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Expression vector for soluble epidermal growth factor fusion protein and application thereof

An epidermal growth factor and fusion protein technology, applied in epidermal growth factor, growth factor/inducing factor, animal/human protein, etc., can solve the problem of easy mismatch, incomplete in vitro renaturation, EGF cannot reach pharmaceutical grade activity, etc. question

Inactive Publication Date: 2016-11-09
PANGO GENE BIOSCI NANJING INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Secreted expression in Pichia pastoris often takes several days and is prone to degradation
Although the expression of Escherichia coli is time-saving and economical, almost all the expression products are inclusion bodies without natural structures, which require complex operations such as "denaturation-desalination-artificial in vitro renaturation", and incomplete in vitro renaturation, easy to mismatch, resulting in The EGF produced by artificial in vitro renaturation often fails to achieve pharmaceutical-grade activity (≥500,000 U / mg EGF)

Method used

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  • Expression vector for soluble epidermal growth factor fusion protein and application thereof
  • Expression vector for soluble epidermal growth factor fusion protein and application thereof
  • Expression vector for soluble epidermal growth factor fusion protein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0053] A method for constructing a soluble epidermal growth factor fusion protein FABP6-EGF expression vector, comprising the following steps:

[0054] Step 1. Artificial synthesis and optimization of DNA encoding human FABP and human EGF. In this embodiment, human FABP6 is taken as an example (SEQ ID NO:8), and human EGF is taken as an example of a mature peptide with 53 amino acids (SEQ ID NO:12 ):

[0055] (1) His-Tag amino acid sequence (SEQ ID NO: 13), FABP6 amino acid sequence (SEQ ID NO: 14) with the initial methionine removed, flexible linker amino acid sequence (SEQ ID NO: 15), EGF amino acid sequence The sequence (SEQ ID NO: 12) is combined into "His-Tag-FABP6-flexible linker-EGF" (SEQ ID NO: 16) in order, but not limited to this sequence, the protein amino acid sequence homology is equal to or greater than 85% limit.

[0056] (2) Input the coding sequence (SEQ ID NO:16) into NCBI software DNAworks (v3.2.3):

[0057] ① Obtain the reverse-translated and codon-optim...

Embodiment 2

[0084] The expression test of embodiment 2 vector:

[0085] (1) The obtained pFABP6-EGF vector DNA plasmid was transformed into competent cells of Escherichia coli expression strain BL21 (DE3) to obtain kanamycin (Kan) resistant colonies.

[0086] (2) Pick a few single colonies, culture them in LB medium, induce with IPTG for 4-12 hours, collect 1 mL of the bacterial liquid, collect the bacterial cells by centrifugation, wash once with 1×PBS, resuspend in 0.5 mL 1×PBS, and sonicate the bacteria body, centrifuged to remove the precipitate.

[0087] (3) Mix an appropriate amount of 20 μL of supernatant with SDS-PAGE loading buffer, heat and denature at 95°C for 10 minutes, collect by centrifugation to the bottom of the tube, and place on ice.

[0088] (4) 10 μL of the sample was taken, electrophoresed with 12% polyacrylamide, stained with Coomassie brilliant blue, and decolorized, and the protein band and the expression of FABP6-EGF protein were observed.

[0089] (5) Recombin...

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Abstract

The invention discloses an expression vector for soluble epidermal growth factor fusion protein and application thereof. The upper stream of the cloning zone of the expression vector contains a coding sequence for human free fatty acid binding protein (FABP6) while the lower stream is a coding sequence for a human epidermal growth factor; a flexible linker coding sequence is located between the coding regions of the two genes; and the amino terminal of FABP6 contains a His-Tag tag protein coding sequence. The expression vector for the soluble epidermal growth factor fusion protein has the characteristics that 1) a ratio of soluble protein to inclusion body protein is greater than 50%; and 2) target protein can be easily obtained in virtue of histidine tag (His-Tag) affinity purification.

Description

technical field [0001] The invention relates to a soluble protein expression technology, in particular to a soluble epidermal growth factor fusion protein expression carrier and application thereof, belonging to the field of molecular biology genetic engineering. Background technique [0002] In 1962, Dr. Stanley Cohen of the United States first discovered a 53-amino acid oligopeptide from the submandibular gland of mice. Because the short peptide has a wide range of cell growth-promoting activities, it is named epidermal growth factor (Epidermal Growth Factor, EGF). After further research, the protein sequence and structure of EGF were clarified. In the 1970s, it was found that human beings have the same function and similar structure of EGF (hEGF). Subsequent studies found that it has a powerful function of promoting cell growth, and its function is realized through the EGF receptor (EGFR) on the cell membrane. The activated receptor activates mitogenic related genes in ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K14/485C07K14/47C07K2319/21
Inventor 李万波陕婧婧卢婵
Owner PANGO GENE BIOSCI NANJING INC
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