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Prokaryotic Expression, Purification and Activity Assay of Human Polypeptide: N-acetylgalactosaminyltransferase2

Author: JiaWei
Tutor: WuShiLiang;ZhouYingHui
School: Suzhou University
Course: Biochemistry and Molecular Biology
Keywords: Peptide : N- acetyl galactosyltransferase 2 Prokaryotic expression GST fusion protein Glutathione - agarose affinity chromatography Reverse-phase HPLC Activity Detection
CLC: Q78
Type: Master's thesis
Year: 2005
Downloads: 134
Quote: 0
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Abstract


Objective: vector pGEX-5X-3 expression in E. coli polypeptide Plains : N- acetyl galactosyltransferase 2 full-length coding sequence of purified refolded its activity were detected. Methods: PCR from cloning vector pDONR201-T2 get ppGalNAc-T2 full-length coding sequence , subcloned into the prokaryotic expression vector pGEX-5X-3, transformed into E. coli BL21, after isopropyl thiogalactopyranoside (IPTG) to obtain the corresponding induced expression product - glutathione-S- transferase (GST) fusion protein , verified by Western blotting . Expression product refolded using glutathione - agarose (Glutathione-Sepharose) 4B affinity chromatography purified . Create enzymatic reaction , high performance liquid chromatography (HPLC) for activity testing. Results : We successfully constructed a recombinant prokaryotic expression vector pGEX-5X-3/T2, protein electrophoresis detected through molecular weight of about 90KD fusion protein expression, and can be verified by Western blotting . Purified by affinity chromatography of the enzyme protein , detected by reverse-phase HPLC that it has a certain activity. Conclusion : In this study ppGalNAc-T2 full-length coding sequence was successfully expressed and purified , and has a certain catalytic activity for the further preparation of its polyclonal antibody and protein crystallization has laid a solid foundation.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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