Recombinant protein expression in double-strand c form

FIELD: chemistry.

SUBSTANCE: polypeptides or proteins in double-strand form are obtained via recombinant expression in E. coli host cells. The polypeptide or protein exhibits its biological activity in form of a double-strand polypeptide or protein; C-terminal amino acid group of the first strand is an Arg or Lys'groups; the second protein/polypeptide strand has in its N-end 1-20 amino acid groups and a VPXGS (PRS) pentapeptide sequence, where X denotes any naturally occurring amino acid, where V - Val, Leu, He, Ala, Phe, Pro or Gly, P - Pro, Leu, He, Ala, Phe, Val or Gly, G - Gly, Leu, lie, Ala, Pro, Phe or Val, and S denotes Ser, Tyr, Trp or Thr. The method of obtaining the disclosed product involves the following steps: modification of the polypeptide or protein on the nucleic acid level, so that said polypeptide or protein in its modified form contains a VPXGS sequence in its loop area, where X, V, P, G and S are as defined above; incorporation of a structure modified at the nucleic acid level into the E. coli cell; culturing and then lysis of host cells and separation of the double-strand polypeptide or protein which is, for example, a botulinum neurotoxin, particularly type A botulinum neurotoxin (BoNT(A)).

EFFECT: obtained proteins and polypeptides provide cytotoxic effect in small dosages.

23 cl, 9 dwg, 7 ex

 

One aspect of the present invention relates to a method of obtaining proteins in double-stranded form by recombinant expression in cells of the host E. coli. Another aspect of the present invention relates to proteins or polypeptides in double-stranded and biologically active form, which can be obtained by the aforementioned method.

An important advantage compared with the corresponding recombinant proteins/polypeptides that do not show signs according to the invention, is that there is no need to expose them to the specific processing protease for directed cleavage of the polypeptide chain, so the retrieval method is greatly simplified. Additional aspects of the present invention are nucleic acids that encode the polypeptides/proteins according to the present invention; vectors which contain such nucleic acid or nucleic acid sequences; cell owners who, in turn, contain the above-mentioned vectors; and, finally, the pharmaceutical compositions that contain double-stranded and biologically active proteins/polypeptides.

Clostridial neurotoxins are strong inhibitors of calcium-dependent secretion of neurotransmitters in neuronal cells. After oral administration of botulinum toxins (BoNT), the example through bad food, there will be clinical picture called botulism, which is characterized by paralysis of various muscles. Paralysis of the respiratory muscles can eventually lead to death of the infected person. In this regard, the signal transmission from the nerve to the muscle is interrupted at the motor end plate, since the motor neurons can no longer secrete acetylcholine. Botulinum neurotoxins develop their inhibitory effect through proteolytic cleavage of proteins involved in the processes of secretion, the so-called SNARE proteins. In this context, the various neurotoxins serotypes have different specificity in relation to the SNARE proteins and sites of cleavage in the corresponding amino acid sequences. BoNT(A) and BoNT(E) split the SNARE protein SNAP-25, while BoNT(C) recognizes as SNAP-25, and syntaxin-1 as a substrate. Also toxins serotypes b, D, F and G, as well as tetanus toxin (TeNT) split VAMP-2 (synaptobrevin-2) (Schiavo et al., 1997).

Clostridial neurotoxins are the strongest known poisons. For example, injected a lethal dose at which half of all mice group dose die from botulism, is only 5 PG. The fact that the toxins of most serotypes are also toxic when administered orally, is a result of the atom complex proteins, in which they are enclosed and which, thus, protect them from destruction by digestive enzymes as they pass through the gastrointestinal tract. They also attribute function in the absorption of toxins through the epithelium of the small intestine (Fujinaga, 1997).

In the last decades of the botulinum toxin serotypes a and b found in therapeutic applications. For example, it is possible by directed injection only minimal doses to relax individual muscles when they are chronic spasm. A special advantage is the long-term effectiveness, for example, BoNT(A) and BoNT(B) during the period from more than three to six months. The first indications were, among other things, dystonia, such as torticollis, blepharospasm and strabismus; were added additional indications, such as hyperhidrosis or cosmetic treatment for wrinkles. The market of botulinum toxin as a therapeutic agent is rapidly growing, not only in connection with the development of additional indications, but also due to more intensive use of already existing applications. In this regard, attempts have been made to improve the properties of neurotoxins in relation to duration, efficiency and antigenic potential. The tests showed that the complex proteins that are contained in commercially available preparations (VOTO is, available from Allergen, and Dysport, available from Ipsen-Beaufort, as the drugs BoNT(A)and Myobloc/Neurobloc, available from Elan, as the drug BoNT(B))does not have positive effects on the duration and efficiency, but due to the higher amount of protein compared to pure drug neurotoxin with the same activity can trigger immunological reactions in the patient, so further injections become ineffective.

Because of the complex proteins are not required in the preparation of the active ingredient and even have shortcomings, and some modifications to improve properties can only be achieved by using genetic engineering technologies, there is a huge need for neurotoxins by recombinant expression of, for example, by expression in Escherichia coli (neurotoxins formed in this way, free from the above-mentioned complex of proteins). New readings should develop, in addition in the direction that botulinum toxin should be given different cell specificity. In this regard, the path through recombinant toxin or a derivative of the toxin is also preferred.

Botulinum toxin and tetanus toxin have a high degree of homology in terms of their amino acid sequences and are what I like, in particular, in relation to their domain structure. They consist of domain-binding receptor (HC), the translocation domain (HN) and catalytic subunit (L), which performs in the nerve cell splitting corresponding SNARE protein. HWithresponsible for specific binding of neurotoxins with motor end plates, whereas translocation domain provides passage L from endosomes into the cytoplasm of neurons. HN(N-end) and HWith(C-end) form a heavy chain 100 kDa, while L represents the light chain and forms the catalytic subunit of 50 kDa. Both polypeptide chains are connected to each other by a disulfide bridge. Among the participating cysteine residues of the linker region or area of the loop (also called a synonym of the linker sequence, or a sequence of loops, or more simply, the linker or loop), the length of which between botulinum and toxin individual serotypes very variable. At the last moment of the release of toxins from Clostridium during cell lysis loop is cleaved clostridiales the endopeptidase, which is still not characterized, where the ratio of the split and unsplit types varies between serotypes. For activity neurotoxins splitting loop to double-stranded toxin is significant is th (Schiavo et al., 1997). For example, in the case of botulinum neurotoxin And Decapeptide is cut out of the loop, i.e. in the sequence loops VRGIITSKTKSLDKGYNKALNDL, which is on the N-end and C-end cysteine residue as a nearest-neighbor balance, not only one peptide bond is cleaved, but there are two events proteolytic cleavage. In this regard, molecular weight biologically active botulinum neurotoxin And in nature lower than the original toxin, broadcast by Clostridium. Because clostridial protease is absent in other organisms hosts, such as Escherichia coli, recombinant botulinum toxins and fragments or derivatives expressed them in the form of single-chain peptides. This is probably also true for any other proteins that perform their normal biological/biochemical activity in the form of double-stranded protein. Typically, such proteins get through recombinant DNA technology in the form of single-chain proteins, their biological/biochemical activity, which they manifest in nature in the form of double-stranded proteins, therefore, hardly present or absent.

Previously with the goal of creating an active protein, in particular of active botulinum toxin was necessary introduction sequence recognition is La is specific to the sequence of the protease, such as thrombin, factor XA AA or kenenisa, so that after cleaning, it was possible to perform splitting and hence activation by adding endoprotease. The use of such endoprotease has two significant drawbacks: on the one hand, it is not always possible to exclude that in the amino acid sequence are present other additional cleavage sites except one cleavage site, which was added by using genetic engineering techniques. Even when these secondary sites of cleavage cut is much less efficient after treatment with protease in the result may be a mixture of different variants cleavage of the toxin, which can be separated only with difficulty. On the other hand, in the case of pharmaceuticals, for reasons of pharmaceutical legislation (regulatory guidance) significant disadvantage is the extra added protein or the possibility of contact of the drug with additional protein, because it must be ensured the complete removal of this protein and its possible impurities in the further processing; this typically requires a significant investment.

Activation by proteolytic cleavage to double-stranded polypeptide with a disulfide bridge is also required in the case of other bacterial toxins, for example the EP pseudomonades of exotoxin or diphtheria toxin to implement the enzymatic domain of toxic action (e.g., by ADP-ribosylate factor elongation and, thus, inhibition of protein synthesis). These toxins used to produce so-called immunotoxins, which are used, in particular, in the treatment of tumors. For this purpose, the cell binding domain of toxin replace protein domain, which has a high affinity binding ofwholesale surface protein (differentiation antigen or an antigen associated with a tumor). Although the classical immunotoxin these protein domains are composed of the monoclonal antibody or its fragment, specific for certain tumor cells can also be given through cytokines, growth factors, and mutated and selected protein families affilinet, proteins with ancyranum repetitions or anticalins, naming just a few examples. When recombinant expression of such fused proteins receive single-chain polypeptides. Although, for example, ricin has no site for processing proteases, with the exception of proteases Ricinus communis, and such site shall be inserted, fragments of diphtheria toxin and fragments pseudomonades of exotoxin as components of immunotoxins split after internalization in endosomes the compartment protease target cells. This occurs in the area of the loop between cysteine residues to which that form a disulfide bridge. However, only a minimal part, but not all of internalized molecules immunotoxin undergo processing in this way (Ogata et al., 1990).

With the aim of obtaining recombinant proteins, in particular, polypeptides smaller in sufficient quantities and in a soluble form in many cases it is necessary to Express them in the form of a fused protein or hybrid protein, for example, glutathione-S-transferase or protein, maltose binding in Escherichia coli. In addition, commercially available various expression systems, by means of which the desired polypeptide is expressed by N-terminal or C-terminal tag for affinity purification, such as labels His, Strep tag or label FLAG. In many situations in the expression plasmid has the sequence of the protease recognition between multiple cloning sites where integrated DNA sequence encoding a desired protein, and the coding sequence for a partner to merge or affinity label. This sequence is designed to ensure that after the expression and purification of fused protein it was possible to separate the desired protein from the additional peptide regions by adding a suitable specific to the sequence of endoprotease (e.g., thrombin, factor XA or generasi). If the two merge partner were related to each other is d covalent bond via a disulfide bridge instead of a peptide bond, perhaps it would be separating from each other after purification by simple recovery tolstushki substances such as β-mercaptoethanol, dithiothreitol (DTT) or restored glutathione. For example, the desired protein can be eluted from the affinity matrix, such as agarose Ni-NTA or separate StrepTactin the above-mentioned reducing agents, whereas the affinity tag remains associated with the matrix. Following purification step to separate the affinity tag or added endopeptidase could thus be eliminated.

Therefore, it would be desirable to develop a method of recombinant expression of proteins/polypeptides in General, in particular neurotoxins, as well as fragments and derivatives of these neurotoxins, as well as fused proteins or hybrid proteins, in particular immunotoxins that are already present after lysis of host cells, in their biologically active double-stranded structure, where the two chains linked by a disulfide bridges. This method of obtaining such proteins and polypeptides proposed in this invention.

Unexpectedly, the inventor has found that as a movie LHNBoNT(a)and full-size neurotoxin And, both obtained by recombinant expression in a single chain, but also showing its normal biological/biochemical actively is to be in double-stranded form disulfide bridges, produced by recombinant expression in double-stranded form, when the fragment LHNor full-toxin, preferably at the level of nucleic acid, is subjected to at least one specific modification. Subsequent tests conducted by the inventor have shown that this remains true also for any other proteins/polypeptides due to the fact that they receive in accordance with conventional recombinant methods in a single chain, but they exert their biological activity in double-stranded form disulfide bridges.

The above-mentioned "at least one modification in the case of BoNT(A) or in the case of fragment LHNBoNT(A) refers to the insert pentapeptidnogo sequence, called PRS (site of the protease recognition). In General protein/polypeptide pentapeptidnogo sequence that is present in this protein/polypeptide that you want to modify (preferably at the level of a nucleic acid)can be modified in this way (for example, by at least one of the replacement amino acid residue, or by inserting only a few amino acid residues PRS, or by deletion of amino acid residues)to match with pentapeptidnogo sequence PRS, built in already present sequence. In the same way which can be inserted hexa/hepta/Oct- (and so on) peptide sequence with or without deletion of any one, or two, or three, or more amino acid residues. In accordance with the invention, the only advantage is that end-expressed polypeptide has PRS (Pentapeptide) sequence in its area of the loop, where the loop region according to the invention is defined as the amino acid sequence that is between two cysteine residues involved in disulfide bridge. When this sequence PRS is present in the area of the loop, the consequence of this is that after cleavage of single-chain polypeptide adjacent to a polypeptide sequence PRS (at the amino acid level, the sequences that are present in nature in two different circuits, also are distributed on two different circuits. In the case of botulinum neurotoxin A (BoNT(A)) this sequence PRS preferably embedded in the loop by deletions of Pentapeptide Asp443-Asp447BoNT(A) (see Fig.3-1). In other proteins/polypeptides (for example, in the case of BoNT(B), BoNT(C1), BoNT(D), BoNT(E)in the case of ricin, in the case RE pseudomonads exotoxins or in the case of diphtheria toxin (DT)) instead, it is preferable to embed the modified loop BoNT(A) in the sequence of the loop (see figure 3-2 - 3-5), where amino acid residues of the natural sequence of the loop can be either deleterows, is because not deleterows. Modified sequence loop figure 3-2 - 3-5 represents a sequence without the two terminal residues Cys, where the Central amino acid sequence PRS can represent not only R, Y, N or Q, but also any other naturally occurring amino acid. In the case of the above-mentioned other proteins/polypeptides is particularly preferable to embed part of the modified loop BoNT(A), in particular the sequence GIITSKTKSLVPXGSKALNDL (X = a naturally occurring amino acid, where the amino acid residues of the natural sequence of the loop can be either deleterows or not deleterows). Modified sequence loop figure 3-2 - 3-5 represent the sequence without the two terminal residues Cys.

For a fragment LHNBoNT(A) or for full-length recombinant toxin this means, therefore, that the modification of the sequence represents the change in the area of the loop between L and HNand this change ensures the presence of sequence PRS. According to the invention the sequence of the PRS, and not only for BoNT(a)constitutes pentapeptidnogo sequence Val-Pro-Xaa-Gly-Ser. Xaa means any naturally occurring amino acid. Regardless of whether a Xaa Arg or any other naturally occurring amino acid is a, on pentapeptidnogo sequence Val-Pro-Xaa-Gly-Ser in any case referred to as pentapeptidnogo sequence. When, however, one of the other four amino acid residues of the sequence PRS replaced, what is really possible in the context of the present invention, in particular, appropriate hydrophilic/hydrophobic or polar unipolar remains, this sequence will be called in this context and in the future as an option PRS-pentapeptidnogo sequence. Options are, for example, when Val replaced by Leu, Ile, Ala, Phe, Pro, or Gly. In addition, options are present when (also or only) Proline in the second position of the PRS in the N-end, replaced by Leu, Ile, Ala, Phe, Val or Gly. Glycine in the fourth position of the PRS can be, for example, replaced by Leu, Ile, Ala, Pro, Phe, or Val; this leads to other options. And when serine in the fifth position of the PRS replaced, for example, Tyr, Trp, Thr, it is also possible Cys or Met, there is another type of option. According to the invention are those sequences that contain at least one of positions 1, 2, 4 and 5 of the PRS sequence amino acid residue which is different from the Val-1, Pro-2, Gly-4, and/or Ser-5, call options pentapeptidnogo sequence.

When a fragment LHNBoNT(A) (or full-toxin) or any other protein/polypeptide, usually produces the text by recombinant expression in the form of a single-chain protein/polypeptide, but has biological/biochemical activity (only) in double-stranded form, contains pentapeptidnogo sequence Val-Pro-Xaa-Gly-Ser (where XAA represents any of the 20 naturally occurring amino acids and four other amino acids may be replaced in accordance with the value given in the previous paragraph), it will be present in the lysate of the cells of the host E. coli (such as E. coli K12, in particular cells of the host E. coli K12 strains M15[pREP4], XL1-BLUE or UT5600) in double-stranded form, where in the case BoNT(A) a light chain linked by covalent bond with HNor full-size heavy chain by a disulfide bridge (Fig.7). Cleavage of the polypeptide chain is made directly after lysis of the cells, or essentially completed after several hours of incubation of the cell lysate. Autoproteolysis through activity protease domains of the toxin or toxin fragment can be excluded, because the mutants are inactive on the protease that is modified accordingly in the area of the loop, are also present in the double-stranded structure after expression and destruction of host cells E. coli. Obviously, the protease of strain-host E. coli responsible for the breakdown pentapeptidnogo sequence PRS.

The following preferred modification in accordance with the paragraph beginning "Suddenly the inventor of the four paragraphs above, is that on the N-end of the sequence PRS at a distance of from 1 to 20 amino acid residues (amino acids towards the N-Terminus, which is located directly next to the valine residue pentapeptidnogo sequence PRS, in the case of 3-2 - 3-5 the leucine residue has a distance of 1 amino acid residue from the sequence PRS), in particular, at a distance of from 3 to 15 amino acid residues, especially at a distance of from 3 to 10 amino acid residues, particularly preferably at a distance of from 3 to 8 amino acid residues, and even more preferably at a distance of 3 amino acid residues present basic amino acid residue, preferably a lysine residue or an arginine residue, where the end of the protease of the host cell E. coli cleaves a sequence loop. After cleavage, thus, produces a polypeptide which is, for example, has a two amino acid residue (defined above where the distance is 3 amino acid residue) - end-from valine residue sequence PRS. In this case, the "modification" does not necessarily imply a modification in the true sense, i.e. insertion or substitution of amino acid residue so that the N-end of the sequence PRS at a certain distance above from 1 to 20 amino acid residues was located basically Amin is an acid residue (for example, the lysine residue). It is important that basic amino acid residue such as lysine residue or a residue of arginine) were present at the N-end of the sequence PRS on the above-mentioned distance.

Another modification, also optional, but preferred, in accordance with paragraph "Suddenly the inventor..." five paragraphs above, is that the sequence of the loop, in which the protease host cells E. coli enzyme, has a length of at least nine amino acid residues. The preferred length of the loop sequences comprise at least 12, at least 15, at least 18, at least 20 and at least 23 amino acid residue. Especially preferred length of the sequence loops range from 15 to 22, in particular from 18 to 22 amino acid residues.

The method according to the invention is a General method of obtaining proteins/polypeptides in double-stranded form, where the two chains linked by a disulfide bridge, by recombinant expression in cells of the host E. coli, where (1) the protein/polypeptide exerts its biological activity in the form of double-stranded protein/polypeptide with a disulfide bridge; (2) the C-terminal amino acid residue of the first chain is a residue of Arg or Lys residue; (3) the second circuit protein/polypeptide has an N-end of the cyst is the new balance as N-terminal from 1 to 20 amino acid residues and pentapeptidnogo sequence VPXGS, designated as PRS, where X is any naturally occurring amino acid, where V is Val, Leu, Ile, Ala, Phe, Pro, or Gly, where P is a Pro, Leu, Ile, Ala, Phe, Val or Gly, where G represents Gly, Leu, Ile, Ala, Pro, Phe, or Val, and where S represents Ser, Tyr, Trp or Thr; and (4) this method includes the following stages: (a) modification of the protein/polypeptide level nucleic acids to a protein/polypeptide in its modified form was within his area of the loop above pentapeptidnogo sequence (VPXGS); (b) the introduction of design, modified at the level of nucleic acid into cells of E. coli; (b) culturing and subsequent lysis of host cells; and (d) isolation of double-stranded proteins/polypeptides.

According to the invention the first circuit protein/polypeptide preferably is a chain, which is encoded by the N-end of the corresponding DNA, whereas the second chain of the protein/polypeptide, respectively, is a chain that is encoded With the end of the respective DNA. Because the expression of 5'-DNA-3' leads to N-polypeptide, in the above preferred case of the invention this means that the expression can be represented as follows: 5' DNA 3' to N is expressed first polypeptide chain With a loop-N-the second polypeptide chain is C. According to the invention loop splitting of aetsa already in situ, so in the end, the polypeptide/protein N-first polypeptide chain From the N-second polypeptide chain according to the invention is formed in a double-stranded structure.

The expression "the second circuit protein/polypeptide has an N-end of the cysteine residue as the N-terminal from 1 to 20 amino acid residues and pentapeptidnogo sequence VPXGS designated as PRS" means that N is not formed, for example, the valine residue pentapeptidnogo sequence VPXGS, but others (any) amino acid residue. Between the latter and the valine residue PRS can be further located from 1 to 19 amino acid residues, but the N-terminal amino acid residue may be linked directly, for example, with the residue of valine, through a peptide bond, i.e. it can represent the closest residue adjacent to a valine residue PRS.

Proteins/polypeptides according to the invention, which can be allocated to them (biologically) active double-stranded structure, are proteins in which the end of the first chain has a basic amino acid residue, in particular the residue is Arg or Lys residue, and in which the second circuit is provided with N-end of 1-20 amino acid residues and pentapeptidnogo sequence VPXGS called PRS, where X, V, R, G, and S are defined as above.

According to the present invention in the case of immunodeficiency is toksinov, based on recombinant ricin, for example, the processing specific to the sequence of a protease, such as thrombin or factor XA to activate is unnecessary. For example, in the case of immunotoxins on the basis of the diphtheria toxin or pseudomonades toxin expected a significant increase in efficiency, and it really is also obtained, since the treatment with protease target cells as limiting the speed stage to move the enzymatic domain of toxins in the cytoplasm is no longer required. Such immunotoxins that are already present in the form of double-stranded polypeptide with a disulfide bridge can be used in small doses and still provide the same cytotoxic effect. This reduces, on the one hand, the cost of therapy and, on the other hand, reduces the risk of antibody formation, which would make immunotoxins ineffective in subsequent applications. A method of obtaining a double-stranded with a disulfide bridge and, therefore, activated immunotoxins proposed by the present invention.

The method proposed in this invention, it is also possible getting fused proteins or hybrid proteins, i.e. proteins with a peptide tag for affinity purification of double-stranded form, the two polypeptide chains are covalently linked by disulfide is the first bridge, and after affinity chromatography or other methods of cleaning may be separated by a simple restore tolstushki substances such as β-mercaptoethanol, DTT or restored glutathione.

Recombinant expression clostridial of neurotoxins and their fragments (e.g., fragment LHNor derived clostridial neurotoxin, for example, with a modified cell specificity) in the expression strains of E. coli, such as M15[pREP4] or BL21(DE3), gives the single-chain polypeptides. In the processing of these polypeptides trypsin cleavage takes place in the field of sequence loops in the transition region proteasome domain translocation. Because trypsin is a protease that is specific to the sequence, probably splitting, usually undesirable, in additional regions of the polypeptide. For example, BoNT(A) optionally cleaved by trypsin between the HNand HWithso get a double-stranded fragment LHNand a fragment of HWith. To ensure selective cleavage in the area of the loop, is desirable in most cases, must be present, possibly after insertion, sequence recognition for specific endoprotease.

Cleavage of recombinant fused protein/hybrid proteins by specific sequence is lnasty of endoprotease, such as thrombin, factor XA, kenenisa etc. is within the scope of the well-known spectrum of methods. Perhaps the Department after cleaning partner mergers, which gives improved solubility of the recombinant protein/polypeptide and/or enhanced expression of, or serves as a peptide tag for affinity purification. For this purpose, the protein solution is incubated with soluble endoproteases in soluble form or in an immobilized form on the matrix.

This technique can also be used for expression of the above-mentioned recombinant proteins/polypeptides that show their normal biological/biochemical activity in the form of double-stranded protein/polypeptide by recombinant DNA technology are obtained in the form of inactive single-chain proteins/polypeptides (e.g., expression clostridial neurotoxins, fragments clostridial of neurotoxins, such as fragments LHNor derivatives clostridial neurotoxins, for example, with a modified cell specificity). Sequence recognition for endoprotease clone in the polypeptide, preferably at the level of nucleic acids, for example, in the area of the loop between L and HNand, in addition, clone N-end or at the C-end of optional sequence recognition for the same Il is more endoprotease, flanked peptide tag for affinity purification. Then single-stranded expressed protein/polypeptide is activated by processing the corresponding endoproteases or endoprotease simultaneously or sequentially by splitting the loop area between L and HNand the peptide tag is removed.

In addition to the cost of the use of such endoprotease and the need, therefore, additional stages of processing, their use in pharmaceutical preparations (for example, the use of recombinant botulinum toxin or derivatives thereof) is highly problematic for reasons of pharmaceutical legislation (regulations). On the one hand, the purity of the used endoprotease should be confirmed experimentally and, on the other hand, the complete removal and, in particular, freedom of the drug from viruses during Protocol treatment must be accurately documented; this generally requires great expenses for the analyses. Because in the future botulinum toxins, for example, with improved properties or modified cell specificity should be made by recombinant expression, there is a great need in the way of expression, which ensures obtaining the above-mentioned recombinant proteins/polypeptides that show their is armalloy biological/biochemical activity in the form of double-stranded proteins/polypeptides, but obtained through recombinant DNA technology in the form of inactive single-chain proteins/polypeptides, in particular, provides the botulinum toxin or derivatives thereof in the form of double-stranded with a disulfide bridge, and therefore, biologically active polypeptides/proteins, without the use of endoprotease.

In the invention, which will be explained in more detail hereinafter, is offered in a very broad sense, the way in which proteins, such as clostridial neurotoxins, as well as their fragments and derivatives can be obtained by recombinant expression in cells of the host E. coli and can be allocated in their double-stranded with a disulfide bridge, and therefore, biologically active form, without their activation requires adding endoprotease.

In the first preferred embodiment of the invention the amino acid sequence region loops BoNT(A) between cysteine residues 430 and 454 (see figure 3-1 - 3-5) is modified in such a way that expressed the toxin or its fragments/derivatives in the cell lysate of the host E. coli are present in the form of double-stranded polypeptide. These two chains are covalently bound to each other with the participation of cystine residue 430 and 454 through a disulfide bridge. In a particularly preferred embodiment of the invention, as explained in figure 3,the Pentapeptide Asp 443-Asn447(DKGYN) can be replaced by a Val-Pro-Arg-Gly-Ser (VPRGS). In additional preferred embodiments of the invention Pentapeptide Asp443-Asn447(DKGYN) can also be replaced by a Val-Pro-Tyr-Gly-Ser (VPYGS), Val-Pro-His-Gly-Sr (VPHGS) or Val-Pro-Gln-Gly-Ser (VPQGS). In this context also remains true that not only the Central amino acid residue may be any naturally occurring amino acid, but also the fact that four other amino acid residue can be replaced, as explained in detail above (replacing at least one of these residues version of the PRS sequence is present in the meaning of the invention). In addition, in both this embodiment and other preferred embodiments which will be explained later, it remains true that, in addition, preferably, when the loop sequence is at the N-end to PRS at a distance of from 1 to 28 amino acids basic amino acid residue, in particular the residue is lysine or arginine.

Specialist in the art it is obvious that additional replacement of individual or several amino acid residues or insertion or deletion of additional amino acid residues in the field described above loops BoNT(A) also leads to the result that the expressed toxin according to the invention or education is consistent from it fragments/derivatives in the lysate are present in the form of double-stranded polypeptides. These options are also covered by the present invention.

Specialist in the art will also readily apparent that the Pentapeptide Asp443-Asn447(DKGYN)present in BoNT(A) wild-type, may be replaced by a Hexapeptide, heptapeptide, oktapeptidom etc. so that expressed and translated into single-stranded form of the polypeptide/protein in the area of the loop was present PRS-pentapeptidnogo sequence or one of its possible variants. As explained above, preferably, when N is the end of this Pentapeptide present basic amino acid residue (preferably lysine).

In addition, the specialist in the art it is obvious that the preferred embodiment of Pentapeptide (Val-Pro-Arg-Gly-Ser) PRS represents a portion of a possible sequence recognition for protease thrombin, which plays an important role in the cascade of blood coagulation and has a high specificity for the sequences. It should be particularly noted that, first, nor in the botulinum neurotoxin type a, or in other polypeptides do not require cleavage by thrombin order to obtain the desired double-stranded form a disulfide bridge, and that, secondly, the sequence recognition of thrombin itself, that is, in its immobilized form, favorable for the split is to be placed by the activity of the lysate of E. coli, but not mandatory. Embodiments pentapeptidnogo sequences PRS, which is built or constructed in the respective polypeptides (better: in their loops that do not contain an arginine residue at the C-end of which thrombin can cleave (instead, there is another naturally occurring amino acid), also lead to the splitting of the loop, as explained above. This cleavage is carried out preferably when the lysine residue of the loop, which is located on the N-end Pentapeptide, as explained above (see also example 2; Figure 3).

Because other serotypes of botulinum toxin, such as BoNT () and BoNT(C1) as neurotoxins long steps and BoNT(E) as neurotoxins short-term actions, as well as completely different polypeptides/proteins that can be expressed by recombinant in a single chain, but exert their biological activity only in the form of double chains, can be used therapeutically, it would be desirable that these neurotoxins, as well as their fragments or derivatives (as well as other polypeptides/proteins) could be also obtained in the form of double-stranded polypeptides/proteins with disulfide bridge from lysates of E. coli. In particular, in the case of BoNT(B) complete cleavage of recombinant toxin in E. coli lysate to double-stranded polypeptide/protein would provide a significant Avenue the property compared to the native neurotoxins, which are secreted into Clostridium botulinum, which generally account for 40 percent of the attendees in the form of single-stranded and, therefore, inactive polypeptide and may not be separated from the active double-stranded form. It is also clear that the area of the loop of neurotoxins serotypes b, C1 and e, and between the cysteine residues involved in disulfide bridge, relative to a hinge BoNT(A) is much shorter (Fig 3 and 4). If in the case of BoNT(A) there are 23 amino acid residue (Val431-Leu453), BoNT () only 8 (Lys438-Ile445), BoNT(C1) 15 (His438-Asp452) and BoNT(E) 13 amino acid residues (Lys413-Ile425) are present in this area. Instead, with the exception of BoNT(B), it was found that these relatively short sections are sufficiently long to allow the splitting of the chain and the formation of disulfide bonds, when they have the PRS sequence in accordance with the present invention. Even though BoNT(B), when the Pentapeptide in the loop replaced pentapeptidnogo sequence PRS (thus, the full-length sequence of the loop are only eight amino acid residues), was split into two chains (light and heavy) within the meaning of the invention, were obtained the best results, that is, in accordance with the invention preferably have a loop of at least 9, at least, and the 15, at least 20, or even at least 22, amino acid residues. One of the last-mentioned embodiments, in which the loop has a 22 amino acid residue, are explained on the examples of sequences Figure 4-1 and 4-2 or comparison between the two.

Also experimentally proved that the replacement of loop regions in subtypes b, C1, etc. or significant areas of the region loops BoNT(A) or its essential parts would be preferred in relation to the splitting of neurotoxins to double-stranded polypeptides/proteins with disulfide bridge, in particular, when in this way the loop is extended until at least 9, preferably up to 15 residues, and/or N-end PRS built-basic amino acid residue (e.g., and preferably, the residue Lys) (as previously N-terminal basic or Lys residue was not present). Especially preferred are replacement, as illustrated in Figure 4 (where the PRS sequence in figure 4 represent the VPRGS, but at the same time, and also preferably represent, however, the sequence VPYGS, VPHGS, VPQGS, VPKGS, VPIGS and VPAGS).

In other embodiments of the invention the amino acid sequence and gene sites, coding, thus, the area of the loop in the botulinum toxin serotypes b, C1, D, E, F and G, as well as tetanus toxin, modified between ostad the mi cysteine, involved in disulfide bridge between L and HNso that downregulation of toxins or formed from them are fragments/derivatives in the cell lysate of the host E. coli are present in the form of double-stranded polypeptides, in which the two chains are covalently linked by a disulfide bridge (the same remains true also for any other polypeptides/proteins, which are formed by recombinant expression in a single chain, but develop biological activity only in double-stranded form). In preferred embodiments of the invention, the full scope of the loop (or their parts) of neurotoxins or fragments derived toxin derived from it, can be replaced by a full-area loop BoNT(A), as described in figure 3, or areas of the field loop BoNT(A), where the Pentapeptide Asp443-Asn447substituted preferably, for example, Val-Pro-Arg-Gly-Ser (VPRGS). In the following preferred embodiments of the invention Pentapeptide Asp443-Asn447can also be substituted Val-Pro-Tyr-Gly-Ser, Val-Pro-His-Gly-Ser or Val-Pro-Gln-Gly-Ser. In especially preferred embodiments of the invention the field of loops or parts of the areas of the loops above neurotoxins and formed from them fragments/derivatives can be replaced by Oligopeptide Arg/Ser-Gly-Ile-Ile-Thr-Ser-Lys-Thr-Lys-Ser-Leu-Val-Pro-Arg-Gly-Ser-Lys-Ala (18-Mer: R/SGIITSKTKSLVPRGSKA). Additional C the exchange, insertion or deletion of individual or several amino acid residues in the region of the above-described sequence of the loop, as shown, for example, in figure 4, which also lead to downregulation of the neurotoxin or its fragment/derivative after expression in E. coli (for example, in the cells of the host E. coli K12 or its derivatives) in the form of double-stranded polypeptide/protein disulfide bridge, right covered by this invention (the same remains true also for any other polypeptides/proteins that can be produced by recombinant expression in a single chain, but possess biological activity only in double-stranded form).

How often repeated above, the method according to the invention in accordance with the following embodiment of the invention can also be obtained fused proteins or hybrid proteins, which have, for example, the following components a, b and C:

- effector domain, which due to its enzymatic activity can, for example, to inhibit the secretion of the target cells or to destroy them (A);

sequence loop, which according to the invention, as explained above, modified and which has a higher pentapeptidnogo PRS sequence VPXGS (for example, a modified sequence loops BoNT(A) or its variants, as illustrated by the figure 3, and to which may be attached to the cysteine residue at the N-end and/or at the C-end (In); and

domain linking cell that gives the cell specificity of the fused protein or hybrid protein (S).

Component In (loop sequence) can also be in both embodiments mentioned directly above, preferably like (1) a modified sequence of the loop, as illustrated in Figure 4, (2) any of the sequences formed from it, due to the fact that the Central residue PRS may be a residue of any naturally occurring amino acid, or (3) variant (the variant definition, see above) (1) or (2). In figure 4 the corresponding sequences of the loop BoNT(B), BoNT(C1 or BoNT(E), except for one or two N-terminal and two C-terminal amino acid residues, deleterow, and deleteregvalue amino acid residues substituted 17-Merom GIITSKTKSLVPRGSKA (Figure 4-2 and 4-6) or 18-Merom RGIITSKTKSLVPRGSKA (Fig.4-4) a modified sequence loops BoNT(A).

In addition to the above components a, b and C merged/hybrid proteins can be broadcast domain (which in the case of botulinum neurotoxins localized between the loop sequence and a domain that binds a cell). This additional domain contributes to the implementation of the effector domain into the cytoplasm of target cells. xpressia such fused proteins in E. coli (such as E. coli K12 or its derivatives) leads to double-stranded polypeptides/proteins, in which one domain is on one circuit and the other two domains are on the second circuit (in the case of botulinum toxin effector domain in the light chain covalently linked by a disulfide bridge with the other two domains on the heavy chain.

These fused or hybrid proteins according to the invention can be a so-called immunotoxins, which, in particular, are used in the treatment of tumors. In this regard, the domain of the toxin give specificity to a specific cell type, in General, tumor cells, by attaching a domain that binds a cell. As the domain of the toxin primarily use the enzymatic domains of diphtheria toxin, pseudomonades toxin and ricin. These toxins belong to the double-stranded toxins AB, in which A-chain, which provides the enzymatic activity, covalently linked through a disulfide bridge with In-circuit, which combines the activity of the translocation and binding activity of the cells. However, in immunotoxin other possible toxins or fragments of toxins, due to the fact that the desired action (for example, the destruction of tumor cells) develops in the target cells. If the first generation of immunotoxins obtained by chemical combination of dominatoin, as, for example, the a-chain of ricin, a monoclonal antibody immunotoxins of the second generation is produced by recombinant expression in the form of Fab toxins, single-chain Fv toxins (scFv toxins) or stabilized by a disulfide bond Fv (dsFv toxins), but also in the form of a fused protein with growth factors or cytokines, primarily in E. coli (Reiter, 2001). In future generations of immunotoxin cell specificity can also be given through a modified polypeptide, which is selected in accordance with the high binding affinity of, for example, tumor-specific surface protein, for example, of the families of proteins affilinet, proteins with ancyranum repetitions or anticlines.

When all the possible options of immunotoxins must be guaranteed that the enzymatic domain of the toxin can be injected into the cytoplasm of target cells with the purpose of development of toxic action. As immunotoxins in E. coli is expressed in the form of a single-chain polypeptide, proteolytic cleavage, as well as the restoration of the disulfide bridge is necessary to distinguish, in relation to the chain, the enzymatic domain of the toxin from the region of the translocation domain and linking the cell. In the case of recombinant fragments of diphtheria toxin and recombinant fragment pseudomonades of exotoxin rasdale the s is carried out after internalization in endosomes the compartment of target cells by cellular protease, such as furin (Williams et al., 1990). The ricin, on the other hand, has no such site processing and therefore requires artificially introduced sequence recognition protease with the purpose to enter it in the form of already stranded immunotoxin with a disulfide bridge. However, in the case of immunotoxins, which is based on diphtheria toxin and pseudomonades the exotoxin, only a minimal part of the internalized fused protein is cleaved in such a way that only the minimum equal part of the enzymatic domains can reach the cytoplasm (Ogata et al., 1990). The following preferred embodiments of the invention described methods and structures, where with the help of these methods options immunotoxins, as described in the previous paragraphs, obtained by recombinant expression in cells of the host E. coli and can be allocated in their double-stranded with disulfide bridges and, consequently, biological (enzymatic) active form without their activation requires cell endoprotease or endoprotease, added in vitro. These immunotoxins is capable of transporting the enzymatic domain of the toxin to the target cell in the form competent for translocation, so that the degradation of the cell protease is not required and significantly reduces the dose of immunotoxin that can be used for achievement of the desired cytotoxic effects.

The following preferred embodiment of the invention includes, respectively additional protein or a hybrid protein, which has the following components a, b and C:

domain of the toxin or its fragment or derivative of (A);

sequence loop, which according to the invention, as described above, modified and which has a higher pentapeptidnogo PRS sequence VPXGS (for example, a modified sequence loops BoNT(A) or its variants, as illustrated in Figure 3) and to which may be attached to the cysteine residue at the N-end and/or at the C-end (In); and

domain linking cell, which can be taken from a representative protein families monoclonal antibodies, their fragments, affilinet, proteins with ancyranum repetitions, anticalins, growth factors (e.g., TGF-alpha, FGF, VEGF or IGF-1) or cytokines (e.g. interleukins IL2, IL4 or IL6) (C).

In accordance with this latter preferred embodiment component (loop sequence) may be similar to (1) a modified sequence of the loop, as illustrated in Figure 4, (2) any of the sequences formed from it, due to the fact that the Central residue PRS may be a residue of any naturally occurring amino acid, or (3) variant (the variant definition, see above) (1) is whether (2).

The domain of the toxin can be an A-chain of ricin, a fragment pseudomonades of exotoxin, such as RE or RE (domains II and III domain or no domain Ib; Figure 2) or a fragment of diphtheria toxin. The aforementioned effector domain or the domain of the toxin and domains linking cell, it should be understood only as examples. All proteins or protein fragments covered by the invention in such a way that, on the one hand, give a fused protein/hybrid protein-specific binding activity to a surface antigen of the target cells, for example tumor cells, and, on the other hand, in the target cell after internalization are defined action, such as the destruction of cells, where the expression of such merged/hybrid proteins according to the invention in E. coli provides double-stranded polypeptides/proteins, in which the domain of the toxin or its derivatives covalent linked by a disulfide bridge with a domain that binds a cell.

To improve the efficiency and specificity of immunotoxins based pseudomonades of exotoxin previously chosen different approaches. For example, a domain that binds a receptor (domain Ia with amino acid residues 1-152), replaced by fragments of the monoclonal antibody, and at the same time, the region of the loop (Figure 2 and 5) in the translocation domain (domain II) between cysteine residues 13 and 35 (numbering relative to domain II) modificirovana thus, that the latter was no longer sensitive to the splitting of the widely popular cellular protease Purina, but instead are sensitive to specific proteases that are expressed to a greater extent and partially are secreted only some tumor cells (U.S. patent 6426075). This modified sensitivity to proteases was intended to give immunotoxins increased cell specificity in addition to replaced by the domain that binds the receptor. However, it is unlikely that enhanced cleavage in the loop and, consequently, improved the efficiency of translocation of the enzymatic domain III will be obtained through other cellular proteases.

According to the following approach for immunotoxin domain that binds the receptor and the N-terminal region of domain translocation was removed up to the arginine residue 27 within the scope of the loop. Necessary cellular specificity in this immunotoxin attached, for example, by inserting a domain VHmonoclonal antibodies, which was associated domain VLthrough a disulfide bridge in the website of domain Ib between domains II and III, or by joining With the end of domain III (U.S. patent 5980895). In such constructions the activation of the protease is no longer required; on the one hand, it will contribute significant is the increased efficiency of transportation. However, on the other hand, we should expect that the translocation domains by linking the receptor is localized on the N-end or the end of the enzymatic domain III, will be reduced like domain of the VHmonoclonal antibody or TGF-alpha. Because these domains, binding the receptor, not separated from enzymatic domain, you should expect negative effects on enzymatic activity and, therefore, toxicity in target cells. Relative to the maximum degree of cytotoxic activity obtained in immunotoxin-based pseudomonades of exotoxin, when, on the one hand, the loop between cysteine residues 13 and 35 is already present in the split form disulfide bridges, and activation of cellular protease, therefore, is not required, and when, on the other hand, a domain that binds the receptor, fused instead of domain I of exotoxin N-end domain translocation in such a way that after the restoration in the cytoplasm is separated from the domains of the toxin and, therefore, may not reduce the enzymatic activity of domain III.

Especially preferred embodiment of the invention includes, therefore, flushed/hybrid protein that contains a domain that binds a cell that can be taken from a representative protein families monoclonal antibodies, their fragments, affilinet, proteins with ankyrin the new replays anticalins, growth factors (e.g., TGF-alpha, FGF, VEGF or IGF-1) or cytokines (e.g., IL2, IL4 or IL6), which is fused at the C-end of the modified fragment RE, which may bear on the far-end signal hold to the endoplasmic reticulum, Lys-Asp-Gly-Leu, or its variants. Modification of the fragment RE is to replace the full sequence of the loop (or only area) between cysteine residues 13 and 35 on pentapeptidnogo sequence PRS VPXGS, preferably a modified sequence loops BoNT(A), illustrated in Figure 3, or its variants, in particular a peptide sequence Arg-Gly-Ile-Ile-Thr-Ser-Lys-Thr-Lys-Ser-Leu-Val-Pro-Arg-Gly-Ser-Lys-Ala (Figure 5) (see above definition of variants). Preferably, in this embodiment also guaranteed that basic amino acid residue localized in the N-end to PRS at a distance of from 1 to 20 amino acid residues, as illustrated in the sequence of Figure 5. Accordingly, the modified fragment RE, and flushed/hybrid proteins that contain this modified fragment, present in the cell lysate of E. coli hosts (e.g., M15[pREP4]) in double-stranded form disulfide bridges.

In contrast pseudomonades the exotoxin of the enzymatic domain of diphtheria toxin A-chain, is present at N-end. On the C-terminal b-chain etc which are present translocation domain and domain binding the receptor. Both chains are connected by a loop sequence, in which the arginine residue 193 when secretion from cells of Corynebacterium diphtheriae is the proteolytic cleavage by the protease (Collier, 2001). Two chains after splitting remain covalently bound to each other by a disulfide bridge between cysteine residues 186 and 201. In this regard, diphtheria toxin similar in its domain structure of botulinum toxin and tetanus toxin.

To obtain recombinant immunotoxins domain that binds the receptor, or its plot has been replaced by, for example, VEGF or IL2 (Arora et al., 1999; Williams et al., 1990) with the aim of giving a fused protein of a new cell specificity. To the a-chain has reached the cytoplasm of target cells, on the one hand, polypeptide chain immunotoxin, expressed as a single chain in E. coli, should be split in the area of the loop between a-chain and b-chain and, on the other hand, disulfide bridge should be restored. Although the latter occurs during the process of translocation, proteolytic cleavage by a cellular protease is incomplete, so that only a minimal part of the A-chain may be released into the cytoplasm (Williams et al., 1990). If immunotoxin was present in double-stranded form disulfide bridges at the moment of introduction, one would expect a significant increase in efficiency is efficiency, since all a-chain would be available in a form competent for translocation.

The following particularly preferred embodiment of the invention includes, therefore, fused or hybrid protein that contains a domain that binds a cell that can be taken from a representative protein families monoclonal antibodies, their fragments, affilinet, proteins with ancyranum repetitions, anticalins, growth factors (e.g., TGF-alpha, FGF, VEGF or IGF-1) or cytokines (e.g., IL2, IL4 or IL6), which is fused at its N-end of the modified fragment of diphtheria toxin. This fragment of the toxin may contain A-chain, and at least one domain translocation of the b-chain (Gly1-Phe389or Gly1-Asn486). Modification of a fragment of diphtheria toxin is that the full sequence of the loop (or its site) between cysteine residues 186 and 201 replaced by a modified sequence loops BoNT(A), illustrated in Figure 3, or its variants, in particular a peptide sequence Arg-Gly-Ile-Ile-Thr-Ser-Lys-Thr-Lys-Ser-Leu-Val-Pro-Arg-Gly-Ser-Lys-Ala (Figure 5) (see above definition of variants). Accordingly, the modified fragment of diphtheria toxin, and fused proteins, which contain this modified fragment, present in the lysate of the cells of the host E. coli, such as M15[pREP4], double-stranded the Orme with disulfide bridges.

Immunotoxins of the first generation on the basis of ricin was obtained by blending the a-chain of ricin with a monoclonal antibody. Previously this was achieved by deriving antibodies with chemical linker molecule that forms a disulfide bridge with a thiol functional group of the cysteine residue localized at the C-end of the a-chain. Such conjugates were heterogeneous due to the omnidirectional receiving derived antibodies. Effective against tumors was low not only because of the size of the conjugate and of the absence of the translocation domain that is localized in the b-chain. When In-circuit in native form is also present as a component of immunotoxin, toxicity increases significantly, but the result is similar to lectins cell-binding properties of the In-circuit takes place nonspecific capture in cells other than the desired target cells. With this conflict targets fought with strategy, according To which the chain has been modified so that the translocation activity remained unaffected, but the binding affinity of for glycostructures on the cell surface was, however, significantly reduced (patent application WO 89/04839). Expressed by recombinant immunotoxins, containing such a modified B-chain have, however, a single-stranded structure is ur, so in the absence of sequence recognition for cellular protease in the linker peptide between the a-chain and b-chain of the release and translocation of the a-chain after the capturing immunotoxins in the target cell is impossible or possible only very inefficiently. In U.S. patent 6593132 documented modifications of this native linker peptide, which represent a sequence of recognition for various proteases with cell specificity. Variants of ricin with such modifications will have the appropriate cellular specificity due to the fact that the corresponding protease that is able to proteolytic cleavage of the modified linker peptide, is expressed only in the desired target cells compared to other cell types in much higher amounts. However, it should be assumed that the cleavage takes place only at a fraction of internalized molecules of toxin and, therefore, only appropriate minimum number of A-chains moves into the cytoplasm. Desirable would be double-stranded immunotoxins based ricin, in which A-chain crosslinked by a disulfide bridge with a modified B-chain, in which the translocation activity remains unaffected, but actinopodidae properties of nonspecific binding of cells n is Dawley, and who merged them With-end with a specific domain that binds the cell. Such immunotoxins would unite the cellular specificity and high toxicity.

The following preferred embodiment of the invention includes, therefore, a protein that has the following components a, b and C:

- A-chain of ricin (A);

the sequence of the loop, which is modified according to the invention, as described above, and which has a higher pentapeptidnogo PRS sequence VPXGS (for example, a modified sequence loops BoNT(A) or its variants, as illustrated in Figure 3, and to which may be attached to the cysteine residue at the N-end and/or at the C-end (In); and

domain linking cell, which can be taken from a representative protein families monoclonal antibodies, their fragments, affilinet, proteins with ancyranum repetitions, anticalins, growth factors (e.g., TGF-alpha, FGF, VEGF or IGF-1) or cytokines (e.g., IL2, IL4 or IL6) (C).

Component In accordance with this latter preferred embodiment may be similar to (1) one of the modified sequences of the loop, illustrated in Figure 4, (2) any sequence formed from it, due to the fact that the Central residue PRS may be a residue of any found in the ode amino acids, or (3) variant (the variant definition, see above) (1) or (2).

In particular, the sequence loops may contain the peptide sequence Ala-Pro-Pro-Arg-Gly-Ile-Ile-Thr-Ser-Lys-Thr-Lys-Ser-Leu-Val-Pro-Arg-Gly-Ser-Lys-Ala-Asp-Val (5-6), that is, the modified loop in the a-chain of ricin. The cysteine residue is preferably additionally provided on the end of the sequence loops. In PRS-sequence Val-Pro-Arg-Gly-Ser contained therein, Arg may, however, be any other naturally occurring amino acid XAA. With both ends of the loop sequence can be extended an additional amino acid residues (e.g. residues of glycine and serine). In addition, the a-chain of ricin can be made with a full In-circuit or parts or variations, the loop sequence, which replaces the amino acid residues between the cysteine residues 259 and 283 sequence Pro-ricin wild-type fully or partially and at least covers the area of the modified loop BoNT(A)described in figure 3, or its variants. In this regard, the disulfide bridge formed by cysteine residues 259 and 283 (relative sequence Pro-ricin wild type). Domain linking cell, fused to a C-end In-circuit and taken from the above families of polypeptides. The corresponding merged/hybrid proteins present in the lysate of cells hozjaeva. coli, for example, cells of strain M15[pREP4], in double-stranded form disulfide bridges.

The following embodiment of the invention relates to recombinant fused proteins, which have the following components a, b and C:

protein or Oligopeptide, which gives flushed best protein solubility, contributes to a higher rate of expression and/or affinity purification (for example, glutathione-3-transferase (GST), protein a, maltose binding (MBP), His label, Strep tag, label, FLAG) (A);

the sequence of the loop, which is modified according to the invention, as described above, and contains defined above pentapeptidnogo PRS sequence VPXGS (for example, a modified sequence loops BoNT(A), illustrated in Figure 3, or its variants) and to which may be attached to the cysteine residue at the N-end and/or at the end; and

- any type of polypeptide (S).

Component In (loop sequence) in accordance with this latter preferred embodiment may be similar to (1) one of the modified sequences of the loop, illustrated in Figure 4, (2) any sequence formed from it, due to the fact that the Central residue PRS may be a residue of any naturally occurring amino acid, or (3) variant (the variant definition, see above) (1) or (2).

In h is particularly the loop may have a peptide sequence Val-Arg-Gly-Ile-Ile-Thr-Ser-Lys-Thr-Lys-Ser-Leu-Val-Pro-Arg-Gly-Ser-Lys-Ala-Leu-Asn-Asp-Leu, where Arg in the center of the PRS may again be a Haa. At both ends it can be extended an additional amino acid residues (e.g. residues of glycine and serine). The expression of such fused proteins in E. coli leads to double-stranded polypeptides/proteins, two chains which are covalently linked by a disulfide bridge and after cleaning can be separated from each other without the addition of protease in the simple recovery tolstushki substances (for example, β-mercaptoethanol, DTT or the reduced glutathione). This expression system is particularly suitable for recombinant proteins, which should be equipped on one of the two ends of the cysteine residue in order to obtain after purification and separation of a merge partner reactive thiol group site for reactions of combinations, for example, with colorectum linker molecules, or changes in, for example, polyethylene glycol.

The invention includes, in addition, all nucleic acids that encode the polypeptides according to the invention described in the preceding sections, taking into account the different possibilities of use of codons. In addition, the invention covers available for sale individually or scone is trueromance cloning and expression plasmids, which contain the coding DNA sequence for the corresponding polypeptides according to the invention and suitable cloning and expression strains of E. coli, which transformed the appropriate expression plasmids and which can Express the corresponding polypeptides according to the invention in their active double-stranded form disulfide bridges. One example of such expression systems is the expression plasmid pQE series in combination with the strain of the host E. coli M15[pREP4].

To a person skilled in the art, which deals in particular with the development of pharmaceutically applicable polypeptides/proteins, strictly adhere to the advantages that are associated with the fact that activation of these polypeptides/proteins do not need to add endoprotease. A huge part of polypeptides/proteins according to the invention described in the preceding sections, specifically designed for pharmaceutical applications. The invention thus also encompasses pharmaceutical preparations which contain one of the polypeptides/proteins according to the invention or a mixture of polypeptides/proteins according to the invention as active ingredients and mineral supplements, which give the drug enough stability and composition of which corresponds to the desired shape of the injection.

The attached gr the specific materials and the sequence listing described below.

Figure 1 shows a schematic illustration of a release of botulinum neurotoxin type a with a loop of wild-type or modified loop according to the invention of Clostridium botulinum or Escherichia coli K12. A: when the lysis of cells of Clostridium botulinum neurotoxin is cleaved in the region of the loop between the light chain (L) and heavy chain (H) clostridiales endoproteases. Both chains are connected to each other by a disulfide bridge. Q: After the expression of recombinant neurotoxin with a loop of the wild-type E. coli and lysis of the cells it is present in single-stranded form. C: When recombinant neurotoxin with a loop, modified according to the invention, is released from cells of E. coli, the splitting in the loop is endoproteases.

Figure 2 shows a schematic illustration of the various recombinant toxins areas loops wild type and area of the loop, modified according to the invention, in comparison after release from cells of E. coli. A: botulinum neurotoxins; B: pseudomonades exotoxin; S: diphtheria toxin.

Figure 3 shows a comparison of the loop of the wild-type sample loop sequences BoNT(A), modified according to the invention. Illustrates the nucleotide sequence and deduced amino acid sequence, which include limiting cysteine residues formed the first chain and the heavy chain. Arrow indicates the site of cleavage for endoprotease in E. coli lysate.

Figure 4 shows a comparison of the loop of the wild-type with an exemplary sequence of the loop, modified according to the invention, botulinum neurotoxins serotypes b, C1 and E, respectively. Illustrates the nucleotide sequence and deduced amino acid sequence, which include limiting cysteine residues of a light chain and heavy chain. Arrow indicates the site of cleavage for endoprotease in E. coli lysate.

Figure 5 shows a comparison of the loop of the wild-type sample loop sequence modified according to the invention, a fragment RE pseudomonades of exotoxin, diphtheria toxin (DT) and ricin, respectively. Illustrates the nucleotide sequence and deduced amino acid sequence, which include limiting cysteine residues of a light chain and heavy chain. Arrow indicates the site of cleavage for endoprotease in E. coli lysate.

Figure 6 shows the combination of oligonucleotides that were used for cloning of recombinant toxins and fragments of toxins. Sequence recognition for restriction endonucleases are underlined.

7 shows the analysis of recombinant fragments LHNBoNT(A) with a sequence of loops, modifica vannoy according to the invention, in polyacrylamide gel with sodium dodecyl sulfate (LTOs). The expression of the fragment LHNcarried out in cells M15[pREP4], which have been transformed with the plasmid pQE-BoNT(A)-Lmod1HN. Tracks 2 and 5: fragment LHNpurified on agarose Ni-NTA; lanes 1 and 4: fragment LHNafter incubation with thrombin; lane 3: molecular weight marker. Application of samples in reducing conditions (lanes 1 and 2) and reducing conditions (lanes 4 and 5).

On Fig shows analysis of the recombinant fragment LHNBoNT(B) with a sequence of loops, modified according to the invention, on a polyacrylamide gel with LTOs. The expression of the fragment LHNcarried out in cells M15[pREP4], which have been transformed with the plasmid pQE-BoNT(B)-Lmod1HN. Tracks 1 and 4: fragment LHNpurified on agarose Ni-NTA; lane 2: molecular weight marker; lane 3: no application. Application of samples in reducing conditions (lane 1) and reducing conditions (lane 4).

Figure 9 shows the analysis of recombinant BoNT(C1) with a sequence of loops, modified according to the invention, on a polyacrylamide gel with LTOs. Expression of the toxin was carried out in cells M15[pREP4], which are transformed by the plasmid pQE-BoNT(C1)-Lmod1HNHC. Tracks 1 and 4: toxin, purified on agarose Ni-NTA; lane 2: marker mo is collaroy mass; lane 3: no application. Application of samples in reducing conditions (lane 1) and reducing conditions (lane 4).

SEQ ID NO: 1 is an example of a nucleic acid (DNA)which encodes a recombinant botulinum neurotoxin type a with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(A)-(mod1).

SEQ ID NO: 2 is an example of a recombinant botulinum neurotoxin type a with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(A)-(mod1).

SEQ ID NO: 3 is an example of a nucleic acid (DNA)which encodes a recombinant fragment LHNbotulinum neurotoxin type a with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(A)-Lmod1HN). This sequence corresponds to SEQ ID NO: 1, where the nucleotides 2620-3888 deleterows.

SEQ ID NO: 4 is an example of a recombinant fragment LHNbotulinum neurotoxin type a with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(A)-Lmod1HN). This sequence corresponds to SEQ ID NO: 2 where amino acid residues 874-1296 deleterows.

SEQ ID NO: 5 is predstavljaet an example of nucleic acid (DNA), which encodes the recombinant fragment LHNHCNbotulinum neurotoxin type a with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(A)-Lmod1HNHCN). This sequence corresponds to SEQ ID NO: 1, where the nucleotides 3286-3888 deleterows.

SEQ ID NO: 6 is an example of a recombinant fragment LHNHCNbotulinum neurotoxin type a with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(A)-Lmod1HNHCN). This sequence corresponds to SEQ ID NO: 2 where amino acid residues 1096-1296 deleterows.

SEQ ID NO: 7 is an example of a nucleic acid (DNA)which encodes a recombinant botulinum neurotoxin type with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(B)-mod1).

SEQ ID NO: 8 is an example of a recombinant botulinum neurotoxin type with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(B)-mod1).

SEQ ID NO: 9 is an example of a nucleic acid (DNA)which encodes a recombinant fragment LHNbotulinum neurotoxin type b consequently the capacity of the loop, modified according to the invention, and the C-terminal getservletinfo label (rBoTN ()-Lmod1HN). This sequence corresponds to SEQ ID NO: 7, where the nucleotides 2623-3915 deleterows.

SEQ ID NO: 10 is an example of a recombinant fragment LHNbotulinum neurotoxin type b with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN ()-Lmod1HN). This sequence corresponds to SEQ ID NO: 8, wherein amino acid residues 875-1305 deleterows.

SEQ ID NO: 11 is an example of a nucleic acid (DNA)which encodes recombinantly botulinum neurotoxin type C1 with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(C1)-mod1).

SEQ ID NO: 12 is an example of a recombinant botulinum neurotoxin type C1 with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(C1)-mod1).

SEQ ID NO: 13 is an example of a nucleic acid (DNA)which encodes a recombinant fragment LHNbotulinum neurotoxin type C1 with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(C1)-Lmod1HN). This sequence according to the corresponds to SEQ ID NO: 11, where nucleotides 2599-3858 deleterows.

SEQ ID NO: 14 is an example of a recombinant fragment LHNbotulinum neurotoxin type C1 with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(C1)-Lmod1HN). This sequence corresponds to SEQ ID NO: 12, where amino acid residues 867-1286 deleterows.

SEQ ID NO: 15 is an example of a nucleic acid (DNA)which encodes a recombinant botulinum neurotoxin type E with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(E)-mod1).

SEQ ID NO: 16 is an example of a recombinant botulinum neurotoxin type E with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rBoTN(E)-mod1).

SEQ ID NO: 17 is an example of a nucleic acid (DNA)which encodes a recombinant 40 kDa fragment pseudomonades of exotoxin containing domains II, Ib, and III, with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (PE40-mod1).

SEQ ID NO: 18 is an example of a recombinant 40 kDa fragment pseudomonades of exotoxin containing domains II, Ib, and III, with a sequence of loops, modified according to what obreteniyu, and C-terminal getservletinfo label (PE40-mod1).

SEQ ID NO: 19 is an example of a nucleic acid (DNA)which encodes a recombinant fragment of diphtheria toxin, containing the a-chain and the N-terminal fragment of the b-chain, with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (DT389-mod1).

SEQ ID NO: 20 is an example of a recombinant fragment of diphtheria toxin, containing the a-chain and the N-terminal fragment of the b-chain, with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (DT389-mod1).

SEQ ID NO: 21 is an example of a nucleic acid (DNA)which encodes a recombinant toxin ricin with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rRicin-mod1).

SEQ ID NO: 22 is an example of a recombinant toxin ricin with a sequence of loops, modified according to the invention, and the C-terminal getservletinfo label (rRicin-mod1).

Examples

Example 1: Cloning and expression of a fragment LHNbotulinum neurotoxin type a with a modified loop

For cloning of the DNA sequences of the light chain and domain translocation chromosomal DNA was isolated from a culture of Clostridium botulinum type a (strain ADS 3502). Through the Yu PCR amplification with primers #1 and #2 (6) was obtained gene fragment, encoding a light chain of BoNT(A), with a modified sequence of the loop and C-terminal His label. The product of PCR amplification were cloned in the expression plasmid pQE-60 restriction sites Nco 1 and Sal 1, so the result obtained plasmid pQE-BoNT(A)-Lmod1. Using PCR amplification with primers #3 and #4 (6) was obtained gene fragment encoding domain, the translocation of BoNT(A). The restriction sites for Stu I and Xho I, he was cloned between the loop sequence and the sequence for His label in pQE-BoNT(A)-Lmod1(plasmid pQE-BoNT(A)-Lmod1HN; sequence #2, Fig 3, No. 2). The expression strain E. coli M15[pREP4] (Qiagen) transformed with plasmid pQE-BoNT(A)-Lmod1HN. Expression of the modified fragment LHNwas carried out by stepwise induction 500 M final concentration isopropylthioxanthone (IPTG) at 25 degrees Celsius during the night. Cells were subjected to lysis in 50 mm phosphate buffer at pH 8.0 300 mm NaCl by treatment with lysozyme and ultrasonic treatment. Centrifuged the lysate was subjected to chromatography on a column of agarose Ni-NTA. Analysis on polyacrylamide gel with LTOs showed that reducing conditions two zones at approximately 50 kDa, as well as the area at 100 kDa was stained, Kumasi, while not reducing conditions observed one band at 100 kDa (7). Thus, odnosno what about the demonstrated that fragment LHNwas freed from bacteria by more than 75 percent in the form of double-stranded polypeptide in which two chains are covalently bound to each other by a disulfide bridge. The result of further processing by thrombin was, on the one hand, the cleavage of single-stranded form and, on the other hand, shortening domain translocation in the double-stranded polypeptide (Fig.7). A two-hour incubation of the E. coli lysate before purification of the fragment LHNled to a complete breakdown in the double-stranded polypeptide.

Respectively, expressed and purified fragment LHNwith the native sequence of the loop (Figure 3, No. 1) showed on polyacrylamide gel LTOs as in not reducing and reducing conditions zone at 100 kDa. Single-chain polypeptide could be turned only after treatment with trypsin in double-stranded fragment LHNwith a disulfide bridge.

Example 2: Cloning and expression of a fragment LHNHCNbotulinum neurotoxin type a with a modified loop and characterization of the cleavage site

Fragment HNHCN(translocation domain with N-terminal half of the domain, receptor binding, BoNT(A)) was constructed using PCR amplification with primers #3 and #5 (6) and cloned by restriction sites for Stu I and Xho I in plasmid pQE-BoNTA)-L mod1(plasmid pQE-BoNT(A)-Lmod1HNHCN; sequence #3). Expression and purification was performed in accordance with the scheme described in example 1. Analysis on polyacrylamide gel with LTOs showed in addition to a weak area, which corresponded to the single-stranded polypeptide and additional non-specific zones, the zone at 50 kDa, and at 75 kDa, which corresponded to a light chain and a fragment of HNHCN. N-terminal sequencing of the first four amino acid residues of the fragment HNHCNgave the sequence Ser-Leu-Val-Pro. The breakdown by activity in the lysate of E. coli occurred, so after Lys440and therefore, in the N-terminal direction of the polypeptide Val-Pro-Arg-Gly-Ser, built-in loop.

Example 3: Cloning and expression of a fragment LHNbotulinum neurotoxin type b with a modified loop

For cloning of the DNA sequences of the light chain and domain translocation chromosomal DNA was isolated from a culture of Clostridium botulinum type b (strain Okra). Using PCR amplification with primers #6 and #7 (6) constructed a gene fragment that encodes the light chain of BoNT () with the modified sequence loops BoNT(A). With primers #8 and #9 (6) constructed a gene fragment encoding domain, the translocation of BoNT(B). Cloning into the expression plasmid pE-60 was carried out first by replacing the fragment of the gene BoNT(A)-L in pQE-BoNT(A)-L mod1the product of amplification BoNT(B)-Lmod1the restriction sites for Nco I and Stu I. Then the product of amplification BoNT(In)-HNcloned followed by restriction sites for Stu I and Xho I, so the result obtained plasmid pQE-BoNT(B)-Lmod1HN(sequence #5). Expression in strain-master M15[pREP4] and purification of the fragment LHNperformed by analogy with example 1. Analysis on polyacrylamide gel with LTOs showed that reducing conditions two zones at approximately 50 kDa and 55 kDa were stained, Kumasi, while not reducing conditions observed area at approximately 105 kDa (Fig). It definitely showed that the fragment LHNreleased from bacteria essentially in the form of double-stranded polypeptide in which two chains of more than 80 percent were covalently linked to each other by a disulfide bridge.

Example 4: Cloning and expression of a fragment LHNbotulinum neurotoxin type C1 with a modified loop and characterization of the cleavage site

For cloning of the DNA sequences of the light chain and domain translocation was obtained preparation of chromosomal DNA from a culture of Clostridium botulinum type C1 (strain S). Using PCR amplification with primers #10 and #11 (6) constructed a gene fragment that encodes the light chain of BoNT(C1) with a modified sequence loops BoNT(A). With what ramarama #12 and #13 (6) constructed a fragment of the gene encoding domain, the translocation of BoNT(C1). Cloning into the expression plasmid pQE-60 was carried out first by replacing the fragment of the gene BoNT(A)-L in pQE-BoNT(A)-Lmod1the product of amplification pQE-BoNT(C1)-Lmod1the restriction sites for Nco I and Stu I. Then the product of amplification BoNT(C1)-HNcloned followed by restriction sites for Stu I and Xho I, so the result obtained plasmid pQE-BoNT(C1)-Lmod1HN(sequence #7). Expression in strain-master M15[pREP4] and purification of the fragment LHNperformed by analogy with example 1. Analysis on polyacrylamide gel with LTOs showed that reducing conditions two zones at approximately 50 kDa and 55 kDa were stained, Kumasi, while not reducing conditions observed area at about 105 kDa. It definitely showed that the fragment LHNwas freed from bacteria by more than 90 percent in the form of double-stranded polypeptide in which two chains are covalently bound to each other by a disulfide bridge. As a result, N-terminal sequencing of the first four amino acid residues of the fragment HNreceived sequence Ser-Leu-Val-Pro. The breakdown by activity in the lysate of E. coli was behind Lys447and thus in the direction of the N-end Pentapeptide Val-Pro-Arg-Gly-Ser, built-in loop BoNT(A). Using site-directed mutagenesis of the arginine residue built the first Pentapeptide replaced by histidine, tyrosine and glutamine. The mutated fragments LHNexpressed in the same way, were present after two hours incubation of the lysate of E. coli by more than 90 percent in double-stranded form a disulfide bridge, where the cleavage efficiency is somewhat lower than for a fragment LHNthat contains a loop BoNT(A), modified by Pentapeptide Val-Pro-Arg-Gly-Ser.

Example 5: Cloning and expression of recombinant botulinum neurotoxin type C1 with a modified loop

By using chromosomal DNA of a strain of Clostridium botulinum C fragment of a gene that encodes a heavy chain, amplified with primers #12 and #14 (6). The restriction sites for Stu I and Xho I, it was cloned in the plasmid BoNT(C1)-Lmod1HNbetween the sequence encoding the light chain and the sequence for the His-tag (plasmid pQE-BoNT(C1)-Lmod1HNHC; sequence #6). Expression of the E. coli strain M15[pREP4] (Qiagen) transformed the appropriate expression of the plasmid. Expression in strain-master M15[pREP4] and the purification was performed by analogy with example 1. Analysis on polyacrylamide gel with LTOs showed that reducing conditions two zones at approximately 50 kDa and 105 kDa was stained, Kumasi, while not reducing conditions observed area at about 155 kDa (Fig.9). Thus, it is defined what about the showed that recombinant neurotoxins released from the bacteria by more than 90 percent in the form of double-stranded polypeptide in which two chains are covalently bound to each other by a disulfide bridge. In the test result on the activity in the analysis of the right or the left dome of the diaphragm is received toxicity, which is comparable to high compared to the native neurotoxin type C1 isolated from Clostridium botulinum. Modification of the area of the loop between the light chain and a translocation domain, thus, has no effect on toxicity.

Example 6: Cloning and expression of recombinant fragment pseudomonades of exotoxin (Re) with a modified loop

By using the chromosomal DNA of the strain Pseudomonas aeruginosa 103 fragment of the gene coding region of domain II, which is localized in the C-terminal direction of the loop between cysteine residues 13 and 36, and domain III, amplified by PCR with primers #17 and #18 (6). The amplification product was cloned into plasmid pQE-BoNT(A)-Lmod1by Nco I and Mlu I, in exchange for a fragment of the BoNT gene(A)-Lmod1(plasmid pQE-PEII3III). The sequence for the region of domain II, which is the N-terminal region of the loop, was built by hybridization of the oligonucleotide #15 and #16 (6) and cloning by restriction enzymes cut sites Nco I and Kpn I in plasmid pQE-PEII3III (plasmid pQE-PEIImodIII; bsos shall egovernance #9). The expression strain E. coli M15[pREP4] (Qiagen) transformed the appropriate expression of the plasmid. Expression in strain-master M15[pREP4] and the purification was performed by analogy with example 1. The result of analysis on polyacrylamide gel with LTOs in reducing conditions was weaker area at 40 kDa, and more strong area at 37 kDa. In non reducing conditions, however, observed only one band at 40 kDa. During incubation of the cell lysate at least for two hours at room temperature before purification by affinity chromatography area at 40 kDa was not detectable in reducing conditions. In the replacement area of the loop between cysteine residues 13 and 36 in domain II fragment ARE modified loop BoNT(a) cleavage of the polypeptide chain, thus, occurred where the above cysteine residues formed a disulfide bridge. N-terminal fragment of approximately 3 kDa was not detectable after recovery in 12% of the LTO-gel.

Example 7: Cloning and expression of recombinant fragment of diphtheria toxin (Dt389) with a modified loop

By using the chromosomal DNA of the strain Corynebacterium reagent grade NCTC 13129 fragment of the gene that encodes the a-chain of diphtheria toxin, amplified by PCR with primers #19 and #20 (6). The restriction sites for Nco I and Stu I prod the CT amplification was cloned into plasmid pQE-BoNT(A)-L mod1(see example 1) (plasmid pQE-DT-Amod1). In the same way the gene fragment encoding the N-terminal fragment of the b-chain, amplified with primers #21 and #22 (6) and cloned by restriction enzymes cut sites Stu I and Xho I in plasmid pQE-DT-Amod1(plasmid pQE-DT389-mod1; sequence #10). The expression strain E. coli M15[pREP4] (Qiagen) transformed the appropriate expression of the plasmid. Expression in strain-master M15[pREP4] and the purification was performed by analogy with example 1. Analysis on polyacrylamide gel with LTOs showed that reducing conditions two zones at approximately 22 kDa was stained, Kumasi, while not reducing conditions observed one zone at about 43 kDa. It definitely showed that recombinant fragment of diphtheria toxin released from the bacteria by more than 90 percent in the form of double-stranded polypeptide in which two chains are covalently bound to each other by a disulfide bridge.

Example 8: Cloning and expression of recombinant ricin with a modified loop

By using mRNA seeds of Ricinus communis fragment of the gene encoding the a-chain of ricin, amplified by PCR with reverse transcriptase (RT-PCR) with primers #23 and #24 (6). The restriction sites for Nco I and Xho I, it was cloned into plasmid pQE-BoNT(A)-Lmod1(see example 1) (plasmid pQE-ricin-A). In the same way the fragment of the gene code is highlighted In the chain, amplified with primers #25 and #26 (6) and cloned in pQE-ricin-A the restriction sites for Kpn I and Xho I (plasmid pQE-ricin-mod1; sequence #11). The expression strain E. coli M15[pREP4] (Qiagen) transformed the appropriate expression of the plasmid. Expression in strain-master M15[pREP4] and purification of the soluble part of the downregulation of ricin was performed by analogy with example 1. Analysis on polyacrylamide gel with LTOs showed that reducing conditions two zones at approximately 19 kDa and 42 kDa was stained, Kumasi, while not reducing conditions observed area at approximately 62 kDa. It definitely showed that the soluble part of recombinant ricin was released from the bacteria by more than 90 percent in the form of double-stranded polypeptide in which two chains are covalently bound to each other by a disulfide bridge.

The scientific literature

Arora et al. (1999), Cancer Res. 59: 183-8

Collier (2001), Toxicon 39 (11): 1793-803

Fujinaga (1997), Microbiology 143: 3841-47

Ogata et al. (1990), J. Biol. Chem 265 (33); 20678-85

Reiter (2001), Adv. Cancer Res. 81: 93-124

Schiavo and Montecucco (1997), The Clostridia: Molecular Biology and Pathogenesis, Academic Press, San Diego: 295-322

Williams et al. (1990) J. Biol Chem, 265 (33): 20673-77

Patent literature

Borgford, US patent 6,593,132

Brown and Jones, WO 89/04839

Fitzgerald et al., US patent 6,426,075

Pastan et al., US patent 5,980,895

1. A method of obtaining a polypeptide or protein having the properties of a toxin in double-stranded form, g is these two chains linked by a disulfide bridge, by recombinant expression in cells of the host E. coli, where
1) polypeptide or protein exerts its biological activity in the form of double-stranded polypeptide or protein with a disulfide bridge,
2) the C-terminal amino acid residue of the first chain is a residue of a basic amino acid,
3) the second circuit protein/polypeptide has at the N-end in the direction from N to s from 1 to 20 amino acid residues and pentapeptidnogo sequence VPXGS called PRS, where X is any naturally occurring amino acid, where V is Val, Leu, Ile, Ala, Phe, Pro, or Gly, where P is a Pro, Leu, Ile, Ala, Phe, Val or Gly, where G represents Gly, Leu, Ile, Ala, Pro, Phe or Val and where S is a Ser, Tyr, Trp or Thr; and
4) the method comprises the following stages:
a) modification of the polypeptide or protein at the level of the nucleic acid such that the polypeptide or protein in its modified form in his field loop has a sequence VPXGS, where X, V, R, G, and S are as defined above, and where the area of the loop is defined as amino acid sequence, localized between Cys residues that form a disulfide bridge;
b) introduction of design, modified at the level of nucleic acid into cells of E. coli;
C) culturing and subsequent lysis of host cells and
g) in the division of double-stranded polypeptide or protein by a disulfide bridge without the preliminary stage of adding protein type endoprotease.

2. The method according to claim 1, where the remainder of the basic amino acid is a residue of Arg or Lys.

3. The method according to claim 1 or 2, where the first chain of the polypeptide/protein is a more light chain of the specified polypeptide/protein, and the second circuit is a more heavy chain of the specified polypeptide/protein, in particular where the protein is a hybrid protein.

4. The method according to claim 1 or 2, where the polypeptide or protein is a botulinum neurotoxin, in particular botulinum neurotoxin serotype A (BoNT(A)), and in particular the fragment LHNBoNT(A).

5. The method according to claim 4, where the PRS sequence VPXGS built between amino acids Leu442and Lys448BoNT(a) deletion of amino acids 443-447, in particular, where built PRS sequence VPRGS, VPYGS, VPHGS or VPQGS.

6. The method according to claim 1 or 2, where the PRS sequence VPXGS built in oktapeptid Lys438-Ile445BoNT(B), 15-Mer His438-Asp452BoNT(C1) or 13-Mer Lys413-Ile425BoNT(E) a deletion of at least one amino acid, in particular, where PRS sequence VPXGS built in form 17-a measure GIITSKTKSLVPRGSKA or 18-measure RGIITSKTKSLVPRGSKA.

7. The method according to claim 3, where the hybrid protein consists of the following components a, b and C:
the effector domain, which through its enzymatic activity capable of inhibiting the secretion of the target cells or to destroy them, or the domain of the toxin (to the ponent (A);
the loop sequence that contains the sequence VPXGS (component b); and
domain linking cell that gives the cell specificity of the fused protein or hybrid protein (component C), and possible component D:
domain translocation.

8. The method according to claim 7, where the domain of the toxin (A) represents a domain of diphtheria toxin, pseudomonades of exotoxin or ricin, in particular, where the domain of the toxin (A) is a fragment RE (domain III, domain II and domain Ib) or a fragment RE (domain III and domain II) pseudomonades of exotoxin or A-chain of ricin.

9. The method according to claim 7, where the domain linking cell (C), is a monoclonal antibody, affilin protein with ancyranum repetitions, anticalin, growth factor, such as a growth factor in T-cell alpha (TGF-alpha), fibroblast growth factor (FGF), growth factor vascular endothelial (VEGF) or insulin-like growth factor 1 (IGF-1) or cytokine, such as interleukin IL2, IL4 or IL6.

10. The method according to claim 3, where the hybrid protein consists of the following components a, b and C:
protein or Oligopeptide, which gives flushed best protein solubility, contributes to a higher rate of expression and/or affinity purification (component a);
the loop sequence containing a sequence VPXGS (component b); and
any type of polypeptide (component C).

11. The method according to claim 10, where component a is a glutathione-S-transferase (GST), protein a, maltose binding (MBP), His label, Strep tag or label FLAG.

12. The method according to any one of claims 1 to 11, where E. coli cells are cells of E. coli K12, in particular cells of E. coli K12 strains M15[pREP4], XL1-BLUE or UT5600.

13. The polypeptide or protein having the properties of the toxin, presented in the form of double-stranded polypeptide/protein disulfide bridge and which is biologically active, characterized by the fact that Since the end of the first chain of the polypeptide/protein represents the balance of the basic amino acid and the second chain of the polypeptide/protein contains the N-end in the direction from N to s from 1 to 20 amino acid residues and pentapeptidnogo sequence VPXGS called PRS, where X is any naturally occurring amino acid, where V is Val, Leu, Ile, Ala, Phe, Pro, or Gly, where R is a Pro, Leu, Ile, Ala, Phe, Val or Gly, where G represents Gly, Leu, Ile, Ala, Pro, Phe, or Val, and where S represents Ser, Tyr, Trp or Thr.

14. The polypeptide according to item 13, where the remainder of the basic amino acid is a residue of Arg or Lys.

15. The polypeptide or protein according to 14, where the first chain of the polypeptide/protein is a more light chain of the specified polypeptide/protein, and the second circuit is a more heavy chain of the specified polypeptide/protein, frequent in the spine, where is the end of the first chain is a Lys residue.

16. The polypeptide or protein at 14 or 15, where the second circuit has N-end pentapeptidnogo sequence VPXGS, Hexapeptide sequence XVPXGS or heptapeptide sequence XXVPXGS.

17. The polypeptide or protein at 14 or 15, where this polypeptide/protein contains botulinum neurotoxin, derivative or fragment of botulinum neurotoxin, in particular the fragment LHNor has biological activity of botulinum neurotoxin, in particular, where this polypeptide/protein contains botulinum neurotoxin serotype A (BoNT(A)) or has biological activity of BoNT(A), in particular, where the polypeptide or protein is a fragment LHNBoNT(A) or has biological activity of BoNT(A).

18. The polypeptide or protein at 14 or 15, where the second circuit has N-end heptapeptide sequence SLVPXGS, in particular, where X represents R, Y, N, or Q.

19. The polypeptide or protein at 14 or 15, where the protein is a hybrid protein, in particular, where the hybrid protein consists of the following components a, b and C:
the effector domain, which through its enzymatic activity capable of inhibiting the secretion of the target cells or to destroy them, or the domain of the toxin (component a);
the sequence loops, Kotor, which contains the sequence VPXGS (component b); and
domain linking cell that gives the cell specificity of the fused protein or hybrid protein (component C); and possible component:
domain translocation as component D.

20. The polypeptide or protein according to claim 19, where the domain of the toxin (A) represents a domain of diphtheria toxin, pseudomonades of exotoxin or ricin, in particular, where the domain of the toxin (A) is a fragment RE (domain III, domain II and domain Ib) or a fragment RE (domain III and domain II) pseudomonades of exotoxin or A-chain of ricin.

21. The polypeptide or protein according to claim 19, where the domain linking cell (C), is a monoclonal antibody, affilin protein with ancyranum repetitions, anticalin, growth factor, such as TGF-alpha, FGF, VEGF or IGF-1, or cytokines, such as IL2, IL4 or IL6.

22. The polypeptide or protein according to claim 19, where the hybrid protein consists of the following components a, b and C:
protein or Oligopeptide, which gives flushed best protein solubility, contributes to a higher rate of expression and/or affinity purification (component a);
the loop sequence containing a sequence VPXGS (component b); and
any type of polypeptide (component C),
in particular, where component a is a glutathione-8-transferase (GST), protein a, maltose binding (MBP), His label, the label Strep or, a label FLAG.

23. F. rmaceuticals composition, containing polypeptide or protein having the properties of a toxin according to any one of p-22 as the active ingredient and mineral supplements.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to production of hormones and can be used for culturing invertebrates. Gonadotrophin, selected from the invertebrate Asterina pectinifera is a peptide with molecular weight 4500-4900, it has two subunits, the protein structure of which is combined with SS-bridges, formed between residues of SH cysteine contained in the subunits.

EFFECT: interfusion and oxidation of these two subunits after synthesis allow producing gonadotrophin having gonadal promoting activity.

4 cl, 8 dwg, 1 tbl, 2 ex

FIELD: biochemistry, in particular, methods and devices for producing coloring substances, possible use in food and cosmetic industry, and also during various biological research.

SUBSTANCE: phycoerythrin protein pigment is produced by extraction from seaweed. It is extracted from seaweed, selected from a group including Galaxaura oblongata, Halymenia ceylanica, Helminthocladia australis and Porphyra dentate.

EFFECT: phycoerythrin has high optical density.

2 cl, 27 dwg, 2 tbl

FIELD: equipment for growing plant tissues.

SUBSTANCE: in accordance to the invention, unit for accelerating growth of plant tissues contains a set of boards, forming matrices of holes. Each hole contains a tissue sample. Support for boards is provided by a rack which contains a set of vertically stacked shelves, containing one or more holding recesses, which forcedly move boards to given positions. Light for tissue samples is provided by a set of matrices of light diodes, mounted on mounting plates. Light diodes emit white light. Each mounting plate is supported by corresponding end comber-type rack connector, so that light diodes are close to boards, supported by shelves, positioned lower. Matrix of light diodes preferably matches matrix of holes, supported by a lower positioned shelf in fixed position, so that each light diode is centered above a corresponding hole.

EFFECT: creation of high capacity system for processing samples of tissues which require light for supporting cell reproduction.

7 cl, 7 dwg

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to the strain Bacillus subtilis 0017 isolated from a sample of typical chernozem (black soil) in Bashkorstan Republic and maintained in the collection of microorganisms of Biology Institute of Ufa scientific center RAS. The registration number of the strain in collection is 0017. The strain exceeds the known natural strains with respect to accumulation level of surfactin. Invention can be used for preparing biogenic surface-active substance surfactin possessing the multiple biological activity.

EFFECT: valuable properties of bacterial strain.

1 tbl, 4 dwg, 6 ex

The invention relates to medicine and relates to humanized antibodies that recognize the verotoxin II, and producing their cell line

The invention relates to a method for cyclosporine And high purity by purification of the crude product containing cyclosporiasis complex by multi-step chromatography on silica gel at high load columns from 10 to 52%, using as eluent a mixture of toluene with acetone in an amount of from 10 to 30 vol.% or toluene with ethyl acetate in an amount of from 10 to 35 vol.%, cyclosporine And high purity with content cyclosporine L, U and D less than 0.05% and the content of cyclosporine and < 0,02% vol., industrial method of purification of cyclosporin a from a crude product containing complex cyclosporiasis

Cyclosporine // 2085589

FIELD: medicine.

SUBSTANCE: biologically active peptide is proposed with the common formula: , where OBu - residue of 2-oxobutyric acid, Abu - residue of 2-aminobutyric acid, Dha - residue of 2,3-didehydroalanine, Dhb - residue of 2,3-didehydroaminobutyric acid, besides residues are in sequence linked with amide links, residues Abu3-Ala7, Abu22-Ala27 and Abu24-Ala31 are also linked pairwise with thioether links with formation of three residues of 3-methyllanthionine, residues Ala11-Ala21 are linked with thioether link to form residue of lanthionine. This peptide has antimicrobial effect at gram-positive bacteria Bacillus subtilis, strains L1 and 1621; Bacillus pumilus, strain 2001; Bacillus globigii, strain I; Bacillus amyloliquefaciens, strain I; Bacillus megaterium, strain VKM41; Mycobacterium smegmatis, strain 1171; Mycobacterium phlei, strain 1291; Micrococcus luteus, strain B 1314; Staphylococcus aureus, strain 209p; Rhodococcus sp., strain SSI.

EFFECT: increased efficiency of peptide antimicrobial action.

4 dwg, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: described is a peptide having antimicrobic action, which has general formula: , where OBu is a 2-oxobutyric acid residue, Abu is a 2-aminobutyric acid residue, Dha is a 2,3-didehydroanaline residue, Dhb is a 2,3-didehydroaminobutyric acid residue, where residues in the sequence are joined by amide bonds, residues Ala7 and Ala11, Ala19 and Ala23 are joined in pairs by thioester bonds to form two lanthionine (Lan) residues, Abu25 and Ala28, Abu29 and Ala32 are joined in pairs by thioester bonds to form two 3-methyllanthionine (MeLan) residues.

EFFECT: high antimicrobic action of the peptide.

4 dwg, 1 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: to obtain microbe-resistant plants plant cells are transformed with vector, which contains polynucleotide structure, which codes defensin polypeptide, which has amino-acid sequence C-x (3)-C-x (7, 9)-C-C, C-C-(8)-C-x-C and C-x-C-x (8, 11)- C.

EFFECT: obtaining polypeptide, which can be also applied as drug or veterinary medication, as well as in forage for animals.

23 cl, 4 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria. The immunogene compositions containing a transferrin-binding fiber and Hsf, and a way of their reception are offered. It is shown, that the combination of these two antigens synergically influences on production of antibodies with high activity in the analysis of bactericidal Serum. The composition can be used in vaccines against Gram-negative bacteria, including Neisseria meningitides, Neisseria gonorrhoeae.

EFFECT: creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria.

56 cl, 10 ex, 1 dwg

FIELD: medicine, microbiology.

SUBSTANCE: invention concerns nucleotide sequence coding TolC, and also to certain amino-acid sequence which is built in in permissive, located from lateral aspect of a membrane area of TolC. In the invention is described a plasmid, containing a nucleotide sequence, for an expression of the merged protein or merged peptide. The invention concerns also the transformed bacterium, in particular enterobacteria, to its use as a part of a pharmaceutical composition for stimulation of the immune response and in a diagnostic set for detection of interesting substance in an organism. The framed transport system provides high efficiency of presentation of a product of an expression in an external cellular membrane of a bacterium.

EFFECT: provision of high efficiency of presentation of product of expression in external cellular membrane of a bacterium.

13 cl, 1 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to polypeptides with established amino acid sequence, which have antimicrobial activity. Amino acid polypeptide sequence has 65-99% of identity with amino acid sequence of polypeptide, originating, particularly, from strain Pszudoplectania nigrella CBS 444.97. Invention also relates to polynucleotide, which has nucleotide sequence, coding said polypeptide, constructions of nucleic acid, recombinant expression vector, which contains said construction, and recombinant host-cell, intended for obtaining said polypeptide. In invention methods of obtaining polypeptide and inhibiting microbe cell growth using said polypeptide are described. According to invention polypeptides can be used for production of veterinary or therapeutic medication for treatment or prevention of microbial infection in humans and animals. According to invention polypeptide with antimicrobial properties ensures stability of antimicrobial activity when used in different forms of antimicrobial medications.

EFFECT: obtaining polypeptides with antimicrobial properties.

19 cl, 3 tbl, 8 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention group refers to compositions containing hapten-carrier conjugate within arranged and repeating matrix, and method of related composition production. Offered hapten-carrier conjugate used for induction of agent-specified immune reaction in case of addiction or abuse, contains cortex particle including at least one first apposition site, where specified cortex particle is virus-like particle of RNA-phage, and at least one nicotine hapten with at least one second apposition site, where specified second apposition site is associated by at least one covalent non-peptide bond with specified first apposition site, thus forming arranged and repeating hapten-carrier conjugate. Offered conjugates and compositions under this invention can include virus-like particles connected to various haptens including hormones, toxins and agent, especially agents causing addiction, as nicotine and can be applied for induction of hapten immune reaction for therapeutic, preventive and diagnostic purposes.

EFFECT: vaccines can induce stable immune reactions for nicotine and fast reduce nicotine availability for brain absorbing.

31 cl, 6 dwg

FIELD: molecular biology, veterinary.

SUBSTANCE: invention proposes isolated DNA sequence (variants) encoding Ehrlichia canis protein of size 30 kDa. Also, invention proposes vector comprising such sequence, recombinant Ehrlichia canis 28 kDa protein encoded by this sequence, a cell-host comprising this sequence, a method for preparing the protein, immunoreactive antibody specific to this protein and a method for inhibition of Ehrlichia canis infection in subject. Recombinant protein of size 28 kDa from Ehrlichia canis shows immune reactivity with respect to serum against Ehrlichia canis. Proposed group of inventions can be used in development of vaccines and serodiagnosticum that shows high effectiveness for prophylaxis of diseases and for carrying out the serodiagnosis.

EFFECT: improved preparing method, valuable medicinal and veterinary properties of protein.

19 cl, 17 dwg, 8 ex

FIELD: biotechnology, medicine, in particular treatment, prevention and diagnosis of diseases, associated with Neisseria meningitides.

SUBSTANCE: Claimed protein includes one or more N. meningitides protein fragments with known amino acid sequences, wherein said fragment contains one or more antigen determinants and has not more than 1977 amino acid from SEQ ID NO:1 and/or not more than 1531 amino acid from SEQ ID NO:2 which are described in specification with the proviso, that general protein sequence is not characterized by amino acid sequences represented in NO:1 and NO:2. Each claimed protein is encoded by nuclear acid (NA) with nucleotide sequence that defines protein amino acid sequence according to gene code. Peptides and nuclear acid of present invention are useful in drug production for treatment and prophylaxis of infections induced neisseria, as well as in production of diagnostic reagent for detection of neisseria or specific antibodies. Aldo disclosed is peptide- or NA-based pharmaceutical composition in effective amount with acceptable carrier. Said composition is useful for treatment of N. meningitides mediated infection. For prophylaxis composition is applied in vaccine form. Invention makes it possible to overcome diversity of neisseria antigen properties.

EFFECT: improved method for neisseria infection prophylaxis and treatment.

24 cl, 2 tbl

FIELD: biotechnology, in particular virulent genes and proteins.

SUBSTANCE: peptide with Neisseria meningitidis antigen activity and polynucleotide encoding the same are used in preparation of drug for prevention or treatment of diseases and conditions associated with Neisseria or gram-positive infections. Antibodies is obtained by using disclosed peptide. Vaccines for prevention or treatment of diseases and conditions associated with Neisseria meningitides contain attenuated mutant strain of Neisseria meningitides having gene mutation, insertion or deletion which disturbs expression of certain nucleotide sequence.

EFFECT: method for prevention or treatment of Neisseria infection with increased effectiveness.

13 cl, 1 tbl

FIELD: medicine.

SUBSTANCE: invention relates to compounds of formula

and their pharmaceutically acceptable salts, inhibiting activity of serine protease, in particular, activity of protease of hepatitis C virus HC3-HC4A. Claimed invention also relates to pharmaceutical compositions, which contain said compounds, method of inhibiting serine protease activity.

EFFECT: elimination or reduction of infection with hepatitis C virus.

27 cl, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula (I)

or pharmaceutically acceptable salts thereof, in which: R1, R2, R3, R4, A and E are as described in the claim, and to pharmaceutical composition containing said compounds, and a method of treating and application in order to treat conditions mediated by antagonistic activity towards acid pump, such as gastrointestinal diseases, gastrooesophageal diseases, gastrooesophageal reflux disease (GERD), laryngopharyngeal reflux disease, peptic ulcers, gastric ulcers, duodenal ulcers, NSAID- induced ulcers, gastritis, Helicobacter pylori infection, dyspepsia, functional dyspepsia, Zollinger-Ellison syndrome, nonerosive reflux disease (NERD), viscerogenic pain, cancer, heartburn, nausea, oesophagitis, dysphagia, hypersalivation, disorders of the respiratory channel or asthma.

EFFECT: possibility of using compounds to treat different diseases.

9 cl, 1 tbl, 16 ex

Up!