Extracellular transport of pseudoazurin of Alcaligenes faecalis in Escherichia coli using the COOH-terminal domain of Serratia marcescens serine protease.
A large COOH-terminal domain of Serratia marcescens serine protease (SSP) is essentially required for the excretion of the mature protease region through the outer membrane in Escherichia coli. For investigation of the possibility of transporting foreign periplasmic proteins by utilizing the function of the COOH-terminal domain of SSP, the pseudoazurin (PA) gene of Alcaligenes faecalis was fused to three different lengths of the COOH-terminal domain via a BglII linker sequence (AGATCT) which would be translated into Arg-Ser, one of the cleavage sequences of the OmpT protease. The outer membrane fractions of E. coli JM105 harboring each of the fused genes on a multicopy plasmid was found to contain the PA-SSP hybrid protein without the signal sequence as well as the COOH-terminal domain of SSP processed at the Arg-Ser sequence. Trypsin-treatment of intact and cold osmotically shocked cells suggested that the COOH-terminal domain of SSP in the hybrid protein was rigidly integrated in the outer membrane, while the PA part of the hybrid protein was exposed on the cell surface. The localization of the PA part was confirmed by immunolabelling of intact cells with the anti-PA antibody. Furthermore, exogenous addition of purified SSP to the intact E. coli cells containing the hybrid protein liberated the PA part from the cells into the medium, probably by cleavage at or very near the site processed during the normal secretion of intact SSP.(ABSTRACT TRUNCATED AT 250 WORDS)
Shimada
K
K
Department of Biotechnology, University of Tokyo.
Ohnishi
Y
Y
Horinouchi
S
S
Beppu
T
T
D000421:Alcaligenes / Q000378:metabolism*; D000595:Amino Acid Sequence; D001400:Azurin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism* / Q000502:physiology; D005110:Extracellular Space / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D012706:Serratia marcescens / Q000201:enzymology*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C047163:pseudoazurin; D001400:Azurin; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease