CN101172996A

CN101172996A - Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation

Info

Publication number: CN101172996A
Application number: CNA2007101034433A
Authority: CN
Inventors: 孙九如; 黄阳滨; 张翊; 任军; 梁光军; 姚惠珍
Original assignee: Shanghai New Life Origin Medical Co Ltd; Shanghai Newsummit Biopharma Co Ltd
Current assignee: Shanghai New Life Origin Medical Co Ltd; Shanghai Newsummit Biopharma Co Ltd
Priority date: 2006-09-29
Filing date: 2007-05-10
Publication date: 2008-05-07

Abstract

The invention discloses connection titanium having the following amino acid sequence of X-Arg-Y-Asp-Asp-Asp-Asp-Lys. The invention also discloses fusion polypeptide with the connection titanium and objective polypeptide. The invention also provides a method for preparing the objective polypeptide. The connection titanium can be cut by enterokinase, Kex2 enzyme and carboxypeptidase B after being connected with the objective polypeptide in series, thereby forming the objective polypeptide without any connection titanium sequence.

Description

Connecting peptide for polypeptide fusion expression and polypeptide fusion expression method

Technical Field

The invention belongs to the field of biotechnology and genetic engineering; more particularly, the present invention relates to a linker peptide for fusion expression of a polypeptide of interest, a fusion polypeptide prepared using the linker peptide, and a method of preparing a polypeptide of interest.

Background

The protein fusion expression has wide application in the field of genetic engineering, especially small molecular polypeptide. After isolating a gene, researchers have naturally studied the protein encoded by it, namely expression: the exogenous gene product is purposefully synthesized. In the early days of the development of recombinant DNA technology, it was thought that a strong promoter and an initiation codon in front of the gene was sufficient to obtain good expression in E.coli. Subsequently, it was realized that the conditions required to obtain efficient translation are much more complex, that in addition to a strong promoter and start codon, good expression requires the presence of a ribosome binding site in the mRNA encoding the protein of interest, and that the level of expression is influenced by codon preference and by other, currently unknown factors in the coding sequence. It is often helpful to solve the problem by altering the sequence preceding the start codon, or by altering the 5' terminal coding sequence using codon degeneracy without altering the protein sequence.

In general, fusion expression between two genes can solve these problems more quickly. In this manner, the gene of interest is introduced at the 3' end of a highly expressed protein sequence (fusion tag), such as a sequence of E.coli, or any gene that is highly expressed in E.coli, which provides the necessary signals for good expression, and the N-terminus of the expressed fusion protein contains the fragment encoded by the fusion tag. fusion tag may encode the entire functional protein or a portion thereof. Such as 6 XHis Tag, beta-galactosidase and trpE fusion proteins, glutathione S-transferase (GST) fusion proteins, thioredoxin (Trx) fusion proteins, and the like.

In other cases, the polypeptide expression of small molecules is usually performed by tandem fusion expression, i.e. a plurality of polypeptide genes are connected end to end and transferred into an expression vector for expression; thus being beneficial to the high expression of the small molecular polypeptide, preventing the degradation of the small molecular polypeptide and being beneficial to the purification after expression; the strategy is adopted to express the small molecular polypeptide, so that the cost can be reduced, and the yield of the target polypeptide can be improved. Therefore, fusion expression is also frequently used in the expression of small molecule polypeptides.

In the fusion expression of the polypeptide, connecting peptides are required to be introduced between the target polypeptides and between the target polypeptide and the leader peptide. The linker peptide is a site for cleavage of the fusion polypeptide, typically a specifically cleavable amino acid site, such as Met ↓ (cyanogen bromide), Trp ↓ (BNPS-3-methylindole), Asn ↓ Gly (hydroxylamine), Asp-Asp-Asp-Asp-Lys ↓ (enterokinase), Lys-Arg ↓ (Kex2 enzyme), and the like. These specific cleavage sites are generally introduced between the desired polypeptides before gene expression, and after the expression of the fusion polypeptide, the fusion polypeptide is cleaved at the specific sites using a specific cleavage method (chemical or enzymatic), thereby obtaining the desired monomeric polypeptide having biological activity.

The key to the technology of polypeptide tandem expression is how to process and excise the connecting peptide between the target polypeptides, and at present, no method is yet suitable. Several methods for site-specific cleavage of fusion proteins have been established internationally, including chemical cleavage and protease methods. Wherein, chemical cleavage such as cyanogen bromide (Met ↓), BNPS-3-methylindole (Trp ↓), hydroxylamine (Asn ↓ Gly) and the like is not only cheap and effective, but also can react under the denaturation condition, and is widely applied in the early stage; however, chemical methods are gradually replaced by mild enzymatic methods due to low specificity of cleavage sites, unnecessary modifications of target proteins, strong toxicity of chemicals, limited application in production, and the like.

The enzymatic method is relatively mild in reaction conditions and has high specificity. Among the most commonly used enzymes are: factor Xa, thrombin and enterokinase. Factor Xa, thrombin and enterokinase all have longer substrate recognition sequences (e.g., 7 amino acids in chymosin), thereby reducing the possibility of cleavage at other unrelated sites in the protein; the Xa factor and enterokinase cut polypeptide is identical to natural product, so the application range is very wide.

Among the methods of site-specific cleavage of polypeptides, chemical cleavage has been gradually replaced by enzymatic cleavage. The discovery of proteases with strong specificity is increasing at home and abroad, and the research on the proteases is also deepening.

There are a large number of documents at home and abroad reporting methods for cleaving fusion polypeptides using various proteases. For example, patent PCT/JP00/02736 (patent name: method for producing recombinant insulin from a novel fusion protein) reports fusion expression of an insulin-encoding gene and leader and linker peptides in an expression vector, followed by cleavage of tandem proteins using thrombin; patent 200510023428.9 (patent name: preparation method of recombinant human parathyroid hormone PTH 1-34) reports the gene engineering expression method of fusion protein Trx-PTH (1-34), after the expression of fusion protein, enterokinase is used for enzyme digestion; patent PCT/JP2002/004735 (patent name: method for producing peptide) reports a method for producing a target peptide or a salt thereof, in which a precursor protein to which enzymatic or chemical cleavage sites are added at the N-terminus and C-terminus of the target peptide and which is repeatedly linked is cleaved enzymatically or chemically; patent WO200017336-A (patent name: New DNA cassette comprising peptide encoding a biologically active peptide and linker used for producing the biologically active peptide) reports a DNA element comprising a plurality of DNA sequences encoding a biologically active polypeptide and a linker polypeptide, wherein the linker polypeptide is recognized by a protease or a chemical agent. The invention can be used for preparing bioactive polypeptide, and is particularly suitable for preparing short peptide by using genetic engineering bacteria.

However, the existing methods have the following problems that (1) the chemical method has low specificity, is easy to generate unnecessary chemical modification, has strong toxicity of chemical reagents and is difficult to popularize and apply; (2) the existing enzyme cutting method often has one or more non-target polypeptide constituent amino acids at the tail end, which greatly limits the application of the polypeptide produced by the method, especially the application in pharmacy. The above problems directly affect the use of the method for the expression of polypeptide fusions.

Therefore, there is an urgent need in the art for further improved methods for fusion expression of polypeptides and for the development of new linker peptides more suitable for fusion expression of polypeptides to overcome the drawbacks of the prior art.

Disclosure of Invention

The invention aims to provide a connecting peptide suitable for fusion expression of a polypeptide.

It is another object of the present invention to provide a fusion polypeptide comprising the connecting peptide and a polypeptide of interest.

Another object of the present invention is to provide a method for producing a polypeptide of interest using the linker peptide.

In a first aspect of the present invention, there is provided a linker peptide having the amino acid sequence:

X-Arg-Y-Asp-Asp-Asp-Asp-Lys；

wherein X is selected from Lys or Arg;

y is a sequence of no or 1-100 amino acids.

In another preferred embodiment of the present invention, Y is a sequence consisting of no or 1 to 50 amino acids. More preferably, Y is nothing or a sequence of 1-15 amino acids; further preferably, Y is a sequence of no or 1 to 10 amino acids; for example, Y may be a sequence of 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids.

In a second aspect of the invention, there is provided the use of said linker peptide for linking 2 or more polypeptides of interest to produce a fusion polypeptide; with the additional condition that: the target polypeptide does not contain enzyme cutting sites of enterokinase, Kex2 enzyme and carboxypeptidase B.

In a third aspect of the invention, there is provided a DNA molecule encoding said linker peptide.

In a fourth aspect of the invention, there is provided a fusion polypeptide comprising the structure:

Z0-(Z-Z1)n；

wherein Z0 represents a first polypeptide of interest;

z represents the connecting peptide;

z1 represents a second polypeptide of interest, which may be the same or different;

n＝1-99；

the first polypeptide of interest and the second polypeptide of interest are the same or different and do not contain enterokinase, Kex2 enzyme and carboxypeptidase B enzyme sites.

Preferably, n is 1-49; more preferably, n is 1 to 19.

In another preferred embodiment of the present invention, the fusion polypeptide is cleaved by enterokinase, Kex2 enzyme and carboxypeptidase B enzyme to form the target polypeptide with the linker sequence removed.

In another preferred embodiment of the present invention, the fusion polypeptide is obtained by recombinant expression.

In a fifth aspect of the invention, there is provided a method of preparing a polypeptide of interest, said method comprising the steps of:

adding enterokinase, Kex2 enzyme and carboxypeptidase B to a system containing the fusion polypeptide, and allowing the fusion polypeptide to be digested by enterokinase, Kex2 enzyme and carboxypeptidase B to form a mixture of the target polypeptide and the connecting peptide fragment, and

isolating the polypeptide of interest from the mixture.

In another preferred embodiment of the present invention, enterokinase, Kex2 enzyme and carboxypeptidase B are added simultaneously to a system containing the fusion polypeptide to cleave the fusion polypeptide; alternatively, enterokinase, Kex2 enzyme and carboxypeptidase B were added to the system containing the fusion polypeptide, respectively, to cleave the fusion polypeptide.

In another preferred embodiment of the present invention, the temperature of the enzyme cleavage is 4-60 ℃. Preferably, the temperature of the enzyme is 10-30 deg.C, more preferably 25-30 deg.C.

In another preferred embodiment of the present invention, the pH of the enzyme is 4.0-11.0. Preferably, the pH value of the enzyme digestion is 6.0-9.0; more preferably, the pH value of the enzyme is 7.0-8.0; most preferably 7.3-7.5.

In another preferred embodiment of the invention, the enterokinase, the Kex2 enzyme and the carboxypeptidase B are added according to the enzyme activity: enterokinase Kex2 enzyme carboxypeptidase B ═ 1-4: (1-4): (0.5-4). More preferably, the enterokinase, the Kex2 enzyme and the carboxypeptidase B are added according to the enzyme activity: enterokinase Kex2 enzyme carboxypeptidase B2: 1.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 shows a schematic of plasmid construction of the fusion polypeptide.

FIG. 2 shows SDS-PAGE electrophoretic identification of expression of pTHiohisa-insulin fusion protein, and electrophoretic identification after enzyme cleavage of the fusion protein; wherein,

M：Marker(97.4KD，66.2KD，42.7KD，31.0KD，20.1KD，14.4KD)；

1: carrying out electrophoretic identification on the pTHiohisa-insulin fusion protein;

2: the pTHiohisA/insulin fusion protein is identified by the electrophoresis after the enzyme digestion of EK;

3: the pTHiohisA-insulin fusion protein is cut by EK enzyme, KEX2 enzyme and CPB enzyme and then purified to obtain the result of insuli.

FIG. 3 shows SDS-PAGE electrophoretic identification of pET30a (+) -PTH fusion protein expression, and electrophoretic identification of the fusion protein after enzyme digestion; wherein,

M：Marker(97.4KD，66.2KD，43KD，31.0KD，20.1KD，14.4KD)；

1: electrophoretic identification of pET30a (+) -PTH fusion protein;

2: the pET30a (+) -PTH fusion protein was digested with EK enzyme, KEX2 enzyme and CPB and purified to obtain PTH results.

FIG. 4 shows SDS-PAGE electrophoretic identification of pET30a (+) -C peptide fusion protein expression, and electrophoretic identification of the fusion protein after enzyme digestion; wherein,

M1：Marker(97.4KD，66.2KD，42.7KD，31.0KD，20.1KD，14.4KD)；

M2：Marker(16949D，14404D，10700D，8159D，6214D，2512D)；

1: electrophoretic identification of pET30a (+) -C peptide fusion protein;

2-4: the pET30a (+) -C peptide fusion protein is digested by EK enzyme, Kex2 enzyme and CPB, and then purified to obtain C peptide result.

Detailed Description

The present inventors have conducted intensive studies and, for the first time, have designed a linker peptide which is particularly suitable for the fusion expression of a polypeptide of interest. The linker peptide can be cleaved by Enterokinase (EK), Kex2 enzyme and carboxypeptidase B (CPB)3 enzymes after being expressed in tandem with the target polypeptide, thereby forming the target polypeptide without any linker peptide sequence. In addition, the connecting peptide contains hydrophilic amino acids Asp, Lys and Arg, and is easy to be exposed on the surface of the fusion polypeptide, thereby providing convenience for enzyme digestion. The present invention has been completed based on this finding.

Linker peptide

The invention provides a connecting peptide, which has the following amino acid sequence:

X-Arg-Y-Asp-Asp-Asp-Asp-Lys；

wherein X is selected from Lys or Arg;

y is nothing or 1-100 sequences consisting of any amino acid; preferably, Y is nothing or 1-50 sequences consisting of any amino acid; more preferably, Y is nothing or a sequence of 1-15 amino acids; further preferably, Y is a sequence of no or 1 to 10 amino acids; for example, Y may be a sequence of 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids.

In the connecting peptide, Arg, Lys and Asp contained in the connecting peptide belong to hydrophilic amino acids, so that the connecting peptide is easily exposed on the surface of the fusion polypeptide when the fusion polypeptide containing the connecting peptide is in an aqueous solution, and is easily digested.

The connecting peptide contains an enterokinase enzyme cutting site sequence Asp-Asp-Asp-Asp-Lys ↓; the sequence Lys-Arg ↓orArg ↓ofthe enzyme cutting site of Kex 2; and carboxypeptidase B enzyme cutting sites ↓Argand/or ↓Lys.

The invention also provides a gene, and the gene can code the connecting peptide.

In addition, the invention also provides a recombinant vector containing the coding gene of the connecting peptide, and a gene engineering cell containing the recombinant vector or the coding gene of the connecting peptide integrated in the genome.

In a preferred embodiment of the present invention, the amino acid sequence represented by Y contains hydrophilic amino acids; preferably, the amino acid sequence shown by Y contains at least 20 percent of hydrophilic amino acids; more preferably, Y represents an amino acid sequence comprising at least 50% hydrophilic amino acids. The hydrophilic amino acid is beneficial to exposing the connecting peptide on the surface of the spatial structure of the fusion polypeptide, thereby facilitating enzyme digestion.

Enzyme

In the invention, the enzyme digestion of the fusion polypeptide is realized by combining three genetic engineering enzymes, so that the target polypeptide does not contain a sequence of a connecting peptide after enzyme digestion.

Enterokinase (Enterokinase, EK): is a protease that recognizes the sequence Asp-Asp-Asp-Asp-Lys, hydrolyzing the polypeptide specifically at the C-terminus of Lys. In general, enterokinase can be used at a wide range of temperatures and pH, e.g., temperatures of 4-45 ℃ and pH ranges of 4-10.

The Kex2 enzyme, encoded by the gene KEX2, is capable of selectively recognizing and hydrolyzing Lys-Arg or Arg-Arg bond residues at the carboxy terminus of a polypeptide. The preferred temperature range for Kex2 enzyme action is 4-45 deg.C; the pH value is in the range of 4.5-9.5.

Carboxypeptidase B (CPB) belongs to the class of carboxypeptidases, which is capable of selectively hydrolysing peptide bonds with Arg and Lys residues at the carboxy-terminal end. The preferred temperature range for the action of carboxypeptidase B is 4-60 ℃; the pH value is 5-11.

In the present invention, the above-mentioned genetically engineered enzyme may be used under the same conditions (e.g., temperature or pH); alternatively, the skilled person can also use these enzymes individually with appropriate adjustment of the enzymatic conditions to exert the optimal properties of the enzymes, while maintaining the activity of the polypeptide of interest.

The fusion polypeptide containing the connecting peptide is subjected to enzyme digestion by combining the enterokinase, the Kex2 enzyme and the carboxypeptidase B, so that the enzyme digestion efficiency is high, and the target polypeptide generated after enzyme digestion does not contain any constituent amino acid of non-target polypeptide and is completely consistent with the natural target polypeptide.

Polypeptide of interest

The target polypeptide (target protein) suitable for use in the present invention is not particularly limited as long as it can be expressed in tandem by the linker peptide of the present invention and does not contain the cleavage sites for enterokinase, Kex2 enzyme and carboxypeptidase B in its amino acid sequence. Typically, the polypeptide of interest is between 1-500aa in length; preferably, the length of the target polypeptide is between 5 and 200 aa; more preferably, the length of the polypeptide of interest is between 5-100 aa; further preferably, the length of the polypeptide of interest is between 10-80 aa; most preferably, the length of the polypeptide of interest is between 10-50aa, such as 15aa, 20aa, 25aa, 30aa, 35aa, 40aa, 45 aa.

In a preferred embodiment of the present invention, the target polypeptide is a small molecule polypeptide. The small molecule polypeptide is, for example: insulin (Insulin), parathyroid hormone (pTH), Osteogenic Growth Peptide (OGP), or β -chorionic gonadotropin (β -HCG), etc.

As a preferred embodiment of the present invention, the target polypeptide itself may also be a fusion protein, such as: insulin A chain-Lys-Arg-insulin B chain.

Fusion polypeptides

The invention provides a fusion polypeptide (fusion protein), which contains the following structure:

Z0-(Z-Z1)n

wherein Z0 represents a first polypeptide of interest;

z represents the connecting peptide;

z1 represents a second polypeptide of interest which may be the same or different.

That is, the fusion polypeptide contains, for example, the following structure: target polypeptide 1-connecting peptide-target polypeptide 2-connecting peptide-target polypeptide m (m is a positive integer, m is less than or equal to 100).

The fusion polypeptide can be cut by enterokinase, Kex2 enzyme and carboxypeptidase B enzyme to form a target polypeptide with a connecting peptide sequence removed.

In practice, the type of polypeptide of interest may be selected as desired in the art. In the fusion polypeptide, the target polypeptide can be a polypeptide having the same amino acid sequence, i.e., the same polypeptide; alternatively, the polypeptides of interest may be polypeptides having different amino acid sequences, i.e., different species of polypeptides. These may vary depending on the actual operating requirements.

That is, the first polypeptide of interest and the second polypeptide of interest can be the same or different, and the second polypeptide of interest can be the same or different.

Generally, the amount of the polypeptide of interest in the fusion polypeptide will depend on the length, nature of the polypeptide of interest, the type of expression vector, the type of engineered cell, and the like. Typically, in the fusion polypeptide, the number of polypeptides of interest is 2-100 (n-1-99); more preferably, the number of polypeptides of interest is 2 to 50 (n-1-49); most preferably, the number of polypeptides of interest is 2-20 (n-1-19).

As a preferred mode of the present invention, the fusion polypeptide is exemplified by:

(I) Arg-Ser-Asp-Asp-Asp-Asp-Lys-insulin B chain-Lys-Arg-Ala-Asp-Asp-Asp-Asp-Lys-insulin A chain-Lys-Arg-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys-insulin B chain-Lys-Arg-Ala-Asp-Asp-Asp-Lys-insulin A chain-Lys-Arg-Asp-Asp-Asp-Lys-insulin A chain-Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Asp-Lys-insulin B chain-Lys-Arg-Ala-Asp-Asp-Asp-Asp-Asp-Lys-insulin A chain; or

(II) Arg-Ser-Asp-Lys-PTH-Lys-Arg-Gly-Ser-Asp-Lys-PTH-Lys-Arg-Gly-Ser-Asp-Lys-PTH-Lys-Arg; or

(III) Arg-Ser-Asp-Asp-Asp-Lys-C-peptide-Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Lys-C-peptide-Arg-Gly-Gly-Gly-Ser-Asp-Asp-Lys-C-peptide-Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys-C-peptide.

The fusion polypeptide containing the connecting peptide and the target polypeptide prepared by the invention does not have any more amino acids at the N end and the C end of the target polypeptide under the combined enzyme digestion action of enterokinase, carboxypeptidase B and Kex2 enzymes. The target polypeptide is prepared by expressing the fusion polypeptide, so that the production efficiency and the product quality of the target polypeptide can be greatly improved.

The invention also provides a gene encoding the fusion polypeptide, a recombinant vector containing the encoding gene of the fusion polypeptide, and a genetically engineered cell containing the recombinant vector or the encoding gene of the fusion polypeptide integrated in the genome.

Methods for the preparation or expression of fusion polypeptides are well known to those skilled in the art and may, for example, employ the following steps:

(1) connecting the gene for coding the target polypeptide and the gene for coding the connecting peptide in series according to the required copy number to form a fusion gene; wherein the ligation can be performed by designing appropriate primers, or can be artificially synthesized to synthesize the desired fusion gene;

(2) inserting the fusion gene prepared in the step (1) into a multiple cloning site of an expression vector to obtain the expression vector inserted with the fusion gene;

(3) transferring the expression vector inserted with the fusion gene obtained in the step (2) into host cells to obtain transformed host cells;

(4) culturing the transformed host cell, thereby expressing the fusion polypeptide;

(5) and separating to obtain the fusion polypeptide.

In a preferred embodiment of the present invention, the conventional method for preparing a fusion polypeptide can be performed as follows: by adopting a gene recombination technology, firstly, a gene for coding a target polypeptide is recombined and connected with a gene for coding a connecting peptide, and then, the sequence of the recombined gene is connected in series to form a plurality of copies; after sequencing analysis and verification, the fusion gene is inserted into an expression vector, transferred into a host cell, induced to express and purified.

Methods well known to those skilled in the art can be used to construct expression vectors containing the sequence of the gene encoding the fusion polypeptide and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The sequence of the gene encoding the fusion polypeptide may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector may also include a ribosome binding site for translation initiation and a transcription terminator.

The host cell is any cell suitable for expressing the fusion polypeptide, and may be, for example, a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast and the like.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers, host cells, and the like.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the fusion polypeptide of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell.

The fusion polypeptide may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, the fusion polypeptide can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Process for producing target polypeptide

The present invention provides a method for preparing a polypeptide of interest, said method comprising the steps of:

(a) preparing a fusion polypeptide comprising the structure:

Z0-(Z-Z1)n

wherein Z0, Z, Z1 and n are as defined above.

(b) Cleaving the fusion polypeptide prepared in (a) with enterokinase, Kex2 enzyme and carboxypeptidase B to form a mixture of the polypeptide of interest and the linker peptide fragment.

(c) Isolating the polypeptide of interest from the mixture.

In step (B), enterokinase, Kex2 enzyme and carboxypeptidase B may be simultaneously added to the system containing the fusion polypeptide prepared in (a); alternatively, enterokinase, Kex2 enzyme and carboxypeptidase B may be added to the system containing the fusion polypeptide prepared in (a), respectively. When used for enzymatic cleavage, the present invention is not particularly limited with respect to the order of addition of enterokinase, Kex2 enzyme and carboxypeptidase B to the fusion polypeptide-containing system.

In one embodiment, enterokinase, Kex2 enzyme and carboxypeptidase B may be added sequentially to the fusion polypeptide containing system, and the order of cleavage of the linker peptide from the fusion polypeptide by these enzymes may be as follows:

alternatively, Kex2 enzyme, enterokinase and carboxypeptidase B may be added sequentially to the fusion polypeptide containing system, and the order of cleavage of the linker peptide from the fusion polypeptide by these enzymes may be as follows:

when used for enzymatic cleavage, the amounts of enterokinase, Kex2 enzyme and carboxypeptidase B added are generally determined by the number of copies of the linker peptide contained in the fusion protein, and are not particularly limited in the present invention. For example, when the fusion protein contains 1 copy of the linker peptide, the enterokinase is added in an amount of 1-5 units per 50. mu.g of the fusion polypeptide; the amount of Kex2 enzyme added was 1-5 units/50. mu.g of fusion polypeptide; or carboxypeptidase B is added in an amount of 0.5-3 units per 50ug of fusion polypeptide.

As a preferred mode of the invention, the temperature of enzyme digestion is 4-60 ℃; preferably, the temperature of enzyme cutting is 10-30 ℃; more preferably 25-30 deg.C.

In a preferred embodiment of the present invention, the pH of the enzyme is 4.0 to 11.0. Preferably, the pH value of the enzyme digestion is 6.0-9.0; more preferably, the pH value of the enzyme is 7.0-8.0; most preferably 7.3-7.5.

In a preferred embodiment of the present invention, three enzymes, enterokinase, carboxypeptidase B and Kex2, are added to the same digestion reaction system to digest the pTH-Lys-Arg-Ala-Asp-Asp-Asp-Asp-Lys-pTH-Lys-Arg-Ala-Asp-Asp-Asp-Asp-Lys-pTH-Lys-Arg-Ala-Asp-Asp-Asp-Asp-Asp-Lys-pTH fusion protein. The reaction temperature was: at 25-30 ℃, the reaction pH is: 7.4, the buffer system is: 20mM Tris.Cl100mM NaCl.

Isolation of the polypeptide of interest

After enzymatic cleavage, the polypeptide of interest can be isolated from the mixed system using a variety of protein isolation techniques well known to those skilled in the art. The isolation method is generally determined according to the properties of the polypeptide of interest. For example, the separation can be carried out according to the difference in isoelectric points between the polypeptide of interest to be separated and other peptides in a mixed system. Examples of other methods include, but are not limited to: molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC) or a combination of these methods.

The main advantages of the invention are:

(1) by introducing the connecting peptide provided by the invention between the fusion expressed polypeptides, multienzyme combined enzyme digestion can be used after the fusion polypeptides are expressed, so that the production efficiency of the target polypeptides can be greatly improved, and the target polypeptides obtained after the expressed fusion polypeptides are subjected to enzyme digestion do not contain any non-target polypeptide-constituting amino acid and are completely consistent with natural polypeptides.

(2) The connecting peptide contains a plurality of hydrophilic amino acids, and is easy to be exposed on the surface of the fusion polypeptide, so that the enzyme digestion processing efficiency is particularly high.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

Example 1 fusion expression and Combined enzyme cleavage processing of Insulin (Insulin)

In this example, the desired polypeptides of interest to be prepared are the Insulin A chain (Insulin A chain, GenBank accession No. NM-000207) and the Insulin B chain (Insulin B chain, GenBank accession No. NM-000207) using the following linker peptide sequences:

Arg-Ser-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 1), and AGA TCT GAT GAC GAT GAC AAA (SEQ ID NO: 2) as the sequence of the corresponding coding gene;

Lys-Arg-Ala-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 3) and the corresponding gene sequence of AAG AGA GCTGAT GAC GAT GAT AAG(SEQ ID NO：4)；

Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 5) and the corresponding sequence of the coding gene is AAG AGA GGT GGA GGTGGA TCT GAT GAC GAT GAC AAA(SEQ ID NO：6)。

1. Preparation of fusion polypeptide

The fusion polypeptides were designed as follows: Arg-Ser-Asp-Asp-Asp-Asp-Lys-insulin B chain-Lys-Arg-Ala-Asp-Asp-Asp-Asp-Lys-insulin A chain-Lys-Arg-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys-insulin B chain-Lys-Arg-Ala-Asp-Asp-Asp-Lys-insulin A chain-Lys-Arg-Asp-Asp-Asp-Lys-insulin A chain-Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Asp-Lys-insulin B chain-Lys-Arg-Ala-Asp-Asp-Asp-Asp-Asp-Lys-insulin A chain;

with 3 copies of the insulin B chain and 3 copies of the insulin A chain.

First, the following sequence (SEQ ID NO: 7) containing an insulin A chain, a linker peptide and an insulin B chain-encoding gene was synthesized by an artificial synthesis method:

AA CAC TTG TGC GGT TCC CAC TTG GTT GAG

GCT TTG TAC TTG GTT TGC GGT GAA AGA GGT TTC TTC TAC ACT CCT AAG ACT AAG

AGA GCT GAT GAC GAT GAT AAG GGT ATT GTC GAA CAA TGC TGT ACC TCC ATC TGC

TCC TT

next, primers were designed as follows:

a forward primer: ggaAGA TCTGAT GAC GAT GAC AAA TTC GTT AAC C (SEQ ID NO: 8, with the Bgl II cleavage site underlined);

reverse primer: cg (cg)GGA TCCACC TCC ACC TCT CTT GTT GCA GTA GTT TTC CAATTG GTA C (SEQ ID NO: 9, with the Bam HI cleavage site underlined).

And carrying out PCR reaction by taking the prepared sequence containing the insulin A chain, the connecting peptide and the insulin B chain coding gene as a template to obtain the target gene which can code an insulin B chain-Lys-Arg-Ala-Asp-Asp-Asp-Asp-Lys-insulin A chain sequence and has enzyme cutting sites at two ends.

Next, in order to synthesize a fusion polypeptide containing 3 copies of the insulin B chain and 3 copies of the insulin A chain, the following operations were carried out (see FIG. 1):

(1) the target gene obtained above was digested with Bgl II and Bam HI, and the digested product was cloned into Bgl II-digested pET30a (+) vector (purchased from Novagen) (Bgl II and Bam HI are isocaudarner enzymes), to construct a recombinant vector containing 1 copy of the target gene.

(2) And (3) similarly, carrying out enzyme digestion on the obtained target gene by Bgl II and Bam HI, and cloning the enzyme digestion product into the recombinant vector obtained in the step (1) subjected to enzyme digestion by Bgl II to form a recombinant vector containing 2 copies of the target gene.

(3) And (3) similarly, carrying out enzyme digestion on the obtained target gene by Bgl II and Bam HI, and cloning the enzyme digestion product into the recombinant vector obtained in the step (2) subjected to enzyme digestion by Bgl II to form a recombinant vector containing 3 copies of the target gene.

After the above ligation, the obtained fusion gene was sequenced and analyzed correctly, and the gene encoding 3 copies of insulin A chain and 3 copies of insulin B chain ligated with the gene encoding the linker peptide was obtained as follows (SEQ ID NO: 10, this sequence was also synthesized by conventional artificial synthesis techniques):

ccAGA TCT GAT GAC GAT GAC AAA TTC GTT AAC CAA CAC TTG TGC GGT TCC CAC TTG GTT

GAG GCT TTG TAC TTG GTT TGC GGT GAA AGA GGT TTC TTC TAC ACT CCT AAG ACT AAG

AGA GCT GAT GAC GAT GAT AAG GGT ATT GTC GAA CAA TGC TGT ACC TCC ATC TGC TCC

TTG TAC CAA TTG GAA AAC TAC TGC AAC AAG AGA GGT GGA GGT GGA TCT GAT GAC GAT

GAC AAA TTC GTT AAC CAA CAC TTG TGC GGT TCC CAC TTG GTT GAG GCT TTG TAC TTG

GTT TGC GGT GAA AGA GGT TTC TTC TAC ACT CCT AAG ACT AAG AGA GCT GAT GAC GAT

GAT AAG GGT ATT GTC GAA CAA TGC TGT ACC TCC ATC TGC TCC TTG TAC CAA TTG GAA

AAC TAC TGC AAC AAG AGA GGT GGA GGT GGA TCT GAT GAC GAT GAC AAA TTC GTT AAC

CAA CAC TTG TGC GGT TCC CAC TTG GTT GAG GCT TTG TAC TTG GTT TGC GGT GAA AGA

GGT TTC TTC TAC ACT CCT AAG ACT AAG AGA GCT GAT GAC GAT GAT AAG GGT ATT GTC

GAA CAA TGC TGT ACC TCC ATC TGC TCC TTG TAC CAA TTG GAA AAC TAC TGC AAC AAG

AGA GGT GGA GGT GGA TCC

next, the double digestion was carried out with Bgl II and Bam HI, and the digested product was cloned into pTHiohishA vector (purchased from Invitrogen) which had been subjected to the same double digestion. And transforming the formed recombinant expression vector into escherichia coli BL21(DE3), constructing an engineering bacterium BL21(DE3)/pTHiohisa/insulin3 fusion gene, inducing expression, identifying that the insulin3 copy fusion protein is expressed (see figure 2, lane 1), and purifying to obtain the insulin3 copy fusion protein.

2. Enzyme digestion

Adding enterokinase, carboxypeptidase B and Kex2 enzyme 3 enzymes into the same enzyme digestion reaction system to carry out enzyme digestion on the prepared fusion polypeptide, wherein the enterokinase is used at a concentration of about: 12 units/50. mu.g fusion protein (each fusion protein contains 6 enterokinase cleavage sites), carboxypeptidase B was used at a concentration of: 6 units/50. mu.g fusion protein (each fusion protein contains 6 carboxypeptidase B cleavage sites), Kex2 enzyme was used at a concentration: 12 units/50. mu.g of fusion protein (each fusion protein contains 6 carboxypeptidase B cleavage sites).

The temperature of the enzyme digestion reaction is as follows: at 25-30 ℃, the reaction pH is: 7.4, the buffer system is: 20mM Tris.Cl100mM NaCl.

The result of the SDS-PAGE electrophoretic identification of the pTHiohishA/insulin fusion protein after EK cleavage is shown in FIG. 2 (lane 2); the pTHiohisa-insulin fusion protein was digested with EK enzyme, KEX2 enzyme and CPB, and purified to obtain insulin, which was then identified on SDS-PAGE as shown in FIG. 2 (lane 3).

Further characterization shows that the isolated A and B chains of insulin have the same sequence and activity as native insulin.

Example 2 fusion expression and Multi-enzyme Combined enzymatic cleavage processing of PTH

In this example, the polypeptide of interest to be prepared is parathyroid hormone (PTH) (GenBank accession No. NM — 000315), and the linker peptide sequence used is as follows:

Arg-Ser-Asp-Asp-Asp-Asp-Lys(SEQ ID NO：1)；

Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys(SEQ ID NO：5)。

1. preparation of fusion polypeptide

With reference to a similar method as described in example 1, the pTH gene was ligated in tandem using the coding sequence of the aforementioned linker peptide into a fusion gene containing 3 copies of the gene of interest and encoding the following fusion polypeptides: Arg-Ser-Asp-Asp-Asp-Asp-Lys-PTH-Lys-Arg-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Lys-PTH-Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys-Arg-Gly-Gly-Gly-Ser-Asp-Asp-Lys-PTH-Lys-Arg copy.

The sequencing analysis result is correct. The fusion gene containing 3 copies of pTH gene was inserted into pET30a (+) vector (purchased from Novagen), and then the engineered bacterium BL21(DE3)/pET30a (+)/pTH 3 copy was constructed, expression was induced, and the expression of pTH 3 copy fusion protein was identified (FIG. 3, lane 1), and purified to obtain pTH 3 copy fusion protein.

2. Enzyme digestion

Adding three enzymes of enterokinase, carboxypeptidase B and KEX2 into the same enzyme digestion reaction system to carry out enzyme digestion on the pTH 4 copy fusion protein.

The temperature of the enzyme digestion reaction is as follows: at 25-30 ℃, the pH of the reaction is 7.4, and the buffer system is as follows: 20mM Tris.Cl100mM NaCl.

The pET30a (+) -PTH fusion protein was purified from the enzyme EK, KEX2 and CPB after digestion and the results are shown in FIG. 3 (lane 2).

Further characterization showed that the isolated pTH polypeptide has the same sequence and activity as the native pTH polypeptide.

Example 3 fusion expression and processing of C peptide by enzyme-Linked cleavage

In this example, the desired polypeptide of interest to be produced is the C peptide (coding gene GAA GCT GAA GAT TTGCAA GTT GGT CAA GTT GAA TTG GGT GGT GGG CCC GGT GCT GGT TCT TTG CAA CCATTG GCT TTG GAA AAT TCT TTG CAA (SEQ ID NO: 11)), and the linker peptide sequence used is as follows:

Arg-Ser-Asp-Asp-Asp-Asp-Lys(SEQ ID NO：1)；

Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys(SEQ ID NO：5)。

referring to a similar method as described in example 1, the C-peptide gene was concatenated into a fusion gene containing 4 copies of the gene of interest and encoding the following fusion polypeptides using the coding sequence of the aforementioned linker peptide:

Arg-Ser-Asp-Asp-Asp-Asp-Lys-C-peptide-Lys-Arg-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Lys-C-peptide-Lys-Asp-Lys-Arg-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Lys-C-peptide-Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys-C-peptide.

The sequencing analysis result is correct. The fusion gene containing 4 copies of the C peptide gene was inserted into pET30a (+) vector to construct BL21(DE3)/pET30a (+)/pTH 4 copies of the engineered bacteria, expression was induced, and the C peptide 4 copies of the fusion protein were identified to be expressed (FIG. 4, lane 1), and purified to obtain C peptide 4 copies of the fusion protein.

2. Enzyme digestion

Adding three enzymes of enterokinase, carboxypeptidase B and KEX2 into the same enzyme digestion reaction system to carry out enzyme digestion on the C peptide 4 copy fusion protein.

The reaction temperature was: at 25-30 ℃, the reaction pH is: 7.4, the buffer system is: 20mM Tris.Cl100mM NaCl.

The pET30a (+) -C peptide fusion protein was cleaved with EK enzyme, Kex2 enzyme and CPB and purified to obtain C peptide, the results of which are shown in FIG. 4 (

lanes

2, 3 and 4).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai New Source medicine research Co., Ltd

Shanghai Xinshengyuan Biological Medical Co.,Ltd.

<120> linker peptide for polypeptide fusion expression and polypeptide fusion expression method

<130>072677

<160>11

<170>PatentIn version 3.3

<210>1

<211>7

<212>PRT

<213> Artificial sequence

<220>

<221>MISC_FEATURE

<223> linker peptide

<400>1

Arg Ser Asp Asp Asp Asp Lys

1 5

<210>2

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

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<400>2

agatctgatg acgatgacaa a 21

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<213> Artificial sequence

<220>

<221>MISC_FEATURE

<223> linker peptide gene

<220>

<221>MISC_FEATURE

<223> linker peptide

<400>3

Lys Arg Ala Asp Asp Asp Asp Lys

1 5

<210>4

<211>24

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> linker peptide gene

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aagagagctg atgacgatga taag 24

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Lys Arg Gly Gly Gly Gly Ser Asp Asp Asp Asp Lys

1 5 10

<210>6

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aagagaggtg gaggtggatc tgatgacgat gacaaa 36

<210>7

<211>207

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> insulin A chain-connecting peptide-insulin B chain coding gene

<400>7

gatgacgatg acaaattcgt taaccaacac ttgtgcggtt cccacttggt tgaggctttg 60

tacttggttt gcggtgaaag aggtttcttc tacactccta agactaagag agctgatgac 120

gatgataagg gtattgtcga acaatgctgt acctccatct gctccttgta ccaattggaa 180

aactactgca acaagagagg tggaggt 207

<210>8

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<212>DNA

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ggaagatctg atgacgatga caaattcgtt aacc 34

<210>9

<211>48

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<220>

<221>misc_feature

<223> primer

<400>9

cgggatccac ctccacctct cttgttgcag tagttttcca attggtac 48

<210>10

<211>647

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Gene encoding 3 copies of insulin A chain and 3 copies of insulin B chain

<400>10

ccagatctga tgacgatgac aaattcgtta accaacactt gtgcggttcc cacttggttg 60

aggctttgta cttggtttgc ggtgaaagag gtttcttcta cactcctaag actaagagag 120

ctgatgacga tgataagggt attgtcgaac aatgctgtac ctccatctgc tccttgtacc 180

aattggaaaa ctactgcaac aagagaggtg gaggtggatc tgatgacgat gacaaattcg 240

ttaaccaaca cttgtgcggt tcccacttgg ttgaggcttt gtacttggtt tgcggtgaaa 300

gaggtttctt ctacactcct aagactaaga gagctgatga cgatgataag ggtattgtcg 360

aacaatgctg tacctccatc tgctccttgt accaattgga aaactactgc aacaagagag 420

gtggaggtgg atctgatgac gatgacaaat tcgttaacca acacttgtgc ggttcccact 480

tggttgaggc tttgtacttg gtttgcggtg aaagaggttt cttctacact cctaagacta 540

agagagctga tgacgatgat aagggtattg tcgaacaatg ctgtacctcc atctgctcct 600

tgtaccaatt ggaaaactac tgcaacaaga gaggtggagg tggatcc 647

<210>11

<211>93

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> C peptide-encoding gene

<400>11

gaagctgaag atttgcaagt tggtcaagtt gaattgggtg gtgggcccgg tgctggttct 60

ttgcaaccat tggctttgga aaattctttg caa 93

Claims

1. A linker peptide having the amino acid sequence:

X-Arg-Y-Asp-Asp-Asp-Asp-Lys；

wherein X is selected from Lys or Arg;

y is a sequence of no or 1-100 amino acids.

2. The linker peptide of claim 1 wherein Y is a sequence of no or 1-50 amino acids.

3. Use of the linker peptide of claim 1 for linking 2 or more polypeptides of interest to produce a fusion polypeptide;

with the additional condition that: the target polypeptide does not contain enzyme cutting sites of enterokinase, Kex2 enzyme and carboxypeptidase B.

4. A DNA molecule encoding the linker peptide of claim 1.

5. A fusion polypeptide comprising the structure:

Z0-(Z-Z1)n；

wherein Z0 represents a first polypeptide of interest;

z represents the linker peptide of claim 1;

n＝1-99；

6. A method for producing a polypeptide of interest, said method comprising the steps of:

adding enterokinase, Kex2 enzyme and carboxypeptidase B to a system containing the fusion polypeptide of claim 5, such that the fusion polypeptide is enzymatically cleaved by the enterokinase, Kex2 enzyme and carboxypeptidase B to form a mixture of the polypeptide of interest and the linked peptide fragments, and

isolating the polypeptide of interest from the mixture.

7. The method of claim 6, wherein the temperature of the cleavage is 4-60 ℃.

8. The method of claim 6, wherein the pH of the enzyme is 4.0 to 11.0.

9. The method of claim 6, wherein the enterokinase, the Kex2 enzyme and the carboxypeptidase B are added in a ratio such that the enzyme activities are: enterokinase Kex2 enzyme carboxypeptidase B ═ 1-4: (1-4): (0.5-4).