PMID: 30236207Sep 22, 2018Paper

Expression, purification and binding activity analysis of von Hippel-Lindau (VHL)

Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
Juan LiuWanjun Sun

Abstract

Objective To purify recombinant protein of human von Hippel-Lindau (VHL) and identify its function. Methods VHL gene sequence was amplified from human mammary cDNA using PCR and inserted into the prokaryotic expression vector pGEX-KG. Glutathione S-transferase-VHL (GST-VHL) recombinant plasmid we obtained was converted into BL21(DE3) sensitive bacteria to induce a small amount of GST-VHL protein. The expressed product was detected by SDS-PAGE and Western blot analysis. The recombinant protein was purified by GST beads and its function was verified by GST pull-down assay. Results The obtained recombinant plasmid could be successfully digested by double enzymes. Gene sequencing showed that the VHL sequence was correct and there was no mutation. The recombinant protein with approximately relative molecular mass (Mr) 56 000 was purified by converting recombinant plasmid to BL21(DE3) sensitive bacteria and inducing it in small quantities. GST pull-down assay verified that GST-VHL recombinant protein had the function of binding hypoxia inducible factor-1 α (HIF-1 α) in vitro. Conclusion GST-VHL recombinant protein is purified and can combine with HIF-1α protein in vitro.

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