Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation

A technology for linking peptides and fusion peptides, applied in the fields of biotechnology and genetic engineering, can solve the problems of low specificity of chemical methods, limited use of peptides, and difficulty in popularization and application.

Inactive Publication Date: 2008-05-07
SHANGHAI NEWSUMMIT BIOPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, there are still many problems in the current methods as follows (1) the specificity of the chemical method is low, unnecessary chemical modification is easily produced, and the chemical reagent is highly toxic, so it is difficult to popularize and apply; One or more non-target amino acids at the end of the polypeptide, which greatly limits the use of polypeptides produced by this method, especially in pharmaceutical applications

Method used

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  • Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation
  • Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation
  • Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation

Examples

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preparation example Construction

[0094] The preparation or expression method of the fusion polypeptide is well known to those skilled in the art, for example, the following steps can be used:

[0095] (1) The gene encoding the target polypeptide and the gene encoding the connecting peptide are connected in series according to the required copy number to form a fusion gene; the connection can be realized by designing appropriate primers, or can also be artificially synthesized The method for synthesizing the required fusion gene;

[0096] (2) inserting the fusion gene prepared in (1) into the multiple cloning site of the expression vector to obtain an expression vector inserted with the fusion gene;

[0097] (3) transferring the expression vector inserted into the fusion gene obtained in (2) into a host cell to obtain a transformed host cell;

[0098] (4) cultivating transformed host cells to express the fusion polypeptide;

[0099] (5) Separating and obtaining the fusion polypeptide.

[0100] In a preferre...

Embodiment 1

[0129] Example 1 Fusion expression of insulin (Insulin) and combined multi-enzyme digestion processing

[0130] In this example, the target polypeptides to be prepared are insulin A chain (Insulin A chain, GenBank accession number NM_000207) and insulin B chain (Insulin B chain, GenBank accession number NM_000207), and the linking peptide sequences used are as follows:

[0131]Arg-Ser-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 1), the sequence of its corresponding coding gene is AGA TCT GAT GAC GAT GAC AAA (SEQ ID NO: 2);

[0132] Lys-Arg-Ala-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 3), the sequence of its corresponding coding gene is AAG AGA GCT GAT GAC GAT GAT AAG (SEQ ID NO: 4);

[0133] Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 5), the sequence of its corresponding coding gene is AAG AGA GGT GGA GGT GGA TCT GAT GAC GAT GAC AAA (SEQ ID NO: 6).

[0134] 1. Preparation of fusion polypeptide

[0135] The fusion polypeptide is designed as follows: Arg-Ser-Asp-Asp-Asp-Asp-...

Embodiment 2

[0169] Example 2 Fusion expression of PTH and multi-enzyme combined enzyme digestion processing

[0170] In this example, the target polypeptide to be prepared is parathyroid hormone (PTH) (GenBank accession number NM_000315), and the linking peptide sequence used is as follows:

[0171] Arg-Ser-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 1);

[0172] Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 5).

[0173] 1. Preparation of fusion polypeptide

[0174]Referring to a similar method as described in Example 1, the pTH gene was concatenated into a fusion gene containing 3 copies of the target gene and encoding the following fusion polypeptide by using the coding sequence of the aforementioned connecting peptide: Arg-Ser-Asp-Asp-Asp-Asp- Lys-PTH-Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp-Asp-Lys-PTH-Lys-Arg-Gly-Gly-Gly-Gly-Ser-Asp-Asp-Asp- Asp-Lys-PTH-Lys-Arg copy.

[0175] The sequencing analysis results are correct. Insert the fusion gene containing 3 copies of the pTH gene...

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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

technical field [0001] The present invention belongs to the fields of biotechnology and genetic engineering; more specifically, the present invention relates to a connecting peptide used for fusion expression of a target polypeptide, a fusion polypeptide prepared by using the connecting peptide and a method for preparing the target polypeptide. Background technique [0002] Protein fusion expression is widely used in the field of genetic engineering, especially small molecular polypeptides. After researchers have isolated a gene, the most natural task for researchers to study its encoded protein is to express—that is, to synthesize exogenous gene products purposefully. In the early days of recombinant DNA technology, it was believed that a strong promoter and an initiation codon in front of a gene were sufficient for good expression in E. coli. Subsequently, it was recognized that the conditions required for effective translation were much more complicated. In addition to s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K2/00C12N15/11C07K19/00C12P21/06
Inventor 孙九如黄阳滨张翊任军梁光军姚惠珍
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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