Human platelet-derived growth factor A homologous dimers and production method thereof

Abstract

The invention discloses a human platelet-derived growth factor (PDGF) A homologous dimers and a high-efficiency production method thereof. The method mainly comprises the following steps: cloning a human platelet-derived growth factor A spliceosome 2 gene; constructing an eukaryotic expression vector and transferring the eukaryotic expression vector into an eukaryotic yeast host; screening a yeast transformant for high level secretion expression, wherein the PDGF-As expressed by the yeast are in a glycosylated form and a non-glycosylated form; and performing amplified culture in a shake flask, dialyzing supernate of culture solution, purifying the supernate efficiency by using a chromatography (CM) cation exchange column and G-75 molecular sieve, and obtaining the novel modified protein of the PDGF-AA dimers, of which one PDGF-A monomer is glycosylated and of which the other PDGF-A monomer is not glycosylated, by using a CM cation exchange process and optimizing the pH value of dislysate, wherein the separated and purified new glycosylation modified protein of the PDGF-AA dimers has high uniformity.

Description

technical field [0001] The present invention relates to the application of recombinant DNA technology to produce genetically engineered protein medicine technology, in particular to a highly efficient production of human platelet-derived growth factor A homodimer, and the homologous human platelet-derived growth factor A obtained by the method dimer. Background technique [0002] Mature platelet-derived growth factor (PDGF) is a homologous or heterologous dimer composed of two peptide chains linked by disulfide bonds, and is a glycoprotein with a relative molecular weight of about 30kD. PDGF is composed of four subunits A, B, C, and D, which are aggregated with each other through dithiol bonds to form a dimer. In the body, PDGF contains five isomers: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD. The half-life of PDGF is very short (less than 2min), and the content in the serum of the body is very low. PDGF mainly binds to specific receptors on the cell membrane through ...

AI Technical Summary

Problems solved by technology

The expression product of Escherichia coli usually exists in the form of inclusion bodies, and the active form of PDGF-AA can only be obtained through complex operations of denaturation and renaturation, thereby reducing the yield of active protein; Existing in the form of PDGF-AA, removing the fusion non-PDGF-AA fragment requires complex and high-cost enzyme digestion steps to obtain a protein with the same primary structure as the natural PDGF-AA; third, prokaryotic host expression, especially in Escherichia coli , because of the toxicity caused by the presence of LPS, it is often necessary to analyze and determine the toxicity of the expressed purified product; fourth, the E. coli expression system cannot carry out glycosylation modification on the expressed product, resulting in the expressed product often being biologically active Not high. Finally, to obtain high-purity proteins often requires multi-step purification operations. The more purification steps, the lower the protein yield and the more likely it will lead to the inactivation of the target product.

Although there are foreign reports on the expression and purification of PDGF using Saccharomyces cerevisiae, the expression level of PDGF in Saccharomyces cerevisiae is relatively low. Under high-density fermentation conditions, the pure PDGF product that can be obtained per liter of product is less than 10 mg. The two monomers of the expressed product are modified by different degrees of glycosylation, and the uniformity of the expressed product is poor

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