[Cloning of Human beta2-microglobulin gene and efficient expression in Escherichia coli].
Human beta(2)-microglobulin (beta(2)m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC class I tetramer. The present study was aimed to obtain recombinant human beta(2)m expressed in Escherichia coli (E. coli) for preparing MHC class I tetramers. For cloning of human beta(2)m gene, a pair of specific primers was designed based on the published sequence of this gene. A 300 bp specific DNA fragment corresponding to the encoding region of beta(2)m lack of the signal peptide sequence was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was inserted into the IPTG-inducible expression plasmid pET-17b by Nde I and Bam H I sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pET-beta(2)m was transformed to the competent cells of E. coli BL21 (DE3). The results showed that beta(2)m was expressed in the form of inclusion body and amounted to over 32% of total cell proteins after IPTG induction. After washing with triton X-100 and urea, the inclusion body was dissolved with 4 mol/L urea and then purified with Sephacryl S-200 HR, and the final purity reached above 95%. The denatured protein was renatured by dilution method. Western blot assay indicated that the monoclonal antibody against human native beta(2)m could react specifically with the recombinant protein. In conclusion, the human beta(2)m gene was cloned successfully and expressed efficiently in E. coli BL21 (DE3). This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta(2)m. This provides the basis for the preparation of MHC tetramers.
Sun
Wan-Jun
WJ
Department of Hematology, Affiliated Hospital, Academy of Military Medical Sciences, Beijing 100071, China.
Xu
Dong-Gang
DG
Du
Jian-Fang
JF
Zou
Min-Ji
MJ
Wang
Jin-Feng
JF
Cai
Xin
X
Wang
Jia-Xi
JX
Ai
Hui-Sheng
HS
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015395:Histocompatibility Antigens Class I / Q000235:genetics; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis; D001613:beta 2-Microglobulin / Q000096:biosynthesis* / Q000235:genetics
D015395:Histocompatibility Antigens Class I; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin