[Cloning of human beta-microglobulin gene and its high expression in Escherichia coli].
Human beta2-microglobulin (beta2m) 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 I tetramer. The present study aims to obtain recombinant human beta2m expressed in Escherichia coli (E. coli), for the purpose of preparing MHC class I tetramers. For cloning of human beta2m gene, a pair of specific primers was designed based on the published sequence of this gene and the cDNA of full coding region for beta2m precursor was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was subsequently cloned and its sequence was confirmed by DNA sequencing analysis (the sequence has been deposited in GenBank with accession number of AY187687). The prokaryotic expression vector containing a gene encoding mature beta2m was constructed by inserting the DNA fragment, which was generated by PCR reaction with the cloned beta2m gene as template, into an IPTG-inducible expression vector pET-3c plasmid. The first eight codons for N terminal amino acid residues of beta2m were optimized for its expression in E. coli. The complete sequence of beta2 m gene in the expression vector was verified by DNA sequencing analysis. High-yield expression of beta2m was achieved in E. coli transformed with the expression vector, and most of the recombinant beta2m existed in the inclusion body after IPTG induction. The inclusion body was washed extensively and beta2m in the inclusion body was solublized with 8 mol/L urea. The beta2m was refolded by dialysis and purified by ion-exchange chromatography (Q-Sepharose). Western blotting assay indicated that the polyclonal antibody against human native beta2m could react specifically with the recombinant protein. The purified protein appeared as a single band on both SDS-PAGE and Western blotting, indicating that it was chemical and antigenic pure. This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta2m which is identical to the native protein without any tags fused except for a methionine residue at the amino terminus. This provides the basis for the preparation of MHC tetramers.
He
Xian-Hui
XH
Key Laboratory of Tissue Transplantation and Immunology, Jinan University, Ministry of Education, Guangzhou 510632, China.
Xu
Li-Hui
LH
Liu
Yi
Y
Zeng
Yao-Ying
YY
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
D011994:Recombinant Proteins; D001613:beta 2-Microglobulin