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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Enhancement of the solubility of human tissue inhibitor of matrix metallocroteinase-2 (TIMP-2) in E. coli using a modified in vitro mutagenesis

새로운 유전자 재조합 방법을 이용한 대장균에서의 인간 tissue inhibitor of mtrix metalloproteinase-2 (TIMP-2) 유전자의 가용성 발현

  • Kim, Jong-Uk (Department of Molecular Sciences and Technology, Ajou University) ;
  • Choi, Dong-Soon (Department of Obstetrics and Gynecology, School of Medicine, Ajou University) ;
  • Joo, Hyun (Mitochondrial Signaling Laboratory, Department of Molecular Physiology & Biophysics, College of Medicine, Inje University) ;
  • Min, Churl-K. (Department of Molecular Sciences and Technology, Ajou University)
  • 김종욱 (아주대학교 분자과학기술학과) ;
  • 최동순 (아주대학교 의과대학 산부인과) ;
  • 주현 (인제대학교 의과대학 미토콘드리아 생체신호연구실 생리학교실) ;
  • 민철기 (아주대학교 분자과학기술학과)
  • Published : 2008.06.30
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Abstract

The second family member of tissue inhibitors of matrix metalloproteinases, TIMP-2, is a 21kDa protein which inhibits matrix metalloproteinases 2 (MMP-2). Expression of mammalian proteins in E. coli often forms inclusion bodies that are made up of mis-folded or insoluble protein aggregates. The requirement for the formation of 6 disulfide bonds in the process of the TIMP-2 folding is likely to be incompatible with the reducing environment of E. coli. However, this incompatibility can be often overcome by introducing a mutagenesis that could lead to enhancement of the protein solubility. In this reason, we have attempted to express the soluble TIMP-2 in E. coli by applying a modified staggered extension process (StEP), one of the in vitro PCR-based recombinant mutagenesis methods, and error-prone PCR. C-terminally located CAT fusion protein with respect to mutated TIMP-2 proteins enables us to differentiate the soluble TIMP-2 from the insoluble in E. coli by virtue of chloramphenicol resistance. According to this scheme, E. coli harboring properly-folded CAT fused to TIMP-2 protein was selected, and some of the resulting colonies exhibited an enhanced, soluble expression of TIMP-2 compared to the wild type, implying (i) the StEP technique is successfully employed to enhance the proper folding thereby increasing the solubility of TIMP-2, and (ii) the CAT dependent screening may be a simple and effective method to differentiate the soluble protein expression in E. coli.

암세포의 침윤은 숙주 조직의의 기저막과 세포외 기질을 침투함으로써 일어난다. 침윤과 전이과정에는 단백질가수분해 효소인 matrix metalloproteinases (MMPs)가 깊이 연관되어 있는 것으로 알려져 있으며, MMP의 가수분해 활성은 tissue inhibitors of metalloproteinases (TIMPs)라는 억제 단백질에 의해 억제된다. TIMP-2는 21kDa 크기의 포유류 단백질로 대장균에서 과발현 시 다른 많은 포유류 단백질과 마찬가지로 가용성이 낮은 봉입체 형태로 발현된다. TIMP-2 단백질의 접힘에 6개의 이황화결합이 필요하고, 이는 일반적으로 대장균 환경은 적합하지 않다. 본 연구에서는 대장균에서 불가용성으로 발현되는 TIMP-2 유전자를 유전자셔플링 기법의 한 가지인 StEP (staggered extension process)를 변형하고 동시에 $Mn^{2+}$ 농도 변화와 dGTP 불균형을 이용한 무작위 돌연변이 기법을 혼용하여 대장균에서 가용성 TMP-2 재조합 변이체를 생성하고자 하였다. 무작위로 재조합된 TIMP-2 유전자 중에서 가용성으로 발현되는 TIMP-2 유전자를 선별하기 위해서 chloramphenicol acetyltransferase (CAT)-융합 방법을 도입하였다. CAT 유전자가 가용성으로 발현되는 재조합 TIMP-2 유전자에 융합되면 이를 갖는 E. coli는 높은 chloramphenicol 환경에서 생존이 가능하게 된다. 이러한 in virro mutageuesis 기법과 CAT-융합 방법으로 대장균 가용성 TIMP-2 재조합 변이체를 14가지 얻을 수 있었다. 변이체 TIMP-2의 아미노산서열 분석과 구조 분석 결과 주로 소수성 아미노산이 친수성 아미노산으로 전환되었고, MMP와의 결합이 관여하지 않는 C-말단 부위에 돌연변이가 집중되어 있었다. 본 연구에서 개발된 간편하고 새로운 in vitro 재조합 방법과 CAT을 이용한 스크리닝 기법은 다른 많은 대장균 내 불가용성 단백질의 발현에도 사용될 수 있을 것으로 사료된다.

Keywords

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