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Pichia pastoris로부터 Toll-like Receptor 9의 세포 내 도메인 단백질의 발현과 순수분리 정제
이균영,이곤호,Lee Kyun-Young,Lee Kon-Ho 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.4
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.
Pichia pastoris로부터 Toll-like Receptor 9의 세포 내 도메인 단백질의 발현과 순수분리 정제
이균영,이곤호 Plant molecular biology and biotechnology research 2005 Plant molecular biology and biotechnology research Vol.2005 No.
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 MALDI-TOF mass spectrometry. The proteins have been purified by nickel affinity, cation exchange and gel-filtration chromatography.
조명제,이승규,이곤호,송재영,이우곤,백승철,이광호,윤희상,서지현,강형련 대한미생물학회 2013 Journal of Bacteriology and Virology Vol.43 No.4
Helicobacter pylori, a causative agent of gastroduodenal diseases, is a Gram-negative microaerophilic bacterium. Although H. pylori locates in the microaerophilic mucous layer, the bacteria would come into contact harmful reactive oxygen species generated by host immune system. It has been reported that H. pylori harbors various defense mechanisms which can protect bacterial cells from oxygen exposure. The change of the gene expression profile of sodB-negative isogenic mutant of H. pylori 26695 was analyzed by high resolution 2-DE followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS and microarray analysis. Eighteen genes and 41 genes were upregulated and downregulated respectively, either transcriptionally or translationally. Expression levels of three genes including trxB, yxjE and ribE that were changed both on a mRNA level and on a protein level were confirmed by RT-PCR analysis. However, change of expression levels of other major antioxidants such as KatA, AhpC and NapA were not detected, which means Sod is regulated by different way from that of KatA and AhpC. Mutant study of other antioxidant proteins may give us better understanding for the regulation of stress response in H. pylori.
퉁퉁마디로부터 색소체 외막 단백질 유전자의 분리 및 발현분석
네티엘마와티,신동진,이병현,손대영,이곤호,차준영,양영실,정민희 한국식물생명공학회 2004 JOURNAL OF PLANT BIOTECHNOLOGY Vol.31 No.4
Complementary DNA encoding chloroplast outer envelope membrane protein (OEP) from the halophyte Salicornia herbacea has been cloned and sequenced. The full length cDNA is 596 bp and encodes a polypeptide of 91 amino acid residues with a molecular mass of 8.9 kDa. The expression level of ShOEP increased by salt, drought and ABA treatments. ShOEP expression was largely induced in roots and shoots by high salts. The biological function of ShOEP was examined by yeast complementation. ShOEP can suppress Na+ sensitivity of yeast mutant (cnb ) in the presence of salt. These results suggest that ShOEP is a salt inducible gene and may have functions in the regulation of plant salt stress.
퉁퉁마디로부터 염에 의하여 유도되는 Aldolase 유전자의 분리 및 발현분석
차준영,네티 엘마와티,김순길,이증주,임채오,정우식,이곤호,손대영 Plant molecular biology and biotechnology research 2003 Plant molecular biology and biotechnology research Vol.2003 No.-
Soil salinity is one of the most serious abiotic stresses limiting the productivity of agricultural crops. To cope with salt stress, plants respond with physiological, developmental and biochemical changes, including the synthesis of a number of proteins and the induction of gene expression. Salicornia herbacea is a halophytic plant that grows in salt marches and on muddy seashores. In order to understand the biochemical and molecular mechanisms of salt tolerance in S. herbacea, we isolated several genes that involved in the salt tolerance by mRNA differential display.Here we report the cloning of a cDNA encoding fructose-1, 6-bisphosphate aldolase, named ShADL, which is 1293 bp long and contains an open reading frame consisted of 359 amino acids with calculated molecular mass of 39 kDa. ShADL protein showed 86% identity with Arabidopsis and 78% with aldolase of common ice plant. Northern blot analysis revealed that the transcript of ShADL gene was increased dramatically depending on the NaCI concentrations.
퉁퉁마디로부터 색소체 외막 단백질 유전자의 분리 및 발현분석
네티 엘마와티,차준영,양영실,정민희,신동진,이병현,이곤호,손대영 Plant molecular biology and biotechnology research 2004 Plant molecular biology and biotechnology research Vol.2004 No.-
Complementary DNA encoding chloroplast outer envelope membrane protein (OEP) from the halophyte Salicornia herbacea has been cloned and sequenced. The full length cDNA is 596 bp and encodes a polypeptide of 91 amino acid residues with a molecular mass of 8.9 kDa. The expression level of ShOEP increased by salt, drought and ABA treatments. ShOEP expression was largely induced in roots and shoots by high salts. The biological function of ShOEP was examined by yeast complementation. ShOEP can suppress Na+ sensitivity of yeast mutant (cnbΔ) in the presence of salt. These results suggest that ShOEP is a salt inducible gene and may have functions in the regulation of plant salt stress.