Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis.
A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6x His-esat-6 fusion gene construction. This plasmid was transformed into Escherichia coli strain M15 and effectively expressed. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea or 6M guanidine-hydrochloride at pH 7.4, and the recombinant protein was purified by Ni-NTA column. The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system. The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine-hydrochloride. Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column. These results indicate that urea is more suitable than guanidine-hydrochloride in extracting and refolding the protein. Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.
Wang
Bao-Lin
BL
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, PR China. blwang@fudan.edu.cn
Xu
Ying
Y
Wu
Chao-Qun
CQ
Xu
Yun-Min
YM
Wang
Hong-Hai
HH
D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins; D015153:Blotting, Western; D002498:Centrifugation; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D019791:Guanidine / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000187:drug effects / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D010957:Plasmids; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012015:Reference Standards; D012995:Solubility; D013010:Sonication; D014508:Urea / Q000494:pharmacology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D011993:Recombinant Fusion Proteins; D006639:Histidine; D014508:Urea; D019791:Guanidine