by the procedure in column Elutip-d. After precipitation and drying, the DNA is aged in 30 μl 10 mm Tris, pH 8.

Approximately 15 μg of plasmid pRB164A cut with restrictive enzyme DraIII and XbaI as described above. The top layer corresponding to the vector fragment XbaI/DraIII, stands out from the agarose gel by the procedure in column Elutip-d. After precipitation and drying, the DNA is aged in 30 μl 10 mm Tris, pH 8.

Approximately 5 μl vector DNA pRB16 4A, boiled with XbaI/DraIII, mixed with 5 μl of restrictive fragment XbaI/DraIII from plasmids pBR145 with buffer solution (50 μl) containing 50 mmol Tris-HCl, pH 7,6, 10 mmol MgCl2, 10 mmol DTT, 800 mmol ATP and 6 units of T4DNA ligase. The reaction mixture was incubated at 4oC during the night and used to transform cells of E. Coli K12 RV308, which are transformed into competent cells by standard processing CaCl2.

Transformants are selected on THE agar plates containing 5 μg/ml tetracycline. Plasmids are allocated from resistant to tetracycline colonies and analyzed by digestion with restrictive nucleases XbaI and Bam HI. Builds amino acid sequence of which being the correct sequence is selected for amplification and purification. Thus isolated, transformant E. Coli K12 RV308/pRB172. Expression and accumulation of human proinsulin containing this plasmid, analyzed by visualizing the total cellular protein with subsequent electrophoretic separation in 15% polyacrylamide matrix. Restrictive point and functional map of plasmid pRB172 shown in Fig. 20.

C. Construction of Escherichia Coli RV308/pRB173 and pRB174.

About 20 μg of plasmid pRB172 or pRB145 suspensions in 20 μl of 10X buffer XbaI (150 mmol NaCl, 6 mmol Tris-HCl, pH 7,9, 6 mmol MgCl2, 7 mmol 2-mercaptoethanol, 100 μg/ml BSA), injected 2 μl of restrictive enzyme XbaI (Bochringer Mannheim, 24%), the mixture is gently mixed and incubated at 37oC for 4 h After incubation of the DNA is precipitated, dried and again suspended in 10 µl of 10X buffer solution Bam HI (100 mmol NaCl, 10 mmol Tris-HCl, pH 8, 5 mm MgCl2, 1 mmol 2-mercaptoethanol, 100 μg/ml BSA), 5 μl of restrictive enzyme Bam HI (Bochringer Mannheim, 40%) and 75 μl of H2O. the Reaction mixture was gently mixed and incubated at 37oC for 4 h DNA again precipitated with ethanol and sodium acetate, and subjected to electrophoresis on 1% low melting agarose gel. Ogranichitelem is sushestvennee procedure in column Elutip-d. After precipitation and drying, the DNA is again suspended in 20 μl of 10X buffer solution XmaI (25 mmol NaCl, 10 mmol Tris-HCl, pH 7.5, 10 mmol MgCl2, 10 mmol 2-mercaptoethanol, 10 μg/ml BSA), 10 μl of restrictive enzyme XmaI (New England Biolabs, 10 units ) and 170 μl of water. The reaction mixture was gently mixed and incubated at 37oC for 4 h After incubation of the DNA is precipitated with ethanol and sodium acetate, and subjected to electrophoresis on 1% low melting agarose gel. Larger DNA corresponding restriction fragment XbaI/XmaI about 200 BP, cut from the gel, and this DNA is removed by the procedure in column Elutip-d, as described above. This DNA is aged in 30 μl 10 mm Tris, pH 8.

DNA 51 BP, corresponding restriction fragment Xma/Bam HI gene of human proinsulin, is aligned in the sequence, which is defined below. Two single strand of deoxyribooligonucleotide shown in Fig. 30, synthesized by using a DNA synthesizer, Applied Biosystems (model 380B), and purified by electrophoresis on polyacrylamide gel in the presence of 7 mol of urea. After the isolation of both nucleotide fosfauriliruyutza according to the procedure in the description of E. L. Brown, Belagaje, R.; Ryan, M. J., and Khorana, H. G. (1979). Methods enzymologie 19 µg plasmid pCZR126S (from example 29 A12) suspensions in 20 μl of 10X buffer solution XbaI (150 mmol NaCl, 6 mmol of Tris-HCl, pH 7,9, 6 mmol MgCl2, 7 mmol 2-mercaptoethanol, 100 μg/ml BSA), 2 mg of organic enzyme XbaI (Bochringer Mannheim, 24%) and 178 μl of water. The reaction mixture was gently mixed and incubated at 37oC for 2 h After incubation entered 4 ál of 5 M NaCl and 2 μl of restrictive enzyme Bam HI (New England Biolabs, 20 units), and the incubation continued for another 2 h at 37oC. Then, the DNA is deposited pattern, and sodium acetate standard and subjected to electrophoresis on 1% low melting agarose gel. The top layer corresponding to the vector DNA XbaI/Bam HI, is allocated by the procedure in column Elutip-d. After precipitation and drying, the DNA is aged in 30 μl 10 mm Tris, pH 8.

Approximately 5 μl vector DNA pCZR126S, boiled with XbaI/Bam HI, are mixed with 10 μl of restrictive fragment XbaI/XmaI from pRB145 or pRB172 and 4 ál chineseromance binder XmaI/Bam HI, prepared as described above in a buffer solution (50 μl) containing 50 mmol Tris (pH 7,6), 10 mmol MgCl2, 10 mmol DTT, 800 µmol ATP and 6 units of T4DNA ligase. The reaction mixture was incubated at 4oC during the night and used to transform strain of E. Coli K12 RV308 according to the procedure of example 29 A3. The desired transformants E. the th fragment pRB145) are identified through an analysis of their plasmid DNA and protein before and after induction at 42oC. Restrictive point and functional map of plasmid pRB173 shown in Fig. 21.

Approximately 200 ng of DNA plasmids from the mini-preparative processing are transformed into E. Coli cells 201, and transformants were selected on plates with 2X TU-agar containing 15 μg/ml tetracycline. Proteins expressed by these transformants are analyzed, and the desired transformants are confirmed for production of human insulin. Thus excreted E. Coli L201 pRB173 and E. Coli L201/pRB174.

D. Construction of Escherichia Coli K12 RV308/pRB175, pRB176, pRB177 and pRB178.

Approximately 12 µg plasmid pRB172 suspensions in 15 ál of 10X NdeI buffer (100 mmol NaCl, 50 mmol Tris-HCl, pH 7.5, 10 mmol MgCl2, 1 mmol DTT, 100 μg/ml BSA), and 2.5 μl of restrictive enzyme NdeI (20%) and 133 μl of water. The reaction mixture was gently mixed and incubated at 37oC during the night. After incubation of the DNA is precipitated with ethanol and sodium acetate standard techniques, dried and re-suspended in 15 ál 10X buffer DraIII (50 mmol Tris-HCl, pH 7.5, 20 mmol NaCl, 10 mmol MgCl2, 1 mmol DTT), 5 μl of restrictive enzyme DraIII (20%) and 130 μl of H2O. After careful mixing, the reaction mixture was incubated at 37oC during the night. DNA again aside the m gel. Desired major restrictive fragment cut from the gel and the DNA recovered by the procedure in column Elutip-d. DNA is aged in 30 μl of 10 mm Tris-HCl, pH 8.

Connecting links of the DNA shown in Fig. 31, chemically synthesized using an Applied Biosystems synthesizer (model 380B).

After cleaning each of the oligonucleotide by electrophoresis on acrylamide gel they fosfauriliruyutza as described above and denaturiruty to speed up the stitching and construction of DNA fragments encoding the analogue of the gene of human proinsulin. Connecting the units (i) to (iv) are used to construct plasmids pRB175, pRB176, pRB177 and pRB178 respectively.

About 3 μl of the DNA fragment of the vector pRB172, boiled with NdeI/DraIII, mixed with 4 µl of each kinesiology connecting parts (i-iv) in buffer solution (50 μl) containing 50 mmol Tris-HCl, pH 7,6, 10 mmol MgCl2, 10 mmol DTT, 800 µmol ATP and 6 units of T4DNA ligase. The reaction mixture was incubated at 4oC during the night, and this mixture is used to transform cells of E. Coli K12 RV308 according to the procedure of example 29 A3. The desired transformants of E. Coli K12 RV308/pRB175, E. Li K12 RN308/pRB176, E. Coli K12 RV308/pRB177 and E. Coli K12 RV308/pRB178 are identified and by whom and functional map of plasmid pRB175 shown in Fig. 22.

The analogy of the human proinsulin are given in table. 1.

E. the Original culture of E. Coli K12 RV308/pRB172 is prepared in the form of a permanent source of culture as follows.

Selected colonies are sown on the following phenotypic cups: L-agar + 5 μg/ml Tc (tetracycline), L-agar + Sm (streptomycinresistant), agar medium M-9 (salt medium containing MgSO4, thiamine, HYC ACE and glucose) and agar medium M-9, containing glucose and contains no lactose. One Cup with L-agar/Tc incubated at 42oC, the remaining cups are incubated at 30oC for 24 h Phenotype correct colony, selected with a Cup of L-agar/Tc at 30oC, is used to insulinopenia in a flask containing 50 ml of L-nutrient broth (containing tryptone, yeast extract, NaCl and glucose) plus 5 µg/ml of medium Tc. This flask was incubated at 30oC with shaking for 7 hours the Material in the flask (1.2 ml) is bis-freezing vials and frozen in vapor-phase liquid nitrogen. The material is then thawed and used in quantities of 1 ml for inoculation in a flask containing 50 ml of L-broth plus 5 μg/ml Tc. This flask was incubated at 30oC with shaking for 4 hours Two flasks, the content is related above environments, these flasks are incubated with shaking for 9 h at 30oC.

150-liter fermenter containing 80 l environment (contains citric acid, ferric sulfate, ammonium, K2HPO4, NH4Cl, NaH2PO4, CaCl2, MgSO4, glucose and isotopic inorganic salts), inoculated in 1 l of nutrient broth BC-7. The fermenter is functioning under the 30oC as long, until the rate of carbon dioxide reaches the value of 1 mmol/l/min At this point 50 l of nutrient broth is introduced into the fermentor to obtain the cellular inoculum.

2500-liter fermenter containing 1500 l of the same medium that is used in a 150-liter fermenter, operates at the 30oC as long, until the rate of carbon dioxide reaches the value of 1 mmol/l/min, and then the temperature rises to 40oC. the operation of the fermenter is continued for another 8 hours Then nutrient broth inactivated by heating at 61oC for 7 minutes Of inactivated by heating the broth obtained the following conclusions:

the output cells of 13.1 g/l on dry weight; product efficacy 273 µg DES-formulate/ml of nutrient broth; specific activity (for example, g of product/g of cells) 2, the which the temperature is maintained between 2 and 8oC. Then the whole nutrient broth is centrifuged or filtered to collect E. Coli cells so that the output of the solid products per dry weight was 15-20%. The solid product comprising the collected cells, again suspended in the process water. The cells are then broken using homogenization under high pressure, or other suitable methods so that lysis of cells was 90-99%. Gomogenizirovannogo suspension was diluted to 5% dry weight in the process water. The pH value of the diluted broken cell suspension is brought to values in the range of 7 to 10 by entering the hydrate of sodium oxide. Inclusione education (granules) are separated from the cellular debris by differential centrifugation. The resulting concentrate contains from 15 to 20% solids.

These inclusione education (granules) solubilized at an alkaline pH in the presence of cysteine and urea. These solubilization granules are subjected to cation exchange chromatography on the fluid sepharose SP for separating a mixed disulfide forms of the Pro-insulin analogue of the total volume of granular material.

M is ω C dipeptidylpeptidase. The reaction katerinovka splitting ends by sulfatase tetrathionate potassium and cysteine at pH of 8.8.

After displacing the solvent through Sephadex C-25 received S-sulfonate Pro-insulin analogue is being crimped by incubation at a pH value equal to 10.6, in the presence of cysteine. The process of inflection is suppressed by bringing the pH to 2.4. Proinsulin similar with the proper inflection is separated from the material with the wrong inflection, as well as from contamination remaining after the stage of cleavage of cathepsin C, by chromatographic separation with a hydrophobic interaction resin SP20SS. This separation is performed using acetone gradient, so that the organic solvent must be removed from the main stream by evaporation prior to further operation.

After evaporation of organic solvent proinsulin agent with the correct inflection incubated with trypsin and carboxypeptidase B. These enzymes excision C-peptide of proinsulin analog and form of human insulin, Lys (B28), Pro (B29). This insulin analogue then further purified by cation exchange chromatography on S-sepharose followed by chromatography high RM displacement solvent on Sephadex C-25, and the resulting product is concentrated using a spiral wound ultrafiltration system. The concentrated product is subjected to chromatography exceptions resin Sephadex C-50 and liabilitiesa until dry.

In table. 2 shows the amino-acid analysis of compounds satisfying the above examples.

Physiological effects of insulin analogues, which meets the present invention, shown in the following test system in vivo.

As test animals used normal male rats Sprague Dawley, obtained from Charles River Laboratories. Received rats have weight in the range from 160 to 180 g, and incubated for 1 week in a room in the 75oF (24oC) with a regular light cycle (with a light on from 7 PM to 7 am and with the lights off from 7 am to 7 PM). The diet of rats is cabbage Purina 5001 ad libitum. Rats used for each test are fasting for about 16 DN to use them. When first used, their weight is about 200 g When you reach a weight of about 275 g (during 3 weeks) animals are no longer used. One group of 10 rats-males used the hedgehog is in and the analogue of human insulin). During the week each group is used only once. Ten rats were divided into two groups of five rats each. One group serves as the control, and it introduces only the medium by subcutaneous injection. Another group, consisting of five rats subjected to processing of the test compound. Proteins dissolved in 0.05 N. HCl (pH 1,6), and the result is the initial solution concentration of 100 µg per ml Of him get a number of dilute solutions in normal saline solution, which is administered by subcutaneous injection in the rat organism. 100 μl of the blood sample taken their tail vein in each rat at time zero and after 30 min, 1 h, 2 h, 3 h and 4 h after a meal. Glucose determined by the colorimetric method of glucose oxidase (Sigma Chemical Co). For each rat calculate the change in the content of glucose in the blood from zero time, and the final results are expressed as mean percent change SEM for the experimental group adjusted mean change for the control group for the same day.

Curve dose - response was built according to tests 4-7 different concentrations of the test compounds using the maximum response at a given mo is led by subcutaneous dose of protein, which gives half the maximum hypoglycemic reactions. The results are presented in table. 3.

As mentioned above, insulin analogs, corresponding to this invention have a reduced ability to dimerization or, in other words, the ability to coassociativity high molecular weight forms. So, if you enter one or several of these analogues is achieved very fast initiation activity. Insulin analogs corresponding to this invention, is effective in the treatment of hyperglycemia when entering into the patient in need of this treatment an effective amount of an insulin analog of the formula (I). Under-used term "effective amount" refers to one or more insulin analogues corresponding to this invention, which are necessary to reduce or maintain the level of blood sugar, either therapeutically or prophylactically. This number can typically be in the range of about 10 to 60 units or more per day (or approximately from 0.3 to 2 mg, which is approximately 29 units per milligram). However, it should be borne in mind that the number of analog (analogue) insulin, which is injected into the body, must be determined to decisim physician in accordance with obstojecega insulin, selected to enter into the body, selected parenteral input method, the patient's age, weight and response of each patient and the severity of the patient's symptoms. Therefore, the above dose limits cannot be a restriction of the scope of this invention.

Insulin analogs corresponding to this invention, are introduced into the patient in need of the treatment (that is, of a patient suffering from hyperglycemia), using pharmaceutical compositions containing an effective amount of at least one insulin analog of the formula (I) in a composition with one or more pharmaceutically suitable excipients or carriers. For these purposes, the pharmaceutical compositions can be prepared in such a way that will contain approximately 100 units per ml or similar concentrations with the contents of the effective number of analog (analogue) insulin. These compositions typically (though not necessarily) parenteral in nature and can be prepared in any way with conventional excipients or carriers for input parenteral products that are already known (see , for example, the publication Remington's Pharmaceutical Sciences, 17th ed. , Mack Publishing Company, Easton, PA, USA, 1985). So, is stvorenie desired amount of at least one insulin analog of the formula (I) in a non-toxic liquid carrier, suitable for injection such as aqueous medium and sterilizing the suspension or solution. As a possible variant, the measured amount of coupling can be placed in a vial, and the vial and its contents sterilized and sealed. The ampoule or the media may be prepared for mixing prior to entering into the body. Pharmaceutical compositions suitable for parenteral enter into the body, used diluents, excipients and carriers such as water and miscible with water and organic solvents such as glycerin, sesame oil, peanut oil, aqueous propylene glycol, N,N'-dimethylformamide, etc. Examples of such pharmaceutical compositions are sterile isotonic water-salt solutions of insulin analogs of the formula (I), which can be prepared in the form of a buffer solution with a pharmaceutically suitable buffer and which is free from pyrogenic substances. In addition, the input parenteral pharmaceutical composition may contain a protecting means, such as meta-cresol, and other substances for adjusting the pH of the final product, such as the hydroxide of sodium or hydrochloric acid.

Insulin analogs corresponding to this invention, mogli are described in detail in European patent application N 2200383. Such compositions are prepared together with one or more pharmaceutically suitable diluents, pharmaceutically acceptable salt of an alkali metal, ammonium salts or free acids free from zinc insulin, and optionally amplifying the absorption number of at least one reinforcing absorption agent selected from the group comprising oleic acid or its ester, a liquid form of ester of sorbitol and fatty acids, liquid form polyoxyethylenated derived complex ester of sorbitol and fatty acids, and liquid form of the copolymer exopolysaccharide-polyoxypropylene-polyoxyethylene.

To demonstrate the effectiveness of intranasal input analogue of human insulin, Lys (B28), Pro (B29) in comparison with the human sodium insulin provides the following study. Male Beagle dogs weighing 10 to 12 kg are maintained in good physical condition before and during this study. Dogs kept in starvation mode for 16 h, but they have access to water to prevent unexpected fluctuations in the content of insulin. Throughout the study, dogs anaesthetize pentabarbital of glucose. Test samples of human insulin (that is Lys (B28), Pro (B29) human nitroindoline) dissolved in distilled water, and the pH of the solution is brought to 7.5 by means of hydrate of sodium oxide. The final concentration of insulin is 70 units per ml Human insulin, Lys (B28), Pro (B29) dose of 0.8 units / kg is injected into each of eight dogs, and human nationalin injected dose of 0.8 units per kg in each of the four dogs. All doses are administered through the nose by entering streams through the nostril by means of a dosing dispenser and modified nasal applicator. Blood samples taken from jugular vein for 30, 15 and 0 min prior to entering and after 10, 20, 30, 45, 60, 90, 120, 180 and 240 min after entering to measure the reduction of glucose in the blood. The results of this study are given in table. 4.

When implementing the above method is mapped to the profile of lowering blood glucose in intranasal entering human insulin, Lys (B28), Pro (B29) profile reduction in intravenous and subcutaneous analog input. For intravenous enter the solution of insulin containing analogue of human insulin, Lys (B28), Pro (B29), is injected by the injection of a test dose of 0.1 units / kg) in the subcutaneous veins of the legs of each of the four dogs. Samples cruor insulin, containing analogue of human insulin, Lys (B28), Pro (B29), is injected under the epidermis (or 0.2 units per kg) the other side of the abdomen of each of the six dogs. Blood samples are taken in the same manner as in the case of nasal input, as described above. The results of this comparative study are presented in table. 5.

1. An insulin analog of the formula I listed in the North,

or its pharmaceutically acceptable salt, wherein A21 is asparagine; B1 is phenylalanine; B2 is valine; B3 is asparagine; B9 is serine; 10 is histidine or aspartic acid; B27 is threonine; B28 is any natural L-amino acid, W is L-Proline; B30 is a threonine.

2. The insulin analogue under item 1, characterized in that the B28 is aspartic acid, valine, leucine, isoleucine, norleucine, Proline, arginine, histidine, citrulline, ornithine, lysine, phenylalanine, alanine, or glycine.

3. The insulin analogue under item 1 or 2, characterized in that the B28 is aspartic acid, valine, leucine, isoleucine, norleucine, Proline, arginine, histidine, ornithine or lysine.

4. An insulin analogue according to any one of paragraphs.1-the m that V10 is aspartic acid.

6. An insulin analogue according to p. 1, wherein A21 is asparagine, B1 is a phenylalanine, B2 is valine, B3 is asparagine, B9 is serine, 10 is histidine, B27 - threonine, B28 - lysine, L is L-Proline and B30 - threonine.

7. An insulin analog of the formula I, with activity decreasing the level of glucose in the blood.

 

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Solution: bone // 2055535
The invention relates to medicine, namely to traumatology and orthopedics, dental surgery, and can be used in the treatment of patients with lesions of bone
The invention relates to medicine, namely in otolaryngology

FIELD: medicine.

SUBSTANCE: method involves taking lavage fluid samples from injured bronchi in preoperative period in making fiber-optic bronchoscopy examination. Microflora colonizing bronchial mucous membrane and its sensitivity to antibiotics is determined. Therapeutic dose of appropriate antibiotic and therapeutic dose of immunomodulator agent like leykinferon is introduced in endolymphatic way 40-60 min before operation. Smears are taken from outlying bronchi in doing operation. Sputum or fluid in retained pleural cavity are taken in 1-2 days after the operation. Prophylaxis effectiveness is determined on basis of bacteriological study data. Therapeutic dose of antibiotics and leykinferon are introduced in 6-8 and 20-24 h after the operation in endolymphatic way. The preparations are introduced at the same doses in endolymphatic way making pauses depending on selected antibiotic elimination half-time once or twice a day until the drains are removed mostly during 48-72 h after operation.

EFFECT: enhanced effectiveness of antibacterial protection; high reliability of antibiotic prophylaxis.

FIELD: medicine.

SUBSTANCE: the present innovation deals with insulin preparations applied in therapy of diabetes mellitus. This innovation could be applied in medicinal industry for insulin manufacturing. To obtain insulin one should apply reindeer's pancreas to be homogenized in solution of hydrochloric acid ethanol followed by extraction with subsequent clarification of solution and obtaining the supernatant which then should undergo ion-exchange chromatography and isoelectric deposition by obtaining insulin. The latter should be purified due to high-performance reversed-phase liquid chromatography. Insulin obtained is competitive for the bond with insulin receptor at concentration of above 100 ng/ml due to causing high increase of receptor binding, moreover, it has higher hydrophoby against standard insulins, thus, it has certain differences in the structure of its molecule.

EFFECT: higher efficiency of insulin manufacturing.

2 cl, 2 dwg, 2 ex

FIELD: pharmaceutical industry, medicine.

SUBSTANCE: invention relates to human insulin drug with activity of 100 IU/ml, including cartridge forms. Drug contains active ingredient, glycerol as isotonic agent, conserving agent and water, wherein it contains human insulin substance of high purity with residual proteolysis activity not more than 0.005 adsorption units, sodium chloride as additional isotonic agent, m-cresol as conserving agent, and additionally sodium dihydrogenphosphate dihydrate or disodium hydrogenphosphate heptahydrate as substance with buffer capacity and pH 6.9-7.8.

EFFECT: human insulin drug of short action with increased physiological activity and physical and chemical storage stability.

6 ex, 1 tbl

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