Expression, Purification, and Functional Characterization of Recombinant Human-like Type I Collagen Peptide in E. coli
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Abstract:
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.
