The production of recombinant APRP, an alkaline protease derived from Bacillus pumilus TYO-67, by in vitro refolding of pro-enzyme fixed on a solid surface.
Bacillus pumilus TYO-67 has been isolated from tofuyo, a traditional fermented food made from soybean milk in Okinawa, Japan. This bacterium secretes a soybean-milk-coagulating enzyme (SMCE), which can be applied for the production of processed foods from soybean milk. Thus, an easy method of producing the recombinant enzyme was developed in this study. SMCE is an alkaline serine protease belonging to the subtilisin family; its candidate gene, aprP, which encodes a prepro-enzyme, was isolated in a previous study. Recombinant APRP was then produced by in vitro refolding of pro-APRP-His, i.e., N-terminally His-tagged pro-APRP. A large amount of pro-APRP-His was produced in Esherichia coli BL21(DE3) (ca. 8 mg from a 20-ml culture), collected as insoluble protein, dissolved in 6 M guanidine-HCl (pH 8.0), bound to Ni-NTA, and refolded on the resin at pH 10.0 to become mature APRP by autocleavage. Then, 0.16 mg of purified mature APRP was obtained through single-step chromatography from the refolded sample using 10 mg of pro-APRP-His. The N-terminal sequence and the enzymatic properties of refolded APRP were identical to those of SMCE. In addition, the pro-sequence was found to be essential for the production of mature APRP, suggesting that it could function as an intramolecular chaperone.
Takahashi
Masakazu
M
Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Fukui 910-1195.
Sekine
Tomoko
T
Kuba
Naoko
N
Nakamori
Shigeru
S
Yasuda
Masaaki
M
Takagi
Hiroshi
H
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D002364:Caseins / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry*; D004792:Enzyme Precursors; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry; D013025:Glycine max / Q000201:enzymology; D020860:Subtilisin / Q000737:chemistry
D001426:Bacterial Proteins; D002364:Caseins; D017931:DNA Primers; D004792:Enzyme Precursors; D010455:Peptides; D011994:Recombinant Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; D020860:Subtilisin; C087822:alkaline protease; C119936:soybean-milk-coagulating enzyme