Expression, Purification, and Secondary Structure Characterization of Recombinant KCTD1

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Fanghua Mei^1,2#, Jin Xiang^3#, Song Han², Yuan He², Yajing Lu², Jian Xu², Deyin Guo², Gengfu Xiao¹, Po Tien¹, and Guihong Sun^2*

¹The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China

²School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China; fax: 86-27-6875-9142; E-mail: ghsunlab@whu.edu.cn

³College of Pharmacy, Wuhan University, Wuhan 430072, China

^# These authors contributed equally to this work.

^* To whom correspondence should be addressed.

Received March 29, 2012; Revision received May 18, 2012

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Potassium channel tetramerization domain containing 1 (KCTD1) contains a BTB domain, which can facilitate protein–protein interactions that may be involved in the regulation of signaling pathways. Here we describe an expression and purification system that can provide a significant amount of recombinant KCTD1 from Escherichia coli. The cDNA encoding human KCTD1 was amplified and cloned into the expression vector pET-30a(+). The recombinant protein was expressed in E. coli BL21(DE3) cells and subsequently purified using affinity chromatography. To confirm that KCTD1 was correctly expressed and folded, the molecular weight and conformation were analyzed using mass spectroscopy, Western blot, and circular dichroism. Optimizing KCTD1 expression and investigating its secondary structure will provide valuable information for future structural and functional studies of KCTD1 and KCTD family proteins.

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KEY WORDS: KCTD1, circular dichroism spectroscopy, secondary structure

DOI: 10.1134/S0006297912080160

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