Hyperproduction of a recombinant fusion protein of Staphylococcus aureus V8 protease in Escherichia coli and its processing by OmpT protease to release an active V8 protease derivative.
The expression of a recombinant fusion protein including Staphylococcus aureus V8 protease was studied by using Escherichia coli as the host strain. When the mature V8 protease was expressed as a fusion protein with a truncated E. coli beta-galactosidase (beta-gal97S4D), we could not obtain a sufficient amount of the enzyme because of the toxicity resulting from the expressed protease activity. Synthesis of V8 protease was increased by constructing a sandwich-type fusion protein consisting of beta-gal97S4D, a V8 protease derivative with the 56 C-terminal amino acids deleted (V8 delta 56) and a truncated aminoglycoside-3'-phosphotransferase. This fusion protein was successfully produced as inactive inclusion bodies. To release the V8 delta 56 protease from the fusion protein, we developed a novel processing method using an endogeneous E. coli OmpT protease, which can recognize the dibasic amino acid residues located in the linker peptides of the fusion protein. After solubilizing the inclusion bodies with urea, the V8 delta 56 protein was automatically released from the fusion protein by the OmpT protease, which was coprecipitated with the inclusion bodies. The V8 delta 56 protease thus obtained showed the same enzymatic activity as that of the native V8 protease. We demonstrate in this study that the N-terminal prepro sequence and the C-terminal repeated sequence of this enzyme are not necessary for its enzymatic activity and protein folding.
Yabuta
M
M
Suntory Institute for Medicinal Research and Development, Gunma, Japan.
Ochi
N
N
Ohsuye
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*
D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; C057840:omptin outer membrane protease