Expression, Purification And Functional Characterization Of Recombinant Human Type I Collagen Peptide In Escherichia Coli
Keywords
Collagen Recombinant Expression Antioxidant Cell Proliferationcollagen Recombinant Expression Antioxidant Cell Proliferation
Abstract
In this study, the prokaryotic expression vector pET28a-rhC¢ñ encoding the recombinant human type I collagen peptide gene was constructed and transformed into E. coli Rosetta (DE3) for induced expression. The cDNA sequence of the recombinant human type I collagen peptide was amplified by PCR and cloned into the prokaryotic expression vector pET28a; the recombinant plasmid was transformed into E. coli Rosetta (DE3) for IPTG-induced expression, and the expression conditions were optimized. Expression products were detected using SDS-PAGE and Western Blot. A large amount of recombinant protein was expressed, purified by nickel affinity chromatography, and the purified protein was subjected to in vitro antioxidant studies and cell proliferation analysis. The results showed that the recombinant protein with a molecular weight of approximately 40 ku was obtained through induced expression in E. coli Rosetta (DE3), which was consistent with expectations. A relatively pure protein was obtained through nickel affinity chromatography, and the DPPH experiment proved that it has certain antioxidant activity; and the MTT experiment confirmed that the protein can promote the proliferation of mouse fibroblast 3T3 cells, making it useful in food, cosmetics and The application in the medical industry provides a certain theoretical basis. A prokaryotic expression vector, pET28a-rhC I was constructed, with recombinant, human-like type I collagen peptide gene and transformed into Escherichia coli Rosetta (DE3) for inducible expression. The cDNA sequence of recombinant human-like collagen type I peptide was amplified by polymerase chain reaction (PCR) and cloned into the prokaryotic expression vector, pET28a. The recombinant plasmid was transformed into E. coli Rosetta (DE3) for IPTG-induced expression and gene expression conditions were optimized. The expressed products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The expressed recombinant proteins were purified by nickel affinity chromatography. Additionally, in vitro antioxidant activity and cell proliferation assay were conducted on the purified proteins. The results showed that the molecular weight of the expressed recombinant protein induced by of E. coli Rosetta (DE3) was approximately 40 ku, consistent with the expected value. Nickel affinity chromatography produced a collagen peptide with relatively high purity, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH ) free-radical test revealed that the recombinant protein showed antioxidant activity. In addition, the methylthiazolydiphenyl-tetrazolium bromide (MTT) assay showed that the recombinant protein promoted the proliferation of mouse embryonic fibroblasts cells (3T3). These results indicate potential applications of this protein in industries such as food, cosmetics, and medicine.
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Original research was done by Yang Jing
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