Characterization of secretory intermediates of Serratia marcescens serine protease produced during its extracellular secretion from Escherichia coli cells.
The Serratia marcescens serine protease (SSP; 66 kDa) is synthesized as a precursor (preproSSP; 112 kDa) composed of the NH2-terminal signal peptide of 27 amino acids, the mature protease part and a large COOH-terminal domain. When the SSP gene is expressed in Escherichia coli under the control of the tac promoter, the mature enzyme is excreted into the medium through the outer membrane, whereas preproSSP and two proteins, C-1 (40 kDa) and C-2 (38 kDa), processed from the COOH-terminal domain, are accumulated in the membrane fraction. Although treatment of the intact cells with trypsin caused slight truncation of C-1 and C-2, the main parts of C-1 and C-2, both of which are detected in the outer membrane, were resistant to trypsin, even after the cells had been osmotically shocked. Consistent with this, a high content of beta-sheet structure in C-2 was suggested by marked heat-modifiability, as determined by their electrophoretic mobilities on SDS-polyacrylamide gel. These findings suggest rigid integration of C-1 and C-2 in the outer membrane. Upon induction of the tac promoter, rapid excretion of SSP into the medium was first accompanied by the accumulation of C-1 in the outer membrane, which was followed by conversion of C-1 to C-2. PreproSSP was not detected during the accumulation of SSP in the medium, but it was gradually accumulated after the accumulation of SSP had reached a plateau. In addition, preproSSP still containing the intact NH2-terminal signal peptide was completely digested with trypsin when added to osmotically shocked cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Shikata
S
S
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo.
Shimada
K
K
Ohnishi
Y
Y
Horinouchi
S
S
Beppu
T
T
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012706:Serratia marcescens / Q000201:enzymology*
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases