Method for production of amidated peptide

FIELD: biochemistry, gene engineering, in particular amidation of hormones and other peptides used in medicine and agriculture.

SUBSTANCE: peptide sequence obtained in process of triptic digesting of peptidylglycine alpha-amidating monooxygenase (PAM) from fat thyroid encephaloid carcinoma is determined. On the base of obtained data oligonucleotide sequences are synthesized and used for isolation of full-sized PAM encoding DNA. Expression of obtained DNA fragments in heterogeneous systems makes it possible to obtain recombinant PAM useful in amidation reaction of peptide substrate with C-terminated glycine providing the same effect that for native enzyme.

EFFECT: effective method for production of amidated peptides.

12 dwg, 2 tbl, 9 ex

 

The present invention relates to alpha-lidiruyushchim enzymes, the production of alpha-lidirujuwih enzymes and their use in the production of alpha-emitirovannykh products by influencing enzymes elongated glycine substrates. In certain preferred embodiments, the implementation of the alpha lidiruyushey enzyme according to the invention can be used to obtain useful alpha-emitirovannykh hormones and other products used in agriculture and medicine, including calcitonin, factors the release of growth hormones, peptide, associated with the gene calcitonin.

Intracellular transformation (decomposition and/or modification of functional groups) predecessors of native proteins after they are broadcast from an encoding nucleic acid sequences are clearly presented in the various documents.

Typically, mammalian cells or other eukaryotes can perform certain operations postranslational processing, while prokaryotes do not can. Some prokaryotes, such as E-coli, are widely used as hosts for protein mammals using recombinant DNA technology (gdnc), as they can easily grow by fermentation and because they are genetically accurately characterized. However, many proteins in MLEKO is melting, obtained through genetic engineering, require post-translational processing, and this often leads to contact the use of complex chemical operations in vitro, which because of their high cost restrict large-scale industrial application.

One type of change activity involves the amidation of the amino acids located at the carboxyl end of the protein. Many naturally occurring hormones and peptides contain such a change, which is often essential for their biological activity. An example is calcitonin, where the substitution neemalirovannuyu prolinnova balance aminirovanie Proline native form leads to 3000-fold reduction of biological activity.

An agent that affects the amidation of the C-end (alpha), recognizes glycine residue immediately following amino acid subjected to amidation (R-X-gli, where R is the primary sequence of the protein, X is a residue which is subjected to amidation and gli - glycine residue). Glycine is cleaved and actually gives aminosilanes in the penultimate amino acid, resulting in its amidation.

The first authors that reported approximate molecular weight for alpha-ameerega enzyme were Bradbury and others (Narure 298, 1982, 686-88). They suggested clomidonline apparent molecular mass, some on Sephadex G-100, is approximately 60000 daltons (1 Dalton 1,6601×10-27kg).

Subsequent studies showed that the molecular weight of this enzyme was determined by gel-filtration range from 60000 to 70000 daltons. These studies include work Husaini and Tita S. (FEBS. Letters, 152 No. 2, 1983, 277-281), Alper and others (PNAS, 80, 1983, 5144-5148), Gomez and others (FEBS Letters, 167, No. 1, 1984, 160-164) and J. Kaiser. S. and others (PNAS 81, 1984, 3228-3232).

Aber and others (PNAS 80, 1983, s-48) reported that addition of molecular oxygen requires two cofactor for the maximum activity of the enzyme amidation; they are ascorbic acid and copper ion (11).

The chemical reaction leading to the amidation of carboxyl end of the peptide, require a source of amino groups. Bradbury A.F., and other Nature 298, 1982, 686-688) showed that the glycine molecule gives aminosilanes the penultimate amino acid, resulting in the amidation of the latter. The requirement for glycine as a donor of amino groups was confirmed by other authors.

Langimar and others (R 117, No. 1, 1983, 289-293) showed that D-alanine can also serve as a donor of amino groups in the amidation reaction. Conducted by Kaiser and others (PNAS, 81, 1984, 3228-3232) subsequent work showed two different enzyme activity in the brain of rats, which are capable of catalyzing the reaction of alpha is melirovanie. Samples with higher molecular weight (70000 daltons) have specificity, limited glycine at the carboxyl end of the substrate. An enzyme with a lower molecular weight takes the substrate with β-alanine as a C-terminal amino acids.

The optimal pH value for the alpha ameerega enzyme extracted and partially purified from porcine pituitary, according to Bradbury A.F., and Smith DG. (R, 112, No. 2, 1983, 372-377) was approximately 7.0. Apper and others (PNAS 80, 1983, 5144-5148) confirmed these results message about the optimal pH value of 7.0 for the alpha ameerega of the enzyme was partially purified from rat pituitary. They also noted that the enzymatic activity falls rapidly at pH below 6.5 or above 7.5.

In all the above publications (incorporated herein by reference) extracts and partially purified enzyme mixtures additionally contain proteolytic enzymes that can cause the destruction of potential substrates and products, as well as the alpha lidirujuwih enzymes, thus slowing the amidation using such enzymes, peptides and polypeptides, isolated by purification from natural sources or produced using technologies to produce recombinant DNA.

Earlier measurements lidiruyushey activity was based on the Tran is the cation D-substrates, such as tripeptides, D-Tyr-Val-Gli-COOH, D-Tyr-Val-CONH2. However, the use of form D by other researchers was associated with the need to neutralize the presence of strangers proteolytic enzymes in contaminated drugs lidirujuwih enzymes used in these experiments. These extraneous enzymes can have a pronounced proteolytic effect on substrates with L-amino acid, while having little effect on D-substrates. Nobody so far not been able to show that their enzymatic preparations with α-lidiruyushey activity can effectively lidirovat any physiologically relevant substrates, i.e. L-substrates, obtaining biologically active alpha-emitirovannykh L-products.

As shown here, the preparations according to the present invention can effectively lidirovat L-substrates, and in relation to D-substrates to have activity from 60 to 1000 times higher than the highest activity specified in any previously published source, which is known to the applicants.

Enzymatic drugs which can lidirovat carboxyl ends of peptides and proteins, are known from various published sources. For example, Bradbury A.F. (Nature 298, 1982, 686-688) reported that alpha-lidiruyushey activity is present in the pituitary gland of pigs. The prep is at from porcine pituitary was able to convert peptides, that end glycine, in the appropriate bisglycinate peptidyl-amide. Bradbury and others indicate, however, that the drugs will not be lidirovat peptides or polypeptides, isolated by purification from natural sources.

It is noted that the described preparations contain other proteolytic enzymes that destroy natural peptides or polypeptides, and that unnatural D-the tyrosine residue was used specifically to minimize this destruction.

Further, the authors note that despite the use of after homogenization subcellular fractionation and gel filtration chromatography for purification ameerega enzyme, it remains contaminated proteolytic enzyme.

Husain, I. and Tate S. (FEBS Letters, 152, No. 2, 1983, 277-281) describes alpha ameruoso activity in neurosecretory glands of the pituitary gland of the ox.

Aber and others (PNAS, vol 80, 1983, str-5148) reported the presence of alpha-lidiruyushey of enzyme activity in the anterior, intermediate and posterior lobes of the rat pituitary intermediate pituitary bull. However, in this reference also includes an indication of the use of synthetic D-Tyr-Val-Gli substrate for studies of alpha-lidiruyushey activity and the recognition of the existence of impurities derived drugs.

Gomez and others (FEBS Letters 167, No. 1, 1984, 160-164) has determined that the rat hypothalamus also has al the f-lidiruyushey enzymatic activity.

Bradbury A.F., and Smith DG. (Messages of the Eighth American Symposium on peptides, p. 249-252 (1983), editors Hrubý VJ. and rich DH) describe the activity of alpha-ameerega enzyme in the thyroid glands of rats.

Mains and others (Endocrinology, 114, 1984, 1522-1530) reported that cell line, obtained from the anterior pituitary of the mouse (ATT-20)had an alpha lidiruyushey enzymatic activity, which was clearly decreased over time in culture.

Some lower form, for example, sea dog (Squalus acanthias), according to Donohue TL and others (Peptides 3, 1982, 353-395) contain amidarone peptides in extracts of the pituitary gland. Scheller R.H. and others (Cell 32, 1983, 7-22) reported the presence of signal peptide amidation in marine snails. Despite the apparent world-wide spread of this type of activity in nature, there is little information on the physico-chemical properties of enzymes.

This may be due to very low levels in the presence of enzymes in these neuroendocrine organs.

The presence emitirovannykh peptides in certain tissues is not necessarily associated with high levels of alpha-ameerega enzyme. For example, the tissue of the anterior part of the pituitary gland of rats has high alpha lidiruyushey activity, but does not contain suitable substrates (Appert and other PNAS, 80, 5144-5148 /1983/). Fabric rear part of the pituitary gland of rats contents the t amidarone peptides (oxytocin and vasopressin), but has a very low alpha lidiruyushey activity (Alpert and others, Endo, 116, 2497-2504 (1985)/. Therefore, while certain fabrics will not be tested on alpha-lidiruyushey activity, existing or potential levels of the enzyme cannot be predicted.

Summary of the invention

The aim of the present invention to provide alpha-lidirujuwih enzymatic drugs that may provide efficient production of useful alpha-emitirovannykh products, including those from substrates containing L-amino acids, for example, a peptide or polypeptide substrate, a dedicated purification from natural sources or obtained through recombinant DNA technology.

An additional objective of the present invention is to provide an efficient and economical method of manufacture of such drugs.

An additional objective is the creation of monoclonal antibodies specific against alpha-ameerega enzyme, immobilized antibodies, resins for purification columns for carrying out chromatography on immunoreactive and the like that are using these antibodies effectively clean alpha-lidiruyushey enzyme.

An additional objective is the creation of master cells through genetic engineering that are able to provide highly productive Express the human alpha-ameerega enzyme.

An additional objective of the present invention to provide alpha-emitirovannykh products from substrates containing on-end glycine residue by treating them α-lidiruyushchim enzyme according to the invention.

These and other objectives will become apparent from a consideration of the following description. According to the present invention, applicants provide new alpha lidirujuwie enzymatic preparations of sufficient purity to be specific alpha-ameruoso activity at least 25 IU per mg protein, and preferably above 50 or above 150 IU/mg protein. All units of a specific activity, shown here, is given in terms of the transformation of Dansyl-D-Three-Gli-COOH in Dan-power-D-Tyr-Val-N2. One IU is defined as the amount of activity required to convert one nanomole (nmol) Dansyl-D-Tyr-Val-Gli-COOH in one of nanomoles of Dansyl-D-Tyr-Val-Gli-N2per minute at 37°C and pH 7.0 in the presence of an ion of ascorbic acid (final concentration 3 mm), molecular oxygen in a molar excess relative to the substrate and the copper ions in a concentration sufficient to obtain maximum activity (usually about 2 mm, depending on the purity of the enzyme).

Alpha lidirujuwie enzymes according to the invention having the activity of a peptidyl glycine alpha-lidiruyushey monoux genasi, capable of catalyzing the conversion of peptidnogo connection with glycine residue, at least-the end of the peptide chain, the corresponding peptidyl amide compound having an amino group instead of glycine. Used herein, the term "peptide chain" refers to any polypeptide having at least two amino acids connected by peptide linkages. The term "peptidyl compound" includes any compound with a peptide chain. The term "corresponding peptidyl amide" refers to any product of reactions, in which the glycine at the C-end of the peptide chain is substituted by an amino group.

It is advisable that the reaction of alpha-amidation took place in the presence of oxygen and a reducing substance. Such reducing agents may include, but are not limited to, ascorbic acid, salts of ascorbic acid, dihydroxyfumaric, metal compounds cyanide, tetrahydropyran. It was found that certain cofactors contribute to the course of the reaction or slow down the decrease of enzyme activity. These cofactors include, but are not limited to this, catalase, ethanol, potassium iodide and copper ions. Purified enzyme preparations according to the invention is preferably free from proteolytic activity capable of causing the destruction of the alpha-amide the ith enzyme products or reactants of the reaction of alpha-amidation, due to which they can provide the catalysis reaction of alpha-amidation, when the substrate and the product contains L-amino acids.

As will be described in detail hereafter, the applicants spent clearing the huge number of samples of specific proteins, which have alpha-lidiruyushey activity. "Alpha lidiruyushey activity" in the sense it is used here, means any activity, which tends to leave only one amino group in the position previously occupied by glycine at the C-end of the peptide substrate.

"Alpha lidiruyushey enzyme" in the sense it is used here, refers to any drug or an individual compound, which exhibits alpha-ameruoso activity and active homologues and fragments.

Enzyme preparations isolated in accordance with the present invention can be purified to homogeneity. The term "homogeneous" here refers to the enzymatic preparations exhibiting a single clearly defined band after electrophoresis in polyacrylamide gel with PA-DS and showing only the sequence of amino acids in the respective studies.

Certain alpha-lidirujuwie enzymes, purified to a homogeneous state in accordance with the present invention, roessli specific enzymatic activity above 1500 IU/mg protein.

Enzymatic compositions prepared in accordance with the present invention can be used in the production of useful alpha-emitirovannykh peptide products. As the substrate used peptide compound having a glycine residue at at least one end of the peptide chain, where the substitution of glycine at the C-end amino group results in the desired product. The substrate is introduced into the reaction preferably in the presence of oxygen and a reducing agent and contact with an enzyme preparation obtained in accordance with the present invention, in a period of time sufficient to convert the peptide in an appropriate peptidebased. The reaction rate strongly depends on pH, temperature, the affinity of the substrate, the concentration of cofactors and other parameters that can be changed in a known manner, to optimize the reaction. The reaction usually should evolve over time, selected based on the desired percentage conversion of substrate into product). In preferred embodiments, the implementation uses cofactors, such as those discussed above, to promote the reaction and/or increase and stabilization of enzyme activity.

A huge number of useful products, including natural hormones and the like, to the x-alpha-amidation is preferable or necessary can be obtained by the reaction of the elongated glycine peptidergic compounds with alpha-lidiruyushchim enzymatic preparation in accordance with the present invention. These products include, but are not limited to, various calcitonin, factors the release of growth hormones, peptides associated with the gene calcitonin, and the like. Hormones, to which reference is made, include varieties emitirovannykh to-end proteins, which are characterized by the activity of these hormones, as it is clearly well-versed in this area specialists. For example, calcitonin includes all the species that have shown the ability to regulate the absorption of calcium in the bone, which is a known characteristic of calcitonin. Any nucleotide sequence or amino acid sequence, the claimed here include sequence homologues, in which the substitution, addition or deletion does not have a physical impact on the functions performed by the specified sequence. It is advisable that at least 40%, and most preferable that 50% of the amino acids corresponded to those that claimed. In relation to the nucleotide sequence of codons can, of course, be replaced by equivalent codons encoding the same amino acid.

In the definition is the R preferred options for implementation, reaction of alpha-amidation can be facilitated by immobilization of the enzyme on a solid medium which is insoluble in water and resistant to destruction under the reaction conditions, and the transmittance of the substrate through the immobilized enzyme, preferably in the presence of cofactors. In the sense used here, the term "immobilization", it implies the connection of the enzyme with the substrate or carrier. The media, which may be useful for this purpose include, but are not limited to, porous glass or activated absorbent, sepharose activated with bromine cyan. Immobilized so the enzymes can be reused by removing the reaction mixture from the solid-state media, which continues to hold the enzyme for subsequent use.

Applicants have discovered that the enzyme preparations in accordance with the present invention can be obtained and purified using a number of ways. It is established that soft medullary tissue cancer (carcinoma) of the thyroid gland, preferably obtained from a rat cell line it and/or culture of cells of these cell lines are particularly desirable source of native alpha-ameerega enzyme. Contaminated alpha lidiruyushey enzyme m which can be cleaned by exposure to the original raw composition as chromatography exceptions and uninominal chromatography, preferably a strong uninominal chromatography. In the sense used here, the term "strong" uninominal chromatography, it refers to uninominal chromatography carried out on any resin, which keeps a constant average positive charge in the pH range 2-12. In certain preferred embodiments, the implementation of the present invention exclusion chromatography size is preceded by a strong uninominal chromatography, and chromatography exceptions may be preceded another uninominal chromatography. In one preferred embodiment, one strong uninominal chromatography is carried out with mainly pH, while the other is carried out at acidic pH.

When using samples of the enzyme, treated to achieve uniformity in accordance with the present invention, as antigen were obtained monoclonal and polyclonal antibodies specific enzyme. The antibodies thus obtained from any species can be purified and immobilized on a solid medium with the aim of obtaining speakers for carrying out chromatography on immunoreactive. This column can be used for enzymatic purification of raw material.

The enzyme purified in accordance with the present invention, which was b subjected cryptococcoma digestion; the fragments were sequenced by known methods, and the resulting sequence data was used to synthesize oligonucleotide probes. Using labeled oligonucleotide samples thus obtained, the applicants have identified the gene encoding alpha-lidiruyushey enzyme from a cDNA library derived from poly And+RNA extracted from the midline soft tissue carcinoma of the thyroid gland of the rat. This gene, which is more accurately characterized in the section detailed description of this proposal may be inserted into appropriate expression vector and transferred to any cell, the owner, is able to Express. Appropriate hosts include, but are not limited to, E. coli yeast strains as S. cereviside, or cells of higher eukaryotes, such as the cell line from which the enzyme was originally obtained by purification. It can be expected that industrial mass production will be greatly facilitated by the creation of such microorganisms.

Mass industrial production of enzymes can be facilitated and using methods of cleaning, including pressure chromatography, and uninominal chromatography, where forms of the enzyme, held uninominal chromatographic column, suiryudan using a saline solution having a concentration is the Oia about 250 mm and preferably above 350 mm or 500 mm. At high concentration saline solution to even the most solenoceridae types of enzyme will be suirvey. Chromatography exceptions size should be conducted to separate the species with apparent molecular weights of about 58000 to 67000 daltons, and preferably from about 60,000 daltons to about 65,000 daltons. The purified product can lidirovat peptidyl compound, which was obtained by purification from natural sources or produced using recombinant DNA technologies, i.e. peptides contain L-amino acids.

Brief description of drawings

Figure 1-4 refer to example 1 and explain it.

Figure 5-9 refer to example 2 and explain it.

Figure 10 refers to the example 7 and explains it.

11 is for example 4 and explains it.

Detailed description of the individual preferred options

We have shown that the homogeneous alpha-lidiruyushey enzyme (peptidyl-glycine α-lidiruyushey monooxygenase) can be obtained by using multi-stage method of using a combination of chromatography exceptions in size and ion-exchange chromatography of extracts of hard tissues of solid tumors, cell lines, tumors and tissue cultures of these cell lines.

The enzyme was extracted from moskovites carcinoma of the thyroid gland of rats (MTS)derived from rat VI the tar, described by Ruzam VA and others (Endocrinology 1979, 150, No. 1, 27-32). This fabric is deposited as O-10028. The enzyme was also extracted from other sources, in particular from cell lines carcinoma of the thyroid gland of rats. Cell line (CA-77) was obtained from tumors of thyroid carcinomas in rats with the help of a few passages, as described Minskim and others (J 1983, volume 258, str-83). This cell line deposited as O-10029. The line of human cells (NTT 54(34)) was developed by Ruzam medical center Cleveland, Ohio, using moskovites carcinoma of the thyroid gland human primary culture. This line (NTT 54(34)) was deposited as an exhibit A-10031. (See "The recognition of the Deposit of microorganisms for the purposes of patenting", indicating that these depositors, and the Budapest testimony," No. 34, November 3, 1983, included in the present description fully by reference).

It has been shown that certain environments tissue cultures from human cell lines and rats had significant levels of activity of alpha-ameerega enzyme, indicating that part of the enzyme was isolated from the cells. The enzyme can be received and cleared prior to processing the source material uninominal chromatography. The sample, for example, can be placed on preparative anionoobmennykh, such as cartridge with diethylamine is the ethyl (DEAE"), such as CUNO 250 shipped in the sale of the company CUNO Inc.

Removed from the column, the composition having alpha-lidiruyushey activity, then treated by exclusion chromatography on a resin appropriate permissions, for example, ultra slim column Sephacryl S-200 supplied by the firm of pharmacy Fayne chemical.

Having activity selected fraction is then subjected to ion exchange chromatography using strong uninominal matrix. The resin, which may be used is a strong uninominal resin Mono Q R5/5 company Pharmacia fine chemical, and for homogeneous purification of the enzyme may have one or more transmission through the column. Column Mono Q HR5/5 has a particle size of 10 μm, the volume of pores of 40% and a gel, a charged group which CH2-N+(CH3)3and ion-exchange capacity is of 0.28-0.36 mmol/ml

Each stage of the treatment can be monitored in terms of protein content and level of alpha-lidiruyushey activity. This information is used to calculate the specific activity of the enzyme, which is an indicator relative to its purity.

Peptidyl-glycine alpha-lidiruyushey monooxygenase purified in accordance with the present invention, the enzyme obtained from rats U-10032; enzyme derived from human U-10033 has an apparent molecular weight of about 60000-65000 Dalton (when determining gelfiltration).

The enzyme has been purified so that it showed the specific enzymatic activity of at least about 25 IU/mg protein and preferably at least about 50 IU/mg protein. Specific activity above about 150 IU/mg protein especially appropriate. Alpha lidiruyushey enzyme was also purified so that showed a single homogeneous well-defined band after electrophoresis on SDS-PAGE.

Purified peptidyl-glycine alpha-lidiruyushey monooxygenase used for amidation alpha-carboxyl group of the polypeptide having terminal glycine residue, where glycine is employed as a donor of amino groups. The substrate can be isolated by purification from natural sources, synthesized from amino acids or produced using recombinant DNA technology. Polypeptide with glycine the end combines with oxygen in the presence of an effective amount of frumenta. The amount of enzyme that is required depends on several variables that are well known in this area, including in particular, but not limited to, the following: the specific activity of this enzyme preparation, the amount and chemical nature of the converted substrate, the time during which the transformation should occur, the temperature and pH of the reaction mixture. Well-versed in this area is the illusion known other variables, which can affect the exact amount of enzyme required in this situation. Oxygen is usually present in a molar excess in the reaction on the concentration of the substrate. The required concentration of copper ions can be provided by using any salt containing the anion, which has no adverse effect on the reaction. For enzyme having a specific enzymatic activity of only about 1 IU/mg protein, maximum alpha-amidation takes place at relatively high concentrations (about 4 mm) of copper ions. When the purity of the enzyme increases, the requirements for the concentration to exogenous (external) copper ion is reduced. The enzymatic activity can also be enhanced by the presence of ascorutinum, which can be obtained using any salt of ascorbic acid with the cation, which has no detrimental effect on the reaction. For a purified enzyme having a specific enzymatic activity of approximately 50 IU/mg protein, the maximum activity of the alpha-amidation is observed at a concentration of about 5 mm ascorbate. Alpha lidiruyushey activity can be increased by adding catalase. The optimum pH value for conversion of biologically relevant substrate in amidarone products ranges from 6.5 to 7.5.

Monoclonal and polyclonal who antibodies directed against the enzyme were obtained immune response using homogeneous enzyme as antigen in mice and chickens, respectively. Both monoclonal and polyclonal antibodies were obtained and purified by the applicants, as shown in example 8. Antibody specific to alpha-lidiruyushuyu the enzyme according to the invention, are stored in the laboratories of the applicants.

Antibodies can be immobilizovana on a solid matrix which is insoluble in the medium in which it is used. It is advisable that the matrix was resistant to destruction. Immobilization of antibodies facilitates the Department α-ameerega enzyme from natural and/or recombinant sources. It is carried out by mixing the immobilized antibody with the initial preparations of the enzyme. Antibodies will bind only to a molecule of alpha-ameerega enzyme. Contaminating proteins are not connected with antibodies and can be easily removed by elution or light centrifugation. After removing impurities α-lidiruyushey the enzyme can be removed from the immobilized antibodies by changes in ionic strength or pH, or by adding chaotropic ions ("affinity Chromatography: Principles and Methods, Management, Pharmacia fine Uppsala, Sweden) and recovered in a highly purified form.

The enzyme was also cleared enough to determine the amino-acid sequence. This information was used for separation of nucleic acid that encodes the enzyme.

The subsequent introduction of this NC in the corresponding single-celled organism or cell eukaryotic host is carried out using standard methods to obtain recombinant DNA, such as described in Maniatis, etc. (Molecular cloning: a Laboratory manual, cold spring Harbor, 1982, or By P. editor. How fermentology, volume 68. Academic Press 1979, entered into the description by reference). The obtained cells containing heterogeneous DNA encoding alpha-lidiruyushey enzyme, produce sufficient quantities of the enzyme to carry out post-translational alpha-amidation in vitro, as well as theoretically suggest the possibility of their use for the implementation of this modification of the peptide or polypeptide in vivo.

Although the present invention describes the preferred variants of its implementation, many changes and modifications will become apparent sveduûŝego in this area specialists. Preferred embodiments of the invention the following examples.

Comparative examples

A. Comparison of test systems for the determination of the specific activity of the inventive preparations

Previously several systems test activity is. Most of the works mentioned in the prior art, used studies based on conversion of D-Tyr-Val-Gli in D-Tyr-Val-amide. This study is quantitative and uses radioisotope connection (125I-D-Tyr-Val-Gli), which is mixed with excess of its material (D-Tyr-Val-Gli). The measured conversion labeled atom provides the extrapolation relative to its material and this, in turn, allows for the calculation of activity.

Although this study was used by the Applicants, determining the activity of the claimed drugs were based on direct measurement of the conversion of Dansyl-Tyr-Val-Gli in Dan-forces-Tyr-Val-amide.

In order to provide a comparison of the specific activity of the preparations known from the prior art, those drugs that are claimed according to the present invention, experiments were conducted comparing systems research.

Experimental protocols are summarized as follows.

I. Monodecyl L-Tyr-Val-Gli

Alpha lidiruyushey enzyme preparation isolated from tumors carcinoma of the thyroid gland of rats and of the environment of tissue culture, isolated after cultivation, SA-77, was used as the source of enzyme in these experiments. The concentration of enzyme used in all the experience is iontach, remained constant, except in those cases where it is specified.

The reaction mixture for converting the Deputy mondanile contained

5 μl enzyme

5 μl 30 mm ascorbate

5 μl 20 mm CuSO4

5 μl 100 μg/ml pankreaticheskoi catalase bull

5 μl, containing 2 nanomole substrate

25 μl 150 mm S pH 7.0

The samples were prepared in duplicate and incubated at 37°C for 10, 20 and 30 minutes. The enzymatic reaction was terminated by adding 10 μl of 500 mm EDTA. Substrate and product were separated using the inverse-phase chromatography equipment for liquid chromatography high-pressure company Hullet Packard 1090, quantification was carried out using integrator HP-392. Conversion monodecyl L-Tyr-Val-Gli in alpha aminirovanie product was linear in time.

II.125I-D-Tyr-Val-Gli

D-Tyr-Val-Gli and D-Tyr-Val-CONH2were jodirovanic using iodine firm piercy chemical company. Labeled substrate and product were used to calibrate the cation exchange column with soulful-propylene.125I-D-Tyr-Val-Gli was added to 650 μ M D-Tyr-Val-Gli and was used as substrate. The reaction mixture for converting the substrate contained

5 μl enzyme

5 μl ascorbate (30 mm)

5 BC; l 100 μg/ml catalase

5 μl 20 μM CuSO4

5 μl of substrate 600 μM final concentration

25 μl 150 mm S pH 7.0

Samples were incubated for 10, 20 and 30 minutes at 37°C. the Reaction was stopped by adding 500 mm EDTA. The sample was diluted with 10 mm sodium phosphate buffer pH 5.2 and placed on a cation exchange column with soulful-propylene. The substrate is not joined to the column, aminirovanie product was blueraven 500 mm NaCl. The conversion of labeled substrate in the product were linear in time.

III. D-Tyr-Val-Gli

The reaction conditions used for amidation D-Tyr-Val-Gli, were identical to those described for Danilovka and iodirovannoi substrates. The substrate concentration in the reaction mixture was 650 μM Separation of substrate and product was achieved by gradient alyazia after L with reversed phase, using a HP-1090 liquid chromatography. Flowing liquid from the column was monitored at a wavelength of 280 nm. The level of sensitivity for this study is significantly lower than the sensitivity in the study Danilovich or yugirovannykh substrates. In order to meet this lower level of sensitivity, reaction of alpha-amidation was conducted for longer periods of time and/or increased amounts of the om alpha ameerega enzyme.

Data analysis of fractional conversion125I-D-Tyr-Val-Gli in125I-D-Tyr-Val-amide, Dansyl-Tyr-Val-Gli in dansyl-Tyr-Val-amide shows that in each moment of time is approximately 1.55 times more itinerating substrate was converted than Danilovka substrate. Thus, when comparing the research system, known from the prior art system research Danilova substrate used by the Applicant, should be used a conversion factor of approximately 1.5.

In addition, it was used more rigorous kinetic analysis for comparison studies with dansyl-Tyr-Val-Gli (Applicants) to study D-Tyr-Val-Gli (previously known). This analysis showed:

SubstrateKmVmax
D-Tyr-Val-Gli

/formerly known/
3731
Dansyl-Tyr-Val-Gli /Applicants/1,721

As you can see when comparing the maximum speed /Vmax= pmol product/min/μl/ for the two substrates, D-Tyr-Val-Gli (previously known) gives approximately 1.48 activity dansyl-Tyr-Val-Gli /Applicant/. This confirms and is consistent with the above information.

B. comparison of the activity

The all the RT and other /PNAS/ defines on page 5147, 4, Vmax= 39 picomoles/micrograms/hour, which is equivalent to a specific activity of 0.65 IU/mg protein / minute. This is the highest activity, which was previously reported. Dividing this value by the above conversion factor equal to 1.5, obtain the specific activity of 0.4 IU/mg protein per minute. This value can be directly compared with the specific activity of the enzyme obtained by the applicants. Applicants, as indicated earlier, reached activity at least 25 IU/mg protein and more than 1500 IU/mg protein. Applicants, therefore, reached activity in 60-3750 times higher than the activity reported by Alpert (NS).

Example 1

Purification and study of alpha-lidirujuwih enzymes, tumor-derived MTS rats

Frozen tumor tissue MTS rats were crushed and homogenized in aqueous buffer using a homogenizer Polytron. After low-speed centrifugation supernatant was drained and the sediment was re-extracted with fresh buffer. This second homogenate was again subjected to low-speed centrifugation and the new supernatant was connected with the first. Two assembled and connected supernatant was then clarified by high-speed centrifugation and the resulting supernatant was used as source the th material for purification of the enzyme.

Were this fractionation obtained by high-speed centrifugation supernatant ammonium sulfate. It was found that much of the activity of the enzyme was settled in the faction 26-40% of ammonium sulfate, the residue from the fraction was purified additionally, as described below.

Was held chromatography on a column of Sephacryl S-300. Under these conditions, the elution was observed main peak of activity, but it was followed by a small additional peak, which could correspond to the enzyme with a lower molecular weight. The question arises whether there is an enzyme with a low molecular weight in vivo or is formed by partial proteolytic digestion during extraction and purification.

The main peak of activity with a column of S-300 was separated using chromatography on a column of Mono Q at pH 6.0. Elution was carried out using uncool linear salt gradient. It was found four peak alpha lidiruyushey activity of the enzyme, while the elution Nl 160 mm, 200 mm, 220 mm and 240 mm. (Peaks I, II, III and IV, respectively, figure 1). This shows that there are many forms of the enzyme and that these forms have a certain heterogeneity. Analysis of the proteins of the peaks of enzyme activity using polyacrylamide gel electrophoresis shows that peaks II, III and IV include the alpha and deruosi enzyme with approximate molecular weights of the same order (73000-75000 daltons), while peak I contains the enzyme excellent, perhaps a smaller molecular weight. The activity peak III was converted to a homogeneous substance, as follows.

The enzyme peak III was collected and subjected to chromatographic treatment on a column of Mono Q HR 10/10 at pH 8.0 (figure 2). The enzyme suirvey with this column as a single peak at 250 mm Nl, as shown by the analysis in the gel, and was purified to a homogeneous state (figa, the strip 6). The following experimental studies were performed on purified enzyme peak III.

1. The molecular weight of the enzyme peak III was determined by analysis on a 7% polyacrylamide gel and was about 75,000 daltons (pigv).

2. The optimum pH for activity of the enzyme was 5.0-5.5 using N dansyl Tyr-Val-Gli as a substrate (figure 4). However, due to the increased stability of the enzyme at neutral pH, may be the successful implementation of the amidation reaction, and at this pH.

3. It is determined that the amount of copper required as a cofactor for activity of the enzyme, was inversely proportional to the purity of the enzyme. Purified to a homogeneous state of the enzyme required 0,1 μM (or less) si++for maximum activity, whereas the initial preparations of the enzyme required 2 μM Cu++.

4. Isoelectric point (PI) of the enzyme with Talala 4,8.

5. The specific activity of homogeneous enzyme peak III was 2,100 IU/mg protein.

The enzyme peak II was also purified to a homogeneous state. However, this enzyme was found that chromatography on Mono Q at pH 8.0 was insufficient to obtain a homogeneous preparation. Therefore, the enzyme peak II after column Mono Q at pH 6.0 (figure 1) was utilizaban against 1 M Tris pH 7.0 and loaded onto a column of phenyl-separate, equilibrated in the same buffer. A large part of the contaminating protein impurities were removed by passing through the column, and lidiruyushey enzyme eluruumis at the last stage, was essentially pure. Additional purification of the enzyme, collected from the column with phenyl-separate, on a column of Mono Q HR 10/10 pH 8.0 resulted in homogeneous preparation of the enzyme peak II, eluting from the column at 220 mm Nl and above.

The study of enzyme peak II showed that

1. The molecular weight of the enzyme peak II during electrophoresis in 7% polyacrylamide gel was about 73000-75000 daltons. Thus, the enzymes of peaks II and III were indistinguishable when comparing their molecular masses.

2. The optimum pH for activity of the enzyme peak II was 5.0-5.5. And again, this characteristic of the enzyme peak II is the same as for the enzyme peak III.

3. Isoelectric point (PI) peak II was bring the flax 5,8.

Example 2

Purification and study of alpha-ameerega enzyme obtained from the environment of tissue culture cells, SA-77

Cell line CA-77 carcinoma of the thyroid gland of rats were grown as monolayer in the presence of 8% CO2. The culture was maintained in a defined medium consisting of modified Dulbecco eagle medium: F - 10(1:1), 3.7 g/liter Panso3, 5 μg/ml transferrin, 10 μg/ml insulin, 30 nm selenium, 4 μg/ml pentamycin sulfate. Culture grown so could be stored indefinitely, if the medium was replaced every 48 hours. To stimulate the capacity of the cells, they were pre-advanced cultivated and grown in a medium containing serum (5% HP and 2.5 fetal calf), within three days. Cells were then washed twice with saline phosphate buffer followed by the replacement of the specified environment.

The tissue culture medium was collected with respect to the asepsis 48-hour program and were stored at -20°before cleaning. Cultural environment (usually 6 liters) was diluted with 2 liters of deionized water (3:1) and fed at a flow rate of 50 ml/min to anynobody cartridge (Kuno No. 250), which was previously balanced 1,0 liter of 20 mm bis Tris: Hcl pH 6.0 at a temperature of 4°C. alpha lidiruyushey enzyme (alpha-AE") was blueraven stupen the ATO using 50 mm Tris Hcl pH 7.0, containing 500 mm NaCl at a flow rate of approximately 50 ml/min Fractions having the activity of alpha-AE (specific activity of 10-15 IU/mg), two obtained using anion-exchanger drugs were collected and concentrated 4-5 times under reduced pressure using a rotor pre-treatment savant RH-100.

This material was injected directly into the column 5×50 cm from Sephacryl 300 - SF (Farmacia). The mobile phase consisted of 100 mm Tris: Hcl pH 7.0, with a flow rate of 1.0 ml/min All gel-filtration chromatography was performed at 4°With (figure 5).

The drug ameerega of the enzyme at this stage of treatment is free from non-specific proteolytic activity and has an activity of at least 50 IU/mg protein. The drug ameerega enzyme, obtained at this stage, has been used successfully for amidation of recombinant elongated glycine human calcitonin and factor in the release of growth hormone. Since the density of cells 1-1,5×106cells/ml, we gradually got out 200-350 IU activity ameerega enzyme per liter of medium, passing these two stages of treatment. The enzyme is stable and suitable for use in solution or after immobilize it on a solid substrate.

Combined fractions of the column containing the active alpha-AE, were cialisbuy against 6 lit is s 20 mm bis Tris: Hcl pH 6.0. Dialysate was introduced into a column for strong uninominal chromatography Mono Q HR 10/10, pre-equilibrated to 20 mm bis Tris: Hcl pH 6.0. The enzyme was blueraven from the column using a linear gradient of 0-300 mm Nl for three hours at a flow rate of 2.5 ml/min. Four chromatographically distinguishable forms of alpha-ameerega enzyme were divided into these three stages. Peaks were numbered in order of elution from the column (6). Peaks III and IV present form higher molecular weights of the enzymes and correspond to peaks II and III, obtained from tumors MTS. Peaks I and II represent the form with lower molecular weights of the enzymes that can be proteolytic fragments of peaks III and IV.

Four forms of alpha-ameerega enzyme identified in the laboratory differ from each other in their average surface charge, which is evident from their different times of retention during a strong uninominal chromatography (6). Optimal pH values for these four chromatographic excellent forms of the enzyme are also different. The results, shown in Fig.7 show that the peaks III and IV have the same value of pH optimum between 5.0 and 5.5. These results are consistent with the optimum pH determined for peaks II and III, obtained by isolation and purification from tumor MTS. Peaks I and II have wider is the range of activity with a pH optimum of from 5 to 8.5 (Fig.7). These results are consistent with the optimum pH reported by Alpert and others (Peptides 4, 921-28, 1983) and Marty and others (Biochemistry 261, 1815-22, 1986).

Phase enzyme of peaks II and IV isotope PA125I and subsequent electrophoresis in SDS-PAGE confirmed that the enzyme activity peak IV is characterized by an approximate molecular weight 73-75 KD, whereas the enzyme activity peak II had a molecular weight of below 55 KD. The exact molecular weight of the enzyme from the peak II is unknown, because he was not cleared until smooth (a few protein lines appear in the range of 45-55 KD).

Enzymes peaks III and IV can be purified to a homogeneous state with a combination of hydrophobic and strong uninominal chromatography at pH 8.0. The enzyme peak IV (6) was collected, concentrated to about 2 ml in vacuo and directly introduced into the column 1,3×8 cm with phenyltetrazol (Pharmacia), equilibrated with 500 mm Tris: Hcl pH 7.0. Fractions having the activity of alpha-AE, were suirvey equilibrating buffer at a flow rate of 0.5 ml/min (Fig). Fraction peaks with alpha lidiruyushey activity were collected, utilitary against 50 mm Tris: Hcl pH 8.0, and then introduced into the column Mono Q HR 10/10, equilibrated with 50 mm Tris: Hcl pH 8.0. The enzyme was blueraven using a linear gradient of 0-300 mm NaCl for three hours at a flow rate of 2.0 ml/min (Phi is .9). Fractions with alpha AE activity, erwerbende at 240 mm or higher were collected, brought to a content of 0.001% (in volume) Triton X-100 and stored at 4°C. the Specific activity of the purified enzyme was measured approximately 1500 IU/mg protein at pH 7.0. Peak III α-lidiruyushey activity is purified to a homogeneous state with the same operations described for peak IV.

Physico-chemical characteristics of the tumor enzyme peak (from example 1) and enzyme peak IV tissue culture (from example 2), including the molecular weight (73000-75000 daltons), optimum pH (5.0-5.5), the sequence of aminobenzo and conditions of elution (more than 240 mm sodium chloride at a strong uninominal chromatography carried out at pH 8.0) showed that these two peaks may represent the same enzyme.

Example 3

Alpha-amidation of biologically relevant peptide hormones using alpha ameerega enzyme

Several substrates representing recombinant peptides, including those that are designed for salmon and human calcitonin, factor in the release of human growth hormone and peptide linked to the genome of the human calcitonin, have been received and successfully alpha lidirovali drug alpha ameerega enzyme according to the present image is ateneu. For illustration purposes, the following is the procedure used for the production of active recombinant calcitonin salmon, peptide, associated with the gene calcitonin, and factor in the release of human growth hormone.

The same approaches to problem solving can be used for other recombinant peptides.

Calcitonin salmon is a 32-membered peptide hormone that has an alpha aminirovanie polynomy residue at its carboxyl end. Using genetic engineering were created microorganisms for the production of recombinant fused protein containing the amino acid sequence corresponding to the calcitonin salmon. Gene fused protein was constructed so that the sequence of salmon calcitonin was concluded between methioninol balance aminocore and glycine residue at the C-end, which was also finished all of recombinant protein. After binding of the gene calcitonin salmon in plasmid microorganism has been transformed with this plasmid was achieved by the expression of the fused protein. This calcitonine protein was isolated from the lysate of the recombinant microorganism by deposition and its cysteine residue converted to S-sulfonates. Because salmon calcitonin does not contain methionine, CYANOGEN-bromide the splitting of the giving of the recombinant fused protein has led to the creation of a peptide containing a sequence of salmon calcitonin with glycine continuation of the carboxyl end. This peptide was isolated using or liquid chromatography high-pressure reversed-phase, or ionoobmennoi chromatography, and its structure was established using amino acid composition and analysis micropaleontology.

The penultimate polynomy the residue was converted into prolinamide under the action of alpha-ameerega enzyme. The conditions used for alpha-amidation of the peptide, are shown below. Lyophilized peptide substrate (extended glycine is a precursor of calcitonin salmon 200-300 nanomoles) was dissolved in 200 ml of buffer 150 mm Tris: Hcl pH 7.0, containing approximately 750 uE alpha ameerega enzyme. The enzyme can be extracted or tumor MTS, or from the environment of tissue culture cell line of rat MTS SA-77. The enzyme should be cleaned to such an extent that all impurity proteolytic activity was eliminated. Ascorbic acid and copper sulfate were then added to the mixture in amounts sufficient to provide a final concentration of approximately 3 mm and 2 μM, respectively. Catalase (7.5 g/ml), ethanol (1% by volume) and potassium iodide can be introduced into the reaction mixture to increase the release of calcitonin. The resulting solution was cm the Shang and incubated at 37° C for 5-6 hours.

After removal of the S-sulphonate groups using a beta mercaptoethanol processing, recombinant salmon calcitonin was purified using liquid chromatography high-pressure reversed-phase. The final product was characterized by its retention in the process of liquid chromatography high-pressure reversed-phase, quantitative distribution tripticase of parivara and amino acid analysis. In all examples, the recombinant calcitonin salmon did not differ from synthetic calcitonin salmon.

Human peptide that is associated with the gene of calcitonin, is a 37-membered hormone that has an alpha aminirovanie phenylalanine residue at its carboxyl end. Was designed gene fused protein, similar gene calcitonin salmon (see above) in that regard, the sequence of the peptide was made between methioninol residue and glycine residue, which was also graduated from the recombinant protein. Release, treatment, alpha-amidation and study of recombinant precursor peptide linked to the genome of the human calcitonin were carried out in the same way as it was done in connection with the recombinant calcitonin salmon.

The factor in the release of human growth hormone (hGHRF) is a 44-membered peptide hormone, and is Audi alpha aminirovanie Lazenby residue at its carboxyl end. Gene fused protein for hGRF was designed so that the amino acid sequence for the peptide hormone was concluded between the tryptophan residue at its aminocore and glycine residue at its carboxy end, which was also graduated from the recombinant protein. Containing hGRF protein was isolated from the lysate of the recombinant microorganism by means of deposition. Not containing hGRF part of the fused protein was are denatured by converting a cysteine residue to S-sulfonate derivatives. Because hGRF does not contain tryptophan, the chemical digestion of artificial recombinant protein reactive BNPS-skatole was caused by oxidative cleavage of the fused protein, resulting in naudinianus hGRF with glycine elongation carboxyl end. At the same time meinenemy residue at position 27 in the molecule hGRF was oxidized to methionine-sulfoxide. This extended glycine peptide was isolated using gel filtration and liquid chromatography with reversed phase. Its structure was established using amino acid analysis of the peptide fragments obtained by trypsinogen digestion.

The penultimate Lazenby the residue was converted into leucinamide under the action of alpha-ameerega enzyme according to the present invention. Example y is th, used to produce alpha-liderando hGRF, below. Lyophilized peptide substrate (20-40 nanomoles) was diluted in 150 μl of deionized water and mixed with 90 μl (500 μ (E) (pH 7.0) preparation of alpha-ameerega enzyme in tumor MTS or culture medium of the cell line CA-77 rats. The enzyme was purified to remove all kinds of impurity proteolytic activity by use of gel filtration and ion exchange chromatography. Ascorbic acid and copper sulfate were added to the mixture of enzyme and substrate in amounts sufficient so that the final concentration was 3 μM and 2 μM, respectively. The resulting solution was mixed and incubated at 37°C for 4-6 hours. The usual percentage conversion of substrate to product is 95% in terms of amino acid analysis of the fragments that were selected during the digestion by trypsin. Finally, methionyl-sulfoxide balance was restored to methionine 4 M betterlatethannever, buffered at pH 4, 10 mm sodium acetate, at 80°C for one hour. The final product was purified using liquid chromatography high-pressure reversed-phase and investigated according to the retention time, the analysis of the trypsin digestion and amino acid analysis. Recombinant alpha-aminirovanie product b is l also tested in relation to biological activity. In all examples, the recombinant hGRF was indistinguishable from synthetic hGRF.

In addition to the above examples, two industrially important peptide hormone, elongated glycine, were assessed for their ability to be used as the substrate alpha-ameerega enzyme. These materials were the precursors of alpha-melanocyte stimulating hormone and substance P. In both cases, the results show that both peptides are suitable substrates for alpha-ameerega enzyme according to the invention.

Example 4

Sequence analysis of purified alpha-ameerega enzyme Fractions containing purified alpha-lidiruyushey enzyme were obtained or from tumor tissue MTS rats, or supernatants cell cultures SA-77; sulfhydryl group of the enzyme were subjected to recovery, followed by karboksimetilirovaniya. The reaction mixture was then introduced into a column for liquid chromatography high-pressure reversed-phase (particle size 5 μm, the pore size of 33 nm), which was equilibrated with 0.1% aqueous solution triperoxonane acid. The column was washed with this solution to remove excess buffer salts. The desalted enzyme was removed from the column by elution with 80% acetonitrile containing 0,08% triperoxonane key is lots. The resulting stream (effluent) of the column was monitored by UV at a wavelength of 220 nm. The obtained protein fractions were collected, combined and liofilizovane. This material was then diluted in 100 ml of 0.1% SDS and then introduced into the device for the study of protein sequence (Applied Biosystems model A). Operations used to analyze micropaleontology, was carried out as described by the manufacturer. Received phenylthiohydantoin amino acids were analyzed using liquid chromatography high-pressure column Hypersil C18 (particle size 5 μm, pore size 10 nm) by monitoring absorption at the wavelength of 269 nm and 313 nm for the liquid chromatography system Hewllet Packard 1090. Amino-terminal sequence for the main component peak enzyme (peak 111) of the tumor tissue was as follows

123456789101112
NH2Ser-Phe-Ser-Asn-Glu-Cys-Leu-Gly-Thr-Ile-Gly-Pro-

1314151617181920212223242526
Val-Thr-Pro-Leu-AspAla-Ser-AspPhe-Ala-Leu-AspIle-Arg-

2728
Met-Pro-

Aminobenzene sequence for the main component of the enzyme (peak IV) of the supernatant tissue culture cells, SA-77 shows that it is identical to the one that has a place for the enzyme tissue tumors (peak III). However, not the most component was also detected during the analysis of micropaleontology of this enzyme, which, apparently, contains a continuation of aminocore when compared with the General form of alpha ameerega enzyme. The presence of these components, probably due to the difference in post-translational processing of the enzyme. The sequence of aminocore this components was as follows:

123456789101112
NH2Phe-Lys-Gly-Thr-Thr-Arg-Ser-Phe-Ser-Asn-Gly-Cys-
1314
Leu-Gly

Additional data on amino acid sequence would be and obtained for alpha-ameerega enzyme in tumor tissue MTS rats using the following operations. Approximately 400 μg purified enzyme was subjected to restore and carboxymethylamino. After this procedure, the enzyme solution was transferred to the extrusion of dialysis, and dialysis was carried out for 18 hours at 25 mm Tris-Hcl pH 8.0/0.5 m urea. The precipitate was then peresan 1.5 ml centrifuge tube and concentrated to a volume of 600 μl under reduced pressure. To a solution of enzyme was added 2 μl (2 μg) trypsin and the mixture is incubated for one hour at a temperature of 37°C. At this point, added the second dose of trypsin (2 μg) and continued incubation for two hours at 37°C. the Digestion was finished by adding 200 μl 4M urea /10% acetic acid. The product of digestion was then introduced into the chromatographic column for liquid chromatography high-pressure reversed-phase (particle size 5 μm, the pore size of 33 nm), which was equilibrated 0,10% aqueous solution triperoxonane acid. The column was then eleirovania linear gradient of acetonitrile to a concentration of 50% within four hours, and fractions were collected with a two-minute interval. The obtained peaks of the elution liquid chromatography high-pressure reversed-phase product tripticase digesting enzyme plotted in figure 11. Three obtained using trypsin peptide were automates the computerized analysis of sequences described above, and the results obtained are given below. (The peptides identified by their number fraction).

Trypticase peptide No. 65

-Ser-Met-Gln-Pro-Gly-Ser-Asp-Gln-Asn-His-Phe-Ser-Gln-Pro-Thr-

Trypticase peptide No. 58

-Asn-Gly-Gln-Trp-Thr-Leu-Ile-Gly-Arg-

Trypticase peptide No. 86

-Phe-Val-Thr-Gln-Trp-Cly-Clu-

Example 5

Molecular cloning of DNA sequences encoding alpha-lidiruyushey enzyme moskovites carcinoma of the thyroid gland of the rat or cell SA-77.

First of all, you need to find a reliable source of enzyme protein, which in turn can be a source of informational RNA /mRNA/ and ultimately complementary DNA /cDNA/. Isolating enzyme gene or cDNA also requires molecular probes specific to the enzyme. Typically, this molecular test takes one of two forms; it is either an oligonucleotide whose sequence is complementary to part of the gene encoding the enzyme or antibody molecule or collection of molecules, antibodies which are specific recognizes a protein enzyme. The receipt of such molecular probes requires the development of such a method of purification of the enzyme that could be received specific antibody enzyme or could be determined amino acid sequence of the enzyme for subsequent design oligonu etigny samples. These conditions have been fulfilled for the alpha ameerega enzyme according to the present application.

Alpha lidiruyushey the enzyme was purified from tissue moskovites carcinoma of the thyroid gland of the rat and of the conditioning environment CA-77 cells of the rat. It was indicated that these sources will contain encoding the enzyme mRNA. The methods we used to prepare cDNA alpha ameerega enzyme are well known in the field of molecular biology. Special protocols for these different methods can be found in laboratory manuals such as Molecular Cloning /1982/, DNA Cloning /V.1/ a Practical Approach /1985/ or primary sources, such as Gubler Century and Goffman VJ. (gene 25, 262-69 /1983/; or young, R.A. and Davis, W. (PNAS, 80, 1194-98, /1983/). These procedures have General applicability to critical variable, which is the source used mRNA. For the preparation of specific cDNA ameerega enzyme, we used mRNA tissue moskovites carcinoma of the thyroid gland of the rat mRNA and cell SA-77. Samples of double-strand cDNA were synthesized, in turn, was used to prepare a separate gene libraries using well known procedures.

As described above, the identification of specific cDNA mRNA for alpha-ameerega enzyme requires molecular probes that can distinguish these forms from others who Orme particular library. Examples 1 and 2 detail cleaning methods used in the preparation of enzyme required to generate molecular probes. Example 4 describes the use of this protein to determine the amino acid sequence, while example 6 describes the use of purified protein for the preparation of specific antibodies to the enzyme.

Amino acid sequence of example 4 is sufficient to obtain specific selective oligonucleotide samples. In the preparation of oligonucleotide samples several factors are critical in order to make them selective. A full description of these arguments can be found in the Manhole R.J. (Y. Mol. Biol., 183, 1-12, /1985/). As is usually the same amino acid sequence can be encoded by more than one nucleotide sequence /principle, known as the degeneration of the genetic code/any single nucleotide sequence will represent only one of a number of potential genetic sequences. In order to ensure that the oligonucleotide probe is used to identify the gene of interest can be prepared equimolar mixture of all possible sequences that can encode a specific amino acid sequence ameerega enzyme. The complexity is still the mixtures often makes them not completely selective, therefore, to obtain brand-specific selectivity for a given gene should be used oligonucleotide mixture for more than one region amino acid sequence.

An alternative possibility is that the oligonucleotide probe selective for the gene of interest, can be prepared by creating a unique nucleotide sequence of sufficient length that even when not fully complementary to the desired gene, leads to the formation of a stable hybrid. This unique sequence will have a very small probability of forming a stable hybrid with other gene sequences. The unique sequence is composed of the most frequently used codon for each amino acid protein sequence. The frequency of codon usage for the given sample can be determined from a compilation of known gene sequences and corresponding amino acid sequences for protein samples. These methods are well known to molecular biologists.

Another approach that can be used to prepare specific oligonucleotide probes, involves the incorporation of deoxyinosine residues in the oligonucleotide in the position of maximum degeneration. This nucleot is DNA replacement attenuates the degeneration of the reference sample, and thus, may have a beneficial effect on the selection process. /Use deoxy inosine oligonucleotide in the sample, see Otsuka, etc. Y. Biol., Chem., 260, 2605-08 /1985/.

We used the sequence of the protein alpha-ameerega enzyme for designing sets of oligonucleotide probes, suitable for isolation of cDNA ameerega enzyme. Used sequence and we prepared samples are shown in table 2. It should be noted that when the knowledge of the amino acid sequences for isolation of the gene by hybridization sample specialist in molecular biology can be used and alternative strategies.

Thus obtained oligonucleotide samples were used in accordance with known methods (see Molecular Cloning, 1982/ for screening plasmid and ragovoy libraries cDNA and to isolate cDNA alpha ameerega enzyme.

Table 1 illustrates all possible gene sequence corresponding to the selected regions of the molecule alpha-ameerega enzyme. Below the designed sequences shown some of the additional oligonucleotide probes, which are useful for the identification and isolation to DNA (see end of text).

Example 6

A. Receiving murine monoclonal antibodies specific for alpha-ameerega fer the enta

Twelve mice lb/J were immunized and reimmunization purified preparations ameerega enzyme. In these mice was taken as the blood, and the serum was processed and titrated against the purified enzyme. The study was carried out by absorption of the purified enzyme on the polystyrene plate, which was then washed and blocked to prevent unauthorized attachment of antibodies to the plate/ bovine serum albumin /BSA/. Diluted mouse serum /preferably containing antibodies against ameerega enzyme was then incubated on coated lidiruyushchim enzyme plate and washed. Secondary antibody tagged with a marker, alkaline phosphatase, which facilitates the fixing of the binding of the first antibody coated plate enzyme was then incubated in cells. After washing and addition of the substrate solution, the signal was calorimetrically measured using spectrophotometric recording device. "Positive" serum showed the ratio of signal to noise ratio of at least 2:1.

Mouse No. 7, which showed higher titers in ELISA test, was put to death four days after the final reimmunization. The spleen was aseptically removed and minced with getting to 132.6×106the spleen cells that were SL is you 122,8× 106cell myeloma NS-1 using 1,28 Mls PEG /peg/ 4000. Cells were divided into five 24-cellular plates, which were covered in thymocytes and splenocytes of Balb/cJ, which were used as feeder cells. The cells were kept in HAT selective medium that allows for the survival of the only hybrid cells.

Supernatant from 116 cells, which showed clonal growth were skanirovaniya using radioimmunoassay of samples and method LIS to generate antibodies. The radioimmunoassay procedure the sample was similar to the ELISA test described previously, except that the second antibody was labeled with I125and radioactive score was measured using a gamma counter.

Fifty-six of the 116 cells were positive for obtaining antibodies and were skanirovaniya on reactivity towards alpha lidiruyushuyu the enzyme. Twenty-five clones that were positive for alpha-lidirujuwie enzymes were cloned by serial dilution. Primary clones were skanirovaniya against alpha-ameerega enzyme and BSA /as polystyrene plates were blocked with BSA, antibodies that were bound to the plate, probably just stuck or were absorbed BSA - unspecifically/ to determine whether or not they is ecifica for ameerega enzyme. The clones that showed the signal at alpha lidiruyushey enzyme and which was at least doubled in comparison with demonstrate to BS were cloned with a distribution ratio of 1 cell/ 2 cells.

Twenty-one positive hybridoma /see table 2/ has been moved to the stage of tertiary cloning, and twenty of them were characterized with respect to the class of antibodies using techniques. Ouchternoly and LISA. Seventeen clones had the heavy chain lg G2a and three had lgG heavy chain1. All twenty clones gave prints with Kappa light chains.

Each line were individually grown in mass culture, and aliquots of cells were frozen in liquid nitrogen. Mice Balb/cJ an injection was intraperitoneal cells hybridoma (5×106). A week later the mice that have not appeared ascitic tumor, was made of reinforced injection of cells. Ascitic fluid and blood were removed 1-2 weeks later. After the treatment of ascites and serum were skanirovaniya and titrated against alpha-ameerega enzyme, as well as against negative antigens /for example, BSA, egg protein, carbonic carbonic anhydrase and factor release growth hormone/ to ensure the specificity of the antibody.

Table 2

Mnogokanalnye cell line al is a-ameerega enzyme Balb/c mice
Cell lineHeavy chainThe titer of1
4-1-4-20-3lgG2a~1:5,000
4-1-4-18-1lgG2a 
4-5-7-3lgG2a~1:5,000
4-2-3-17-7lgG2a 
3-7-1-20-24lgG2a~1:10,000
4-8-15-2-6lgG2a 
4-7-21-14-9lgG2a 
4-10-45-1-16lgG2a 
54-2-1-39-2lgG2a~1:10,000
4-6-12-3-17lgG2a 
52-1-31-24-2lgG2a 
4-3-11-46-1lgG2a 
3-7-9-9-3lgG2a 
86-1-38-15lgG11:1,000
4-5-6-1lgG11:1,000
4-4-24-3-5lgG2a~1:5,000
4-11-3-1-8lgG1 
8-9-94-4-2lgG2a 
4-10-30-3-1lgG2a 
75-10-17-14-4lgG2a 
92-10-26-32-39Not classed 
1Ascitic fluid was titrated with 100 ng of purified enzyme in solid-phase ELISA test

C. cleaning Procedure for monoclonal antibodies produced in mice

Monoclonal antibodies specific for alpha-ameerega enzyme prepared by the above methods, were purified as follows. Ascitic fluid collected from several mice inoculated with the same clone was used as source of antibodies. Ascitic fluid was diluted /5x/ 10 mm MES pH of 5.6. Diluted ascitic fluid was placed in a 1.5×20 cm column containing 40 μm, AVH-mixed silicon resin /Gutmaker/pre-equilibrated to 10 mm S pH of 5.6 buffer. Monoclonal antibodies were suirvey from the column using a 0-500 mm gradient of sodium acetate pH 7.0. The fractions containing purified antibodies were combined, tested for specific activity and stored at 4°C until further use.

C. Obtaining polyclonal antibodies specific for alpha-ameerega enzyme in chickens.

Nutrion is e, intramuscular and subcutaneous injections were made two chickens (for a total of approximately 50 mg of purified alpha-ameerega enzyme in Ribi Freund per chicken). Ribi adjuvant is a fully metabolized fat emulsion system, which consists of a mitogen for lymphocytes chicken and adjuvant to enhance the reaction of antibodies to antigens in poultry /Ribi immunochemical research, Installation/. After the initial immunization were two reinforced injection with a two-week interval (approximately 50 mg of the enzyme in chicken). The animal was taken from the blood by approximately 21 day 35 day, and serum were processed and examined for the presence of specific antibodies using the following procedure: serum from both chickens, day 0 /preimmune/, day 21 and day 35 were shielded solid-phase ELISA method against 100 ng of purified alpha-ameerega enzyme. BSA was used as negative control for nonspecific adhesion antibodies. Enzyme-specific antibody was detected rabbit marking alkaline phosphatase serum against JgG chicken.

The results described above showed that specific antibodies can be detected in the sera of chicken 257 35 day. Diluted 1-10 .000 serum gave attitude is ignal-to-noise ratio of approximately 4:1. Chicken 258 showed enzyme-specific antibodies on day 21 and 35 day. For both blood samples, diluted 1:10.000 serum gave the signal to noise ratio of approximately 4:1. The collection of eggs from both chickens began on day 56. Insulated polyethylene glycol /PEG/ IgY from preimmunization eggs and postimmunization eggs was analyzed using the techniques Ouchterlony, and enzyme-specific antibodies were skanirovaniya using ELISA-tests.

D. Purification of chicken lgY-antibodies

Polyclonal avian antibodies specific for alpha-medisinaldepot enzyme were produced in chickens, as described above. Eggs from immunized chickens were collected and either immersed in paraffin or frozen until used for purification lgY. Egg whites are separated from the yolks, which contain specific antibodies to alpha-ameerega enzyme. Egg yolks were diluted three-fold with 10 mm sodium phosphate pH 7.5, containing 0.1m NaCl and 0.01% azide. The initial stage of deposition of ED was carried out using a 3.5% final concentration G8000. Deposition occurred within 30 minutes at room temperature followed by centrifugation, and was left supernatant /containing lgY/. To the supernatant was added polyethylene glycol to obtain a final concentration of 12.5%, PEG lqY antibodies, OSA is built in such concentration and of polyethylene glycol, were separated by centrifugation. lgY antibodies this stage of treatment was further purified using two methods:

1/. Besieged lgY antibodies were re-suspended to 10 mm MES pH of 5.6, and then subjected to dialysis overnight at 4°against the same buffer. The sample was then placed in a 1.5×20 cm column containing 40 μm AVH mixed silicon resin /Gutmaker/. The subsequent treatment Protocol was similar to that described for ascitic fluid.

2/. Alternatively, the precipitate containing lgY, was re-suspended in the starting buffer and then re-precipitated using saturated ammonium sulfate /3:1 V/V/.

The precipitate, containing lgY, was re-suspended in a small volume of distilled H2Oh and stored at 4°C until further use. The immobilization procedure for lgY Chicks described in example 10.

Example 7

Isolation of DNA sequences encoding peak III α-ameerega enzyme

Preparation of RNA:

Total RNA was prepared from tissue moskovites carcinoma of the thyroid gland of the rat using the guanidine-thiocyanato procedures. Poly And+RNA was isolated using oligo d T pulp.

Synthesis of cDNA:

Dunaeva cDNA were prepared using well known methods. Using poly a RNA from tissue mozgoved the Oh carcinoma of the thyroid gland of the rat as template and oligo dt 12-18as a seed, synthesis of the first thread was made fermentive reaction with reverse transcriptase. cDNA and RNA were separated and RNA destroyed with alkali. The synthesis of the second cDNA strands was semestrale using DNA polymerase 1 E.coli. Digestion with nuclease S1 was used for removal of hairpin loops in cDNA and for splitting any single-stranded regions of cDNA. After the reaction with DNA polymerase 1 for promoting growth ends on cDNA Dunaeva cDNA was treated with metrazol EcoRI and S-adenosylmethionine for methylation sites EcoRI and protect them from subsequent enzymatic cleavage. Linkers RI were legirovanyh to cDNA. After digesting RI excess linkers were removed and cDNA was fractionated by size on a column with Separate 4B. In the first synthesis were collected molecules larger than 500 BP, while in the second were combined to clone molecules larger than 1000 base pairs.

Construction of cDNA library λgtll:

Following the synthesis of the double helix cDNA of adapted connections molecules were used to create libraries of cDNA in the vector λgt ll. This was done by ligating the cDNA to DNA λgt ll, which was split EcoRI and treated with phosphate to prevent caroligian the vector DNA. After legirovaniem DNA, recombinant DNA was packaged in vitro to form infectious particles of the bacteriophage. (Extracts for packaging are available in the form of industrial products laboratories Libraries or Clonex or can be prepared by standard methods).

After packaging the DNA aliquots packing of the mixture were tested for the presence of recombinant forms in collections. As it was discovered that one of the libraries contained approximately 2.57 m×106infectious particles, approximately 78% of which were explicitly recombinant /giving clear spots on X-Gal plates when grown in the presence of IPTG/. Other library had some 2.75×106units forming plaques, and approximately 81% obvious recombinant.

Screening of the library.

In order to identify recombinant bacteriophage, which included cDNA alpha ameerega enzyme, phage was investigated using radiometric oligonucleotide probes, designed based on the specific amino acid sequence of alpha-ameerega enzyme. /See example 5, table 2/. Screening was performed by inoculation of samples of bacteriophage and transfer of phage on nitrocellulose filters. Procedures for immobilization of phage on nitrocellulose filters are well known. Two filter from each plate were the guy who idisovna R 32the oligonucleotide AE 9. Hybridization was carried out at 37°C for 20-24 hours at 6x PET, 0,5% N40, 5x Demanovska solution, 100 μg/ml DNA salmon sperm with oligonucleotide breakdown of 0.3-0.4 pmols/ml. After hybridization the filters were washed in 6x SCC at 44-45°within a few hours and exposed to x-ray film. Positively hybridities phage was identified as matching spots on duplicate filters. They were purified by successive enrichment through several stages of sowing and hybridization. Of the approximately 4-5×104analyzed phages 18 were identified using AE 9.

To confirm the specificity of selection was performed by hybridization with a second oligonucleotide alpha ameerega enzyme. This test found that at least four of the eighteen phages were carrying cDNA for sequences of alpha-ameerega enzyme. This discovery was confirmed by further hybridization with the 4 th and 5 th, and analysis of DNA sequences.

The expression α-ameerega enzyme

Peak III αAE /alpha ameerega enzyme/how we determined protein sequence had a molecular weight of about 75,000 daltons. If the average molecular weight of amino acids taken as 120 daltons, lidiruyushey enzyme is, p is at least 625 amino acids. The gene for 625 amino acids must contain at least 1875 base pairs. All four cDNA that we isolated as specific to ameerega enzyme, are too large to fully encode the protein alpha-ameerega enzyme. One of the cDNA clones, λAE, consists of approximately 2200 nucleotides. Within the first 50 nucleotides from the end he starts encoding the amino acid sequence, which was identified as the N-end of the enzyme peak III. Therefore, we can conclude that this cDNA contains all the information required for encoding the enzyme peak III.

One of the possible procedures that can be applied further to cDNA λAE1 depicted in figure 10. Namely, the insertion of a cDNA λAE1 size 2200 base pairs isolated after digestion of DNA with recombinant bacteriophage using EcoR1 and agarose gel electrophoresis. She is cloned into the EcoR1 site pBR322 to obtain plasmid RAE-1, RAE-1 contains a unique cleavage site PKK 1 within the cDNA sequence and unique site Hind III inside a sequence of pBR 322. Digestion RAE-1 using Kpn1 and Hind III yields a fragment of about 2.15 KBP, which lost about 62 base pairs of cDNA /encoding the amino terminal end of the cDNA/. For recovery of amino acids found at the amino-end of the ICA III ameerega enzyme, and to adapt cDNA for cloning into a plasmid expression, Kpn1 fragment-HindIII legasuite with oligonucleotide linker adapters. In the above example, for expression in E. coli was used plasmid RCC-2 by the pharmacy. The cDNA fragment size of 2.15 KBP, legirovannye with the linker-adapter included:

AE/+/305' CATGTCATTTTCCAATGAATGCCTTGGTAC3'

and

AE/-/225' CAAGGCATTCATTGGAAAATGA3'

Adapted fragment is then ligated with DNA plasmids RCC-2, which had been previously cleaved with NcoI and Hind III to obtain a linear vector of 4.6 KBP Legirovannye product, RAE contains the cDNA for the alpha ameerega enzyme with that preceded the start codon ATG and the binding site of the ribosome, under the control of hybrid IPTG inducible trc promoter. For gene enzyme gene should 5S RNA and the site of the transcription has been completed. Induced by IPTG, the expression of the recombinant enzyme was performed after transformation of the plasmid DNA pAE12 .oli with genotype laciq. To determine the activity used the same methods as in the case of natural AE.

Partial sequence of the cDNA insert of 2.2 KBP in λE.

The insert size of 2.2 TPO was cut out by digestion λAE1 using EcoR1 and was labelled32P. After the secondary digestion with Hinc 11 the resulting fragments of 1.6 TPO and 0.6 KBP were connected to the HN in sequence by the method of chemical decomposition of Maxima and Gilbert.

Obtained by sequencing MAXIMO-Gilbert DNA sequence EcoRI-end of the fragment S-1 size 600 BP;

Obtained by scanning MAXIMO-Gilbert sequence of the EcoRI end of the fragment SAE-1 size 1600 BP;

The study of the homology sequence of the oligonucleotide samples AE/-/22

Computational study was performed to determine the homology between the amino acids used to obtain samples AE /Leu-Cty-Thr-Ile-Gly-Pro-Val-Thr/ and broadcast partial DNA sequence 600 BP R fragment λAE1.

Was obtained region of perfect homology, it is highlighted by asterisks on the DNA sequence and are indicated in capital letters for amino acids. Amino acids identified as NH2- terminal sequence of purified liderando enzyme enclosed in parentheses. The name of the amino acids that were found different from that prescribed by the DNA sequence, are indicated by a sign "+".

Example 8

Immobilization of enzyme

Cleaning αAE:

Before immobilization of alpha-lidiruyushey the enzyme was purified weak uninominal chromatography and gel-filtration /example 2/. In some cases, the enzyme preparation may further include the cleaning using either chromatography immune affinity or chromatography on perseverate. The procedure of sequential immobilization regardless of the cleanup; however, the specific activity should be at least 25 and preferably 50 mi or above.

Immobilization αAE:

Technology for immobilization of alpha-ameerega enzyme may be based on the simultaneous reaction of three components: enzyme, the water-soluble copolymer of acrylamide /PAN/ and having a low molecular weight reagent cross /TET/. Pre-designed polymer /RAP/ consists of acrylamide and N-aryloxyalkyl, which is polymerized in a solution of THF using thermal initiation azibiza /isobutyronitrile/. Reagent with cross connections can be α,ω-diamine, Triethylenetetramine /TET/, applied. The reaction of the diamine with active ester groups PAN creates a cross-connection chain of the polymer via amide bond and forms an insoluble gel. The amino function of the enzyme /preferably ∈-amino group of lysine/ at the same time reacts with residual active esters on the gel and forms a stable covalent amide bond. The immobilization procedure is carried out in the presence of substrate and cofactors. The presence of the substrate with a high degree of affinity and cofactors in concentrations above Tomprevents reaction between the active ester PAN and nucleotide sequence that is multidisciplinary groups near or at the catalytic site of the enzyme, protecting the enzyme from chemical inactivation.

The immobilization conditions:

Partially cleared α-AE was dissolved in 30 mm HEPES buffer pH 7.0. The ratio of RAP and TET is set so that 15% of the active esters remain unreacted, which serves as binding sites for alpha-ameerega enzyme. Standard solution RUP represents 20% /W/W/. The reactive mixture of alpha-ameerega enzyme consists of 0,2 uSO4, 40 μM of dansili His-Phe-Gly and 10 mm ascorbate and 0.5-2.0 mg α-AE/gram PAN. The concentration of TET is calculated to be equal to 0.85 equivalents of primary amine/equivalent active ester. To determine the optimum conditions, the reaction is run without enzyme to identify the time of gel formation, i.e. the length of time after addition of TET necessary to achieve gel formation. The optimum is obtained for immobilization of alpha-ameerega enzyme, reached when the addition of the enzyme moves closer to the point of gel formation. The General rule is that the less time the enzyme is subjected to PAN before gel formation, the greater is the yield of active immobilized enzyme. For most reactions α-AE /~ 2.0 mg/g RAP/ added 45-60 seconds after TET. The enzyme-containing gel is left at room temperature in ECENA approximately one hour, to complete the reaction of the compound. After the expiration of 60 minutes the gel is homogenized using a mortar and pestle, resulting in obtaining fragments with an average size of 100 μ. These particles are washed with ammonium sulfate to remove unbound reactants and to convert the residual active groups of the ether amides, thereby coating the reactive group. Active alpha-ameerega enzyme after immobilization is expected to be less than 60%. Wash after immobilization may contain from 30 to 40% of the initial activity. Alpha lidiruyushey the enzyme can be recovered from flushing by increasing the concentration of ammonium sulfate to 45%, which leads to the precipitation of energetic alpha ameerega enzyme.

Particles immobilized gel suitable for the reaction of alpha-amidation in a mixture where the particles are in suspension with a mechanical stirrer. After completion of the enzyme reaction, the particles are precipitated and the supernatant containing the product liderando peptide decanted. This procedure is not optimal for larger scale reactions. Particles containing the enzyme gel is not strong enough to about packing in the column and to obtain a reasonable flow rate. To circumvent this problem, there are two alternative approaches:

1. The gel is given the opportunity polymerization in the presence of glass beads; usually mine who have to achieve the "gel" point glass beads are added to the reaction mixture and mixed in a mechanical mixer prior to coating the beads with a layer of mortar PAN. Fluid properties of this composite material are much better than the gel particles.

2. Alternatively, particles RAP mixed with the goal filtration with Celite 545. Usually a mixture of RAP and Celite is prepared in the PAN, forming less than 8% W/W /dry weight/ mixture. To obtain this type of a column of gel particles were weighed in 50 mm Tris:HCl pH 7.0 and with constant stirring added to Celite 545 and mixed for two hours. The column is Packed this sludge /3×40 cm and the column temperature is maintained at 37°to facilitate the amidation reaction. When using this approach achieved a flow rate of 8-10 liters per day.

An attractive alternative to column chromatography is the use of the system with tangential flow. The polymer PAN before the gel point spreads on the sheet polysulfone porous substrate of 0.45 μm After the formation of the gel sheet can be cut for use in ultrafiltration units with tangential flow system Millipor or new Brunswick. In this design the composite sheets of gel, immobilized polysulfone PAN enzyme, stacked layers, leading to a significant increase in surface area. This approach can be directly scaled to the velocity of the flow approaching liters per minute. This type of system also provides the t of the re-transmission of the reaction mixture to maximize amidation of the peptide substrate.

The advantages achieved by immobilization of alpha-ameerega enzyme include the recovery and reuse of the enzyme. Secondly, immobilized matrix increases the stability of the enzyme and provides a working form of the enzyme, suitable for bolsamania reactions amidation /from grams to kilograms substrate by number/ in for long periods of time.

Example 9

Preparation of immune-affinity column for purification of contaminated compositions alpha ameerega enzyme

A. immobilization Procedure:

Used matrix immobilization was CYANOGEN bromide-activated Sepharose 4V /Formation/. The dry gel was first washed with 1 mm Hcl /200 ml./gram resin/ for washing the solid substrate. Approximately 40 mg of purified polyclonal antibodies isolated from egg yolks of eggs, subjected to dialysis using 100 mm Panso3pH 8.3, containing 0.5 M NaCl. The connection is made using 8 ml of pre-washed solid substrate. The reaction was carried out for three hours at room temperature in acetate buffer with a concentration of 100 mm, pH 8.3, containing 500 mm Nl, which was included to reduce nonspecific link protein with solid base. Remaining active groups of the gel were blocked IP is by the use of 0.2m glycine. After blocking gel was washed up to five times by using a series of buffers with high and low pH /buffer high pH; 100 mm Panso pH 8.3+500 mm NaCl buffer with low pH; 100 mm acetate with pH 4.0+500 mm Nl/. These stage wash to remove any unbound protein and blocking reagent /glycine/ from resin. Resin with immune affinity was placed at 4°in the basic pH of the buffer containing merthiolate as a bacteriostatic agent. All subsequent chromatography with immune affinity is at 4°C. a Similar method can be used for immobilization of purified monoclonal antibodies.

C. Immune-affinity chromatography

Column immune affinity is used as an alternative stage high efficiency in cleaning α-AE. The environment of tissue culture cells, SA-77 /see example 2/ is diluted with distilled water and under pressure is passed through D-anion exchange pre-balanced 20 mm Bis Tris-HCl pH to 6.0. Alpha lidiruyushey the enzyme eluted with 50 mm ris:Hcl pH 7.0 containing 500 mm NaCl. The fractions containing αAE activity or subjected to dialysis against buffer ris:HCl. pH 7.0 or further purified using gel-filtration chromatography /example 2/ before chromatography of immunoreactive. Samples containing αAE, are passed through immunoadsorption is Olonne with a neutral pH. The specific antibodies will bind alpha-ameruoso enzyme, while contaminating proteins will not be connected and will be removed in aliante. Activity αAE can be eleirovania from the column using 100 mm glycine Hcl buffer with pH 3.0, /can be used other desorption agents, including urea, dioxane, ethylene glycol, and N1/. Fractions are collected in 1.0 M Tris:HCl pH 7.0 for neutralization buffer systems, thereby preserving the activity αAE.

The method of obtaining liderando peptide, comprising contacting the precursor peptide with C-terminal glycine, with peptidylglycine-alpha-lidiruyushey monooxygenases, which received expression in the cell host DNA sequences encoding peptidylglycine-alpha-ameruoso the monooxygenase and is able to gibridizatsiya at least two probes selected from the group consisting of:

oligonucleotide probe AE

oligonucleotide probe AE

oligonucleotide probe AE

oligonucleotide probe AE

oligonucleotide probe AE

oligonucleotide probe AE

oligonucleotide probe AE 9

oligonucleotide probe AE

and oligonucleotide probe AE

Convention priority is selected from 14.08.1987 in accordance with the application 86161, filed in the U.S. Patent office.



 

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