Cloning and expression of the ccdA-associated thiol-disulfide oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production.
Brevibacillus choshinensis (Bacillus brevis) is a protein-hyperproducing bacterium with a useful host-vector system for the production of recombinant proteins. Here, we cloned the ccdA-catA (cmacr;cdA āssociated thioredoxin-like tmacr;hiol-disulfide oxidoreductase) locus of B. choshinensis HPD31-S5. CatA protein (molecular weight, 19664) contains a thioredoxin-like motif, Cys-Gly-Pro-Cys. It was successfully expressed in B. choshinensis extracellularly ( approximately 100 microg x ml(-1) culture) using the secretion vector pNCMO2, and in Escherichia coli intracellularly ( approximately 350 microg x ml(-1) culture) with an amino-terminal His-tag. Both recombinant proteins showed thiol-disulfide oxidoreductase activity. Incubation of non-native human epidermal growth factor (hEGF) containing incorrect disulfide bonds with B. choshinensis cells secreting CatA protein resulted in the stimulation of the conversion of non-native hEGF to the native form. Furthermore, co-expression of CatA protein with recombinant hEGF in the B. choshinensis production system increased the yield of native hEGF.
Tanaka
Ryoichi
R
Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
Mizukami
Makoto
M
Ishibashi
Matsujiro
M
Tokunaga
Hiroko
H
Tokunaga
Masao
M
D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000145:classification / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; C104882:CcdA protein, Bacillus; D008565:Membrane Proteins; D011994:Recombinant Proteins; C442131:ScoC protein, Bacillus subtilis; D014157:Transcription Factors; D004815:Epidermal Growth Factor; D011490:Protein Disulfide Reductase (Glutathione)