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Collagen-like protein-human basic fibroblast growth factor fusion protein and preparation method thereof

A fibroblast and collagen-like technology, which is applied in the fields of collagen-like-human basic fibroblast growth factor fusion protein and its expression and purification, can solve the problems of discarding and waste of fusion protein tags, etc.

Inactive Publication Date: 2016-08-17
ZHEJIANG UNIV
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Problems solved by technology

However, such fusion protein tags are then discarded, resulting in some waste

Method used

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  • Collagen-like protein-human basic fibroblast growth factor fusion protein and preparation method thereof
  • Collagen-like protein-human basic fibroblast growth factor fusion protein and preparation method thereof
  • Collagen-like protein-human basic fibroblast growth factor fusion protein and preparation method thereof

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Embodiment Construction

[0028] The present invention is further described by steps below:

[0029] Step 1, total synthesis of human basic fibroblast growth factor hbFGF and collagen-like Scl2 gene: according to the original gene sequence of human basic fibroblast growth factor hbFGF and collagen-like Scl2 derived from Streptococcus pyogenes, according to Escherichia coli The codon analysis table was used to optimize the codons of the original gene sequence, and entrusted Shanghai Jierui Biotechnology Co., Ltd. to obtain the pUC19-hbFGF and pET28a-Scl2 plasmids. The optimized hbFGF gene sequence is shown in SEQ NO.2, and its amino acid sequence is shown in SEQ NO.3; the optimized Scl2 gene sequence is shown in SEQ NO.4, and its amino acid sequence is shown in SEQ NO.5.

[0030] Step 2. Acquisition of collagen-like protein Scl2-M: According to the integrin binding site (GERGFPGERGVE), heparin binding site (GRPGKPGKQGQK) and RGD site and Scl2 gene sequence, design 3 pairs of primers:

[0031] f Scl2 :...

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Abstract

The invention provides collagen-like protein-human basic fibroblast growth factor fusion protein. The whole length of the fusion protein is 488 amino acids, and the nitrogen end is modified collagen-like protein Scl2-M of a streptococcus pyogenes source; the protein length of the Scl2-M is 327 amino acids, the carbon end is a human basic fibroblast growth factor hbFGF, and the protein length of the hbFGF is 155 amino acids. An enterokinase restriction enzyme cutting site exists between two peptide fragments. The preparation method comprises the steps of establishment of an expression vector of the collagen-like protein-human basic fibroblast growth factor fusion protein, fermentation of the fusion protein, purification of recombined human basic fibroblast growth factors and collagen-like protein and measurement of bioactivity. By means of the method, the human basic fibroblast growth factors and the collagen-like protein can be efficiently produced at the same time, and therefore a foundation is laid for scale preparation of the human basic fibroblast growth factors and application of the collagen-like protein.

Description

technical field [0001] The present invention relates to a collagen-like-human basic fibroblast growth factor fusion protein and its expression and purification, in particular to a method for simultaneously and efficiently producing recombinant human basic fibroblast growth factor and collagen-like protein using a collagen-like fusion tag The method belongs to the field of biotechnology. Background technique [0002] The collagen-like protein Scl2 of Streptococcus pyogenes can form a stable triple helix structure without hydroxylation modification and has a Tm of 35-39°C (Chunying Xu, Zhuoxin Yu, et al. Biomacromolecules, 2010, 11: 348-356). The collagen-like protein Scl2 derived from Streptococcus pyogenes has been studied more. The recombinant Scl2 protein consists of an N-terminal globular domain (V) and a collagen domain (CL). The recombinant CL protein can promote cell adhesion and is non-toxic to cells And it can form biomaterials through glutaraldehyde cross-linking a...

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Application Information

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IPC IPC(8): C07K19/00C07K1/22C07K1/18C12N15/62C12N15/70C12P21/06
CPCC07K14/315C07K14/503C07K2319/00C12N15/70C12N2800/101C12N2800/22C12P21/06
Inventor 徐志南唐云平胡斌蔡谨黄磊
Owner ZHEJIANG UNIV
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