title: Expression and purification of thioredoxin (TrxA) and thioredoxin reductase (TrxB) from Brevibacillus choshinensis.
abstract: Brevibacillus choshinensis (formerly Bacillus brevis) is a protein-hyperproducing bacterium and has been used for commercial protein production. Here, we cloned thioredoxin (trxA) and thioredoxin reductase (trxB) genes from B. choshinensis, and expressed the gene products in Escherichia coli with an amino-terminal hexa-His-tag for purification and characterization. His-TrxA and His-TrxB were purified to homogeneity with one-step Ni-NTA affinity column chromatography, and the two recombinant proteins showed identical specific activity with or without removal of the amino-terminal His-tag, indicating that the extrasequence containing the hexa-His-tag did not affect their enzymatic activities. The E. coli expression system used here resulted in a 40-fold increase in production of His-TrxB protein compared to the level of native TrxB produced in non-recombinant B. choshinensis cells. TrxA and TrxB proteins with carboxy-terminal His-tag (TrxA-His and TrxB-His) were successfully expressed in B. choshinensis and were purified by Ni-NTA column chromatography. Co-expression of TrxA-His with recombinant human epidermal growth factor (hEGF) in B. choshinensis promoted the extracellular production of hEGF by up to about 200%.
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Tanaka
authors.forename: Kotaro
authors.initials: K
authors.lastname: Kosugi
authors.forename: Makoto
authors.initials: M
authors.lastname: Mizukami
authors.forename: Matsujiro
authors.initials: M
authors.lastname: Ishibashi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tokunaga
authors.forename: Masao
authors.initials: M
authors.lastname: Tokunaga
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D001671:Biochemistry / Q000379:methods*; D002845:Chromatography; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013880:Thioredoxin-Disulfide Reductase / Q000096:biosynthesis* / Q000737:chemistry*; D013879:Thioredoxins / Q000096:biosynthesis* / Q000737:chemistry*; D013917:Thrombin / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D013879:Thioredoxins; D004815:Epidermal Growth Factor; D004247:DNA; D013880:Thioredoxin-Disulfide Reductase; D013917:Thrombin