[Cloning and expression of HLA-A*0201-BSP].
High-yield production of HLA-A*0201 heavy chain is a prerequisite to the preparation of HLA-A2 tetramer. The present study was aimed to construct the expression vector of recombinant HLA-A*0201-BSP fusion gene for preparing HLA-A2 tetramers. The extracellular region HLA*0201 was cloned by RT-PCR from HLA-A2(+) donor, and a 15-amino acid biotin-protein ligase (BirA) substrate peptide (BSP) for BirA-dependent biotinylation was added to the COOH-terminus of HLA-A*0201 heavy chain. Then the fusion gene was cloned into pBV220 vector at EcoRI and Bam HI sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pBV220-HLA-A*0201-BSP was transformed to the competent cells of E.coli DH5alpha. The results showed that the HLA-A*0201-BSP fusion protein was successfully expressed in the form of inclusion body and amounted to over 28% of total cell proteins via induction at 42 degrees C. After washed with triton X-100 and urea, the inclusion body was dissolved with 8 mol/L urea and then purified with Sepharcyl S-300 HR, and the final purity reached above 90%. It is concluded that the HLA-A*0201-BSP fusion gene was cloned successfully and expressed efficiently in E.coli DH5alpha. This work establishes a convenient approach for purification of large quantity of recombinant HLA-A*0201-BSP. This provides the basis for the preparation of HLA-A2 tetramers.
Sun
Wan-Jun
WJ
Department of Hematology, Affiliated Hospital, Academy of Military Medical Sciences, Beijing 100039, China.
DU
Jian-Fang
JF
Xu
Dong-Gang
DG
Zou
Min-Ji
MJ
Wang
Jin-Feng
JF
Cai
Xin
X
Wang
Ying
Y
Wang
Jia-Xi
JX
Ai
Hui-Sheng
HS
D001710:Biotin / Q000096:biosynthesis / Q000235:genetics*; D019731:Carbon-Nitrogen Ligases / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D015234:HLA-A Antigens / Q000096:biosynthesis / Q000235:genetics*; D015789:HLA-A2 Antigen; D006801:Humans; D008025:Ligases / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
D029968:Escherichia coli Proteins; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001710:Biotin; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli