PMID- 15135395
OWN - NLM
STAT- MEDLINE
DCOM- 20050329
LR  - 20061115
IS  - 1046-5928 (Print)
IS  - 1046-5928 (Linking)
VI  - 35
IP  - 2
DP  - 2004 Jun
TI  - Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia 
      coli.
PG  - 210-7
AB  - 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.
FAU - Piao, Wen-Hua
AU  - Piao WH
AD  - Institute of Basic Medical Sciences, Academy of Military Medical Sciences, 
      Beijing, 100850, PR China.
FAU - Song, Xiao-Guo
AU  - Song XG
FAU - Liu, Mao-Chang
AU  - Liu MC
FAU - He, Yu
AU  - He Y
FAU - Zhang, Heng-Hui
AU  - Zhang HH
FAU - Xu, Wen-Xie
AU  - Xu WX
FAU - Li, Zai-Liu
AU  - Li ZL
FAU - Zhang, He-Qiu
AU  - Zhang HQ
FAU - Ling, Shi-Gan
AU  - Ling SG
FAU - Wang, Gui-Qiang
AU  - Wang GQ
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (DNA Primers)
RN  - 0 (HLA-A2 Antigen)
RN  - 0 (Recombinant Proteins)
RN  - 0 (beta 2-Microglobulin)
SB  - IM
MH  - Base Sequence
MH  - Cloning, Molecular
MH  - DNA Primers
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Escherichia coli/genetics
MH  - HLA-A2 Antigen/*genetics/isolation & purification
MH  - Recombinant Proteins/genetics/isolation & purification
MH  - beta 2-Microglobulin/*genetics/*isolation & purification
EDAT- 2004/05/12 05:00
MHDA- 2005/03/30 09:00
CRDT- 2004/05/12 05:00
PHST- 2003/09/24 00:00 [received]
PHST- 2003/12/30 00:00 [revised]
PHST- 2004/05/12 05:00 [pubmed]
PHST- 2005/03/30 09:00 [medline]
PHST- 2004/05/12 05:00 [entrez]
AID - S104659280400035X [pii]
AID - 10.1016/j.pep.2004.01.002 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2004 Jun;35(2):210-7. doi: 10.1016/j.pep.2004.01.002.