Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli.
Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, beta-2 microglobulin (beta-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the beta-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl beta-d-thiogalactopyranoside IPTG. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein. To address this problem, extracellular fractions of HLA-A0201 and beta-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and beta-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs.
Piao
Wen-Hua
WH
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing, 100850, PR China.
Song
Xiao-Guo
XG
Liu
Mao-Chang
MC
He
Yu
Y
Zhang
Heng-Hui
HH
Xu
Wen-Xie
WX
Li
Zai-Liu
ZL
Zhang
He-Qiu
HQ
Ling
Shi-Gan
SG
Wang
Gui-Qiang
GQ
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015789:HLA-A2 Antigen / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D001613:beta 2-Microglobulin / Q000235:genetics* / Q000302:isolation & purification*
D017931:DNA Primers; D015789:HLA-A2 Antigen; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin