[Efficient and constant expression of cloned human beta2-microglobulin gene in E.coli].
OBJECTIVE
To obtain human beta2-microglobulin (beta2m) gene that is to be efficiently expressed in E.coli.


METHODS
beta2m cDNA including only the coding region for the protein was amplified from Raji cell line by reverse transcriptase-PCR via the primers 5'-GGTGGTCATATGGCTATCCAGCGTACTCCA-3' and 3'-GGTGGTTGCTCTTCCGACATGTCTCGATCC-3'. The product was subsequently cloned into a modified pBV220 vector after digestion with Nde I/Sap I. The recombinant plasmid pBV220-beta2m was transformed into E.coli BL21 after sequence analysis, and the fusion protein was then expressed via induction at 42 for 5 h at D(600) of 0.55-0.60, followed by purification through chitin beads.


RESULTS
The beta2m cDNA was identical with those published in Genbank. The expressed fusion protein was identified in the form of inclusion body at the ratio more than 45 % of the E.coli proteins, and was denatured with 8 mol/L urea, followed by refold and purification to a high purity, displaying a relative molecular mass of 12 000 on 10 % SDS-PAGE gel.


CONCLUSIONS
The human beta2m gene was cloned successfully and expressed efficiently and constantly in E.coli BL21,which lays the ground for engineering MHC-tetramers.
Zhou
Zui-Ming
ZM
Department of Infectious Diseases, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China. zmzhoui@163.com
Guo
Ya-Bing
YB
Wang
Zhan-Hui
ZH
Luo
Kang-Xian
KX
Hou
Jin-Lin
JL
D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis; D001613:beta 2-Microglobulin / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D001613:beta 2-Microglobulin