Involvement of the COOH-terminal pro-sequence of Serratia marcescens serine protease in the folding of the mature enzyme.
The serine protease (SSP) from a Gram-negative organism, Serratia marcescens, is excreted even from Escherichia coli. The protease is synthesized as a 1045-amino-acid precursor (preproSSP) composed of three functional portions, a typical NH2-terminal signal peptide of 27 amino acids, the mature protease part of 618 amino acids, and a large COOH-terminal part of 400 amino acids. After the mature part (Ala28 to Asp645) has been liberated into the medium, most of the COOH-terminal part (Phe717 to Phe1045) remains stably in the outer membrane. When a mutated gene encoding the prepro-SSP with deletion of the junction region (Ser646 to Gly716) between the mature protease and the processed COOH-terminal protein was expressed in E. coli, the SSP protein was not detected in any fractions of the cells nor in the medium, whereas a processed COOH-terminal protein was found in the outer membrane. However, when the outer membrane fraction prepared from the transformant exhibiting the junction region (Ser646 to Gly716) probably on the cell surface was co-cultured with the transformant, SSP was found in the medium. Furthermore, a significant portion of the SSP protein denatured with guanidine hydrochloride was correctly refolded in vitro into the active protease, only in the presence of the outer membrane preparation from the transformant exhibiting the junction region. These results suggest that the junction region is exposed outside the cells, and it plays a role for guiding the folding of SSP in the correct conformation.
Ohnishi
Y
Y
Department of Biotechnology, University of Tokyo, Japan.
Nishiyama
M
M
Horinouchi
S
S
Beppu
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D011489:Protein Denaturation; D017510:Protein Folding; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D012706:Serratia marcescens / Q000201:enzymology*
D009838:Oligodeoxyribonucleotides; D012697:Serine Endopeptidases