A method for highly expressing multi-copy human epidermal growth factor

Abstract

The invention provides a method for efficiently expressing a multi-copy human epidermal growth factor. The method comprises the steps that restriction enzyme cutting sites are introduced to the upstream and downstream of a gene fragment, a tandem repeat sequence of a mature peptide gene of the human epidermal growth factor is obtained after enzyme cutting and connection are conducted, and the problems that the human epidermal growth factor is low in molecular weight and insufficient in expression quantity are solved; meanwhile, an kex2 enzyme cutting site is introduced to the upstream of the gene fragment of the human epidermal growth factor, part of nucleotide bases are modified at downstream, a carboxyl terminal ELR of an original amino acid sequence is changed into a saccharomycete kex2 enzyme cutting site, it is guaranteed that recombinant expressed target protein is cut into monomers through kex2 enzyme inherent to saccharomycetes, the expression quantity is obviously increased, a natural N-terminal is achieved, the difference that only one amino acid residue exists at the C-terminal is achieved, redundant amino acids do not exist, and the activity is not changed; due to the fact that the human epidermal growth factor is naturally synthesized in pichia pastoris, original native state spatial conformation of the protein can be maintained, and therefore efficient large-scale preparation of the human epidermal growth factor is achieved.

Description

technical field [0001] The invention belongs to genetic engineering technology, in particular to a method for highly expressing multi-copy human epidermal growth factor. Background technique [0002] With the development of genetic engineering technology, some small molecular polypeptides with biological activity have been highly valued. Human epidermal growth factor (human epidermal growth factor, hEGF), also known as urogastrone, is a polypeptide of about 6kD composed of 53 amino acids, mainly produced in the duodenal Brunner's gland. The main applications of hEGF in the field of clinical medicine are: (1) to promote the healing of surgical wounds and difficult-to-heal wounds. In recent years, hEGF has been widely used in human burns, wounds, diabetic skin ulcers, bedsores, and varicose skin ulcers, and has achieved good results. (2) Promote the healing of corneal wounds. EGF can promote the proliferation of corneal epithelial cells to treat corneal injury, ulcer, acid-...

AI Technical Summary

Problems solved by technology

There have been a large number of reports on the preparation of recombinant human epidermal growth factor by genetic engineering methods, but the expression in the past is a single copy, even in the copy-rich strains, because the molecular weight of human epidermal growth factor is very small, the expression level cannot achieve satisfactory results. Causes waste of host expression potential and lower yield

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