Cloning，Prokaryotic Expression and Purification of ANKIB1

Abstract

Abstract: Ankyrin repeat and IBR domain-containing protein 1（ANKIB1）contains RING-IBR-RING domain, ankyrin repeat（ANK）motif and ubiquitin-interacting motif（UIM）. It is likely that ANKIB1 has E3 ubiquitin ligase activity and specific catalytic mechanism. However, the function of ANKIB1 remains unclear. For the first time we cloned human ANKIB1 cDNA using nestled PCR and segmental amplication. Full length of ANKIB1 coding DNA sequence was constructed by gene recombination technology. The cDNA was ligated into prokaryotic expression vector pGEX-5X-3, and the recombinant plasmid pGEX-5X-ANKIB1 was transformed into E.coli BL21（DE3）. Using culture medium cotaining 200 mmol/L ZnCl ₂, the stabilizer for RING-type protein, expression of soluble GST-ANKIB1 fusion protein with a molecular weight of 148.5 kD was successfully induced by 0.1 mmol/L IPTG at 15℃ for 10 h. The recombinant protein was purified by affinity chromatography using Glutathione Sepharose 4B.

Key words: ANKIB1 gene, Cloning, Expression, Purification

Xie Yunfei,Yang Zishan,Ma Zhenling,Xie Bohong,Liu Shirao. Cloning，Prokaryotic Expression and Purification of ANKIB1[J]. Biotechnology Bulletin, 2013, 0(7): 189-194.

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