Cloning,Expression,Purification And Crystallization Of Znf-bed3

Znf-bed3 is a novel protein containing a zinc-finger domain which can interact with Axin and activate the Wnt/β-catenin signaling. Its cDNA has been cloned and sequenced. In order to get high quality of crystal to determine the crystal structure of Znf-bed3, a high expression E. coli strain pHisZnf-bed3/BL21-CodonPlus(DE3)-RP was constructed by inserting the Bed3 gene fragment, which was PCR-amplified from plasmid ?72a and digested with NdeⅠand EcoRⅠ, into pET28c at NdeⅠand EcoRⅠsites and transforming the reconstructed vector into E.coli BL21-CodonPlus(DE3)-RP. The expression of Znf-bed3 fusion protein was about 80% in total cellular protein.The expressed Znf-bed3 fusion protein had an extended N-terminal containing a polyhistidine-tag, which could be used to purify the protein through Ni2+-nitrolotriacetate-agarose column. But the result shew this method had limitation for separating the Znf-bed3 fusion protein. More than 80% expressed Znf-bed3 fusion protein formed inclusionbody, so the method...