FOOD SCIENCE ›› 2013, Vol. 34 ›› Issue (9): 114-120.doi: 10.7506/spkx1002-6630-201309025

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Expression and Purification of Putative Substrates of the Site-2 Protease Homologs from Synechocystis sp. PCC6803

QIN Chun-yan,CHEN Gu*   

  1. College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
  • Received:2013-03-18 Revised:2013-04-24 Online:2013-05-15 Published:2013-05-07
  • Contact: CHEN Gu E-mail:chengu@scut.edu.cn

Abstract:

Nearly all cyanobacterial species contain genes encoding site-2-protease (S2P) homologs. Some S2P homologs
have been reported to play essential roles in regulating stress response through intramembrane proteolysis of membranebound
anti-sigma factors. However, the mechanism of action and substrates of S2P in Synechocystis sp. PCC6803, Sll0862,
Slr0643, Sll0528 and Slr1821 remain unsolved. In this study, we focused on the four sigma factor-anti sigma factor pairs, SigEChlH(
Slr1055), SigI(Sll0687)-Sll0688, SigG(Slr1545)-Slr1546 and SigH(Sll0856)-Sll0857. Using pET-30b(+) as vector, we
constructed recombinant plasmids, optimized expression and successfully purified the full length and truncated fragment of
putative substrates, including Slr1055, Slr1055Δ(1–232), Sll0688, Sll0688Δ(1–152), Slr1546, Slr1546Δ(1–174), Sll0857 and
Sll0857Δ(1–101) as well as RseA(1–148), the S2P substrate from E.coli. This work may lay the foundation for constructing in
vitro enzymatic digestion system to justify the relationship of various S2Ps with their putative substrates.

Key words: site-2 protease;substrate;anti-&sigma, factor;prokaryotic expression;purification

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