Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Expression of Human Serine Palmitoyltransferase Genes for Antibody Development

Antibody 제작을 위한 human serine palmitoyltransferase 유전자의 발현

  • Published : 2004.04.01
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Abstract

For antibody development of human serine palmitoyltransferase (SPT, EC 2.3.1.50), SPTLC1 and SPTLC2 genes were subcloned in pRset vector and expressed in E. coli BL21 (DE3)pLys cells. Eucaryotic SPT is a membrane-bound heterodimer enzyme, while all other members are soluble homodimer enzymes. cDNA library were obtained from total RNA from human embryo kidney cell line, HEK293, using RT-PCR and PCR with specific primers was carried out for preparing SPTLC1 and SPTLC2 genes. pRset vector which can express hexahistidine-tag fusion protein was used and the DNA sequences of pRsetB/SPTLC1 and pRsetA/SPTLC2 were confirmed. Recombinant BL21 cells with SPTLC subunits were selected with LB plate containing ampicillin and chroramphenicol. SPTLC1 and SPTLC2 proteins were induced with 1 mM IPTG and seperated on 10% SDS-PAGE gel. Expressed proteins were confirmed by western blotting with His-tag antibody.

사람의 serine palmitoyltransferase(SPT, EC 2.3.1.50)에 대한 항체를 제작하기 위하여 E. coli발현 vector인 pRset vector에 SPTLC1 및 SPTLC2 유전자를 subcloning하고 BL21 (DE3)pLys cell에 발현시켰다. 포유동물의 SPT는 원핵세포의 SPT homodimer와는 달리 SPTLC1 및 SPTLC2 2개의 sub-unit로 된 heterodimer이다. Human embryo kidney cell인 HEK293 cell의 total RNA로부터 RT-PCR을 행하여 cDNA library를 얻은 다음 SPTLC1 및 SPTLC2의 특이적인 primer 들을 이용하여 PCR을 행하였다. SPTLC1 및 SPTLC2 DNA를 hexahistidine fusion 단백질을 발현시킬 수 있는 pRset vector에 cloning하여 pRsetB/SPTLC1 및 pRsetA/SPTLC2를 얻고 염기서열을 확인하였다. 재조합 plasmid를 발현세포인 BL21 cell에 형질전환시킨 다음 ampicillin 및 chroramphenicol 배지에서 선별하여 재조합세포를 얻었다. 1 mM IPTG로서 발현을 유도하였으며 세포 단백질을 SDS-PAGE로 분리한 다음 His-tag antibody로 western blotting을 행하여 SPTLC 및 SPTLC2가 발현되었음을 확인하였다.

Keywords

References

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