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Expression, purification, and secondary structure characterization of recombinant KCTD1

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Abstract

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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Abbreviations

BLAST:

Basic Local Alignment Search Tool

BTB domain:

bric-a-brac, tramtrak, and broad complex domain

CD:

circular dichroism

IPTG:

isopropyl thio-β-D-galactopyranoside

KCTD:

potassium channel tetramerization domain containing

MALDI-TOF:

matrix-assisted laser desorption ionization time-of-flight

ORF:

open reading frame

POZ domain:

pox virus and zinc finger domain

SDS-PAGE:

polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

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Authors and Affiliations

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

    Fanghua Mei, Gengfu Xiao & Po Tien

  2. School of Basic Medical Sciences, Wuhan University, Wuhan, 430071, China

    Fanghua Mei, Song Han, Yuan He, Yajing Lu, Jian Xu, Deyin Guo & Guihong Sun

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

    Jin Xiang

Authors
  1. Fanghua Mei
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  2. Jin Xiang
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  3. Song Han
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  4. Yuan He
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  5. Yajing Lu
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  6. Jian Xu
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  7. Deyin Guo
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  8. Gengfu Xiao
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  9. Po Tien
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  10. Guihong Sun
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Corresponding author

Correspondence to Guihong Sun.

Additional information

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM12-094, July 1, 2012.

These authors contributed equally to this work.

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Mei, F., Xiang, J., Han, S. et al. Expression, purification, and secondary structure characterization of recombinant KCTD1. Biochemistry Moscow 77, 941–945 (2012). https://doi.org/10.1134/S0006297912080160

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  • DOI: https://doi.org/10.1134/S0006297912080160

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