Journal of Plant Biotechnology

  • 제32권4호
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  • Pages.269-273
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  • 2005
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  • 1229-2818(pISSN)
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  • 2384-1397(eISSN)
한국식물생명공학회 (The Korean Society of Plant Biotechnology)
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Pichia pastoris로부터 Toll-like Receptor 9의 세포 내 도메인 단백질의 발현과 순수분리 정제

Expression and Purification of Toll-like Receptor 9 Cytoplasmic Domain in Pichia patoris

  • 이균영 (경상대학교 대학원 응용생명과학부, 식물분자생물학 및 유전자조작연구소, 환경생명과학 국가핵심연구센터) ;
  • 이곤호 (경상대학교 대학원 응용생명과학부, 식물분자생물학 및 유전자조작연구소, 환경생명과학 국가핵심연구센터)
  • Lee Kyun-Young (Division of Applied Life Science, PMBBRC & EB-NCRC, Gyeongsang National University) ;
  • Lee Kon-Ho (Division of Applied Life Science, PMBBRC & EB-NCRC, Gyeongsang National University)
  • 발행 : 2005.12.01
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초록

Methylotrophic 효모 Pichia pastoris 발현시스템을 사용하여 인간 TLR9 단백질의 세포내 TIR 도메인을 발현하였다. TIR 단백질이 P. pastoris에서 발현되어 배지 속으로 분비되는 것을 SDS-PAGE로 확인하였고, 발현된 단백질을 western-blot, MALDI-TOF 질량분석으로 동정하였다. 이를 통하여 TIR 딘백질이 P. pastoris에서 안정적으로 발현됨을 알 수 있었다. 그리고 발현된 단백질을 니켈 친화, 양이온교환수지, 겔 투과 크로마토그라피를 사용하여 순수 분리 정제하였다. P. pastoris를 이용한 단백질의 발현과 정제방법은 대장균에서 잘 발현되지 않는 단백질의 발현에 응용될 수 있을 것이다.

Toll-like receptors (TLR) are important components of innate immunity in the defense against pathogens. TLRs recognize pathogen-associated common molecular patterns. TLRs are similar to the receptors involved in defense responses in plants. TLR protein is a type 1 membrane protein, consisting of an extracellular domain containing leucine-rich repeats and a cytoplasmic domain. The cytoplasmic domain delivers ligand recognition signals that result in production of anti-microbial agents. The cytoplasmic domain (amino acid 858-1032) of toll-like receptor 9 has been expressed using methylotrophic yeast Pichia pastoris. The protein expression was confirmed by Western-blot, N-terminal sequencing and MALDl-TOF mass spectrometry. The proteins have been purified by nickel affinity, cation exchange and gel-filtration chromatography.

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