The Purification of GST-NDPK-A Fusion Protein and Detection of its Interacting Proteins in vitro

Abstract

Abstract: It was to understand the function of nucleoside diphosphate kinase A (NDPK-A) and explore its interacting proteins in vitro through GST-Pull down technique. The target genes were amplified from the templates pBV-NDPK-A and inserted into the prokaryotic expression vector pGEX-4T-2 with glutathione-S-transferase(GST)tag to construct the expression plasmid. After identified by restriction endonuclease digestion and sequencing, the recombinant plasmid was transformed into E. coli BL21 strain and exogenous protein expression was induced by IPTG. After purification using Glutathione Sepharose 4B affinity chromatography, the GST-NDPK-A fusion protein was identified by SDS-PAGE and Western blot then incubated with A549. GST-Pull down assay was performed to explore the interacting proteins in vitro followed by LC-MS/MS identification. Results showed that the prokaryotic expression vector of GST-NDPK-A fusion protein was successfully constructed and expressed effectively in E. coli BL21 strain. The fusion protein with bioactivity was purified using Glutathione Sepharose 4B beads. Furthermore, GST-Pull down assay indicated that NDPK-A could bind to Fussel-18 and Rrp12 in vitro.

Key words: NDPK-A Fussel-18, Rrp12, Expression and purification, Interaction

Lü Fen, Liu Zhong, Yin Xingfeng, Zhao Zhenling, Chen Miaojuan, Zhang Jiaxuan, Chen Wei, Qian Chuiwen, Xiong Sheng. The Purification of GST-NDPK-A Fusion Protein and Detection of its Interacting Proteins in vitro[J]. Biotechnology Bulletin, 2013, 0(5): 137-143.

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