An efficient method for the overexpression and purification of active tyrosinase from Streptomyces castaneoglobisporus.
The melanin-synthesizing gene operon cloned from Streptomyces castaneoglobisporus HUT6202 consists of two genes, designated tyrC and orf378, which encode apotyrosinase (TYRC) and its activator protein (ORF378), respectively. We have suggested that ORF378 may facilitate the incorporation of Cu(II) into apotyrosinase to express tyrosinase activity. To overproduce ORF378 and TYRC in Escherichia coli BL21(DE3)-pLysS, tyrC, and orf378 were independently but not polycistronically placed under the control of a T7 promoter in a vector, pET-21a(+). His(6)-tagged TYRC and His(6)-tagged ORF378 were simultaneously overproduced in an E. coli strain harboring a plasmid, designated pET-mel2, and the two proteins were co-purified with a Ni(II)-bound affinity column. Gel filtration analysis revealed that the two proteins form a heterodimer complex. The complexed protein was retrieved at a high efficiency (11 mg/L). To obtain an active TYRC, which is a Cu(II)-bound form of tyrosinase, we constructed pET-mel3 that carries orf378 without His(6)-tag and His(6)-tagged tyrC. After the cell-free extract from E. coli harboring pET-mel3 was subjected to Cu(II)-bound affinity column chromatography, His(6)-tagged TYRC, eluted from the column, exhibited the tyrosinase activity. The k(cat) and K(m) values for l-3,4-dihydroxyphenylalanine (l-DOPA) of His(6)-tagged TYRC, which catalyzes the oxidation of l-DOPA to dopaquinone, were 880+/-80s(-1) and 8.1+/-0.9 mM, respectively.
Kohashi
Patricia Yumi
PY
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Kumagai
Takanori
T
Matoba
Yasuyuki
Y
Yamamoto
Aiko
A
Maruyama
Masafumi
M
Sugiyama
Masanori
M
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007980:Levodopa / Q000378:metabolism; D008543:Melanins / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology*
D008543:Melanins; D007980:Levodopa; D003300:Copper; D014442:Monophenol Monooxygenase