title: Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli.
abstract: 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.
authors.affiliation: Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing, 100850, PR China.
authors.forename: Wen-Hua
authors.initials: WH
authors.lastname: Piao
authors.forename: Xiao-Guo
authors.initials: XG
authors.lastname: Song
authors.forename: Mao-Chang
authors.initials: MC
authors.lastname: Liu
authors.forename: Yu
authors.initials: Y
authors.lastname: He
authors.forename: Heng-Hui
authors.initials: HH
authors.lastname: Zhang
authors.forename: Wen-Xie
authors.initials: WX
authors.lastname: Xu
authors.forename: Zai-Liu
authors.initials: ZL
authors.lastname: Li
authors.forename: He-Qiu
authors.initials: HQ
authors.lastname: Zhang
authors.forename: Shi-Gan
authors.initials: SG
authors.lastname: Ling
authors.forename: Gui-Qiang
authors.initials: GQ
authors.lastname: Wang
mesh_terms: 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*
chemical_list: D017931:DNA Primers; D015789:HLA-A2 Antigen; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin