Expression, characterization, and reaction of recombinant monkey metallothionein-1 and its C33M mutant.
After we modified the protocol of purification, monkey metallothionein-1 (mkMT-1) and its mutant at position 33 (C33M mutant) were efficiently expressed and purified by using the glutathione-S-transferase fusion protein system. The protein yield has been considerably improved (8 mg/L culture for mkMT-1 and 10 mg/L culture for C33M mutant). The recombinant MT-1 and C33M mutant were characterized by ESI-MS, UV, and CD spectra. The reactions of MI-1 and C33M mutant with 5,5'-dithiobis(2-nitrobenzoic acid) and EDTA also have been carefully studied. The pH titration of MT-1 and C33M mutant has been studied by UV and CD spectra. The mutation of cysteine-to-methionine at position 33 mostly maintains the alpha-domain structure similar to that in wild-type mkMT-1, but the C33M mutant has significant loss of stability and cooperative properties of the domain.
Yu
Wen-Hao
WH
Department of Chemistry, Fudan University, Shanghai, PR China.
Cai
Bing
B
Gao
Yuan
Y
Xie
Yi
Y
Huang
Zhong-Xian
ZX
D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000737:chemistry; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D000882:Haplorhini; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008668:Metallothionein / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D017186:Titrimetry / Q000379:methods
D011993:Recombinant Fusion Proteins; D002104:Cadmium; D008668:Metallothionein; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D005982:Glutathione Transferase