The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli.
The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.
Takahara
M
M
Hibler
D W
DW
Barr
P J
PJ
Gerlt
J A
JA
Inouye
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000494:pharmacology*; D021382:Protein Sorting Signals
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D010455:Peptides; D021382:Protein Sorting Signals; D004262:DNA Restriction Enzymes; D008836:Micrococcal Nuclease