Expression and purification of the calcium binding photoprotein mitrocomin using   ZZ-domain as a soluble partner in E. coli cells.
We constructed a vector for soluble protein expression in the cytoplasm of Escherichia coli cells using the cold induced system. The vector, named pCold-ZZ-P-X, consists of a histidine tag sequence, IgG binding domain of protein A (ZZ domain), the cleavage site of human rhinovirus 3C protease followed by the multiple cloning sites under the controlled of the cold shock protein A (cspA) promoter and the lac operator. Using this expression vector, the calcium binding photoprotein mitrocomin from luminous jellyfish was successfully expressed as a soluble ZZ fusion protein and purified. After removing the ZZ domain by protease digestion, recombinant apomitrocomin was obtained and then regenerated to mitrocomin by incubation with coelenterazine. The luminescence properties of recombinant mitrocomin were characterized and compared to other photoproteins including aequorin, clytin-I and clytin-II.
Yokohama Research Center, Chisso Corporation, 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan. sinouye@chisso.co.jp
Satoshi
S
Inouye
Yuiko
Y
Sahara
D000331:Aequorin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C083952:mitrocomin protein, Mitrocoma celluria; C046165:phialidin; D000331:Aequorin; D002118:Calcium