High yield expression and single step purification of human thionein/metallothionein.
Human metallothionein (MT), isoform 2, was expressed in Escherichia coli as an intein (protein splicing element) fusion protein in the absence of added metals and purified by intein-mediated purification with an affinity chitin-binding tag (IMPACT system). This procedure constitutes a novel and simple strategy to prepare thionein (T), the metal-free form, or MT when reconstituting T with metals in vitro. The yield was 8 mg of T or 6 mg of pure Cd(7)- or Zn(7)-MT from a 1-L culture, significantly higher than yields from any other expression system. Purified recombinant protein is indistinguishable from the native protein on the basis of its metal-binding ability, titration of its sulfhydryls, and UV and CD spectra. The MALDI-TOF mass spectrum is consistent with that of T with a free N-terminus.
Hong
S
S
Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamada-oka, Suita, Osaka 565-0871, Japan.
Toyama
M
M
Maret
W
W
Murooka
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D000882:Haplorhini; D006801:Humans; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D001059:Apoproteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D008668:Metallothionein