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Cloning, Expression and Space Conformation Analysis of Vascular Basement Membrane-Derived Multifunctional Peptide
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Abstract:
Objective:To construct cloning and prokaryotic expression vector of vascular basement membrane-derived multifunctional peptide and to analyze its space conformation. Methods:Vascular basement membrane-derived multifunctional peptide (VBMDMP) sequence obtained by polymerase chain reaction was cloned into vector pUC19. We also subcloned it into prokaryotic expression vector pGEX-4T-1. The recombinant was confirmed by restriction endonuclease digestion and auto-sequencer. pGEX-4T-1-VBMDMP was transformed into E. coli JM109 0.1 mmol/L IPTG can induce high expression of GST-VBMDMP. GST-VBMDMP was obtained by Glutathione Sepharose 4B columns.The space conformation of VBMDMP was predicted by using computer program Antheprot. Results: The recombinant VBMDMP gene confirmed by restriction endonuclease digestion and auto-sequencer was consistent with sequence we designed. The high expression of GST-VBMDMP was induced by 0.1 mmol/L IPTG in E.coli JM109. GST-VBMDMP was highly purified. The analysis of software Antheprot demonstrated that the two domains of tumstatin linked by upper hinge region of IgG3 can extend itself freely. Conclusion: The cloning and prokaryotic expression vector was successfully constructed.
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