The role of the beta-lactamase signal sequence in the secretion of proteins by Escherichia coli.

Abstract

A derivative of pBR322 has been constructed that contains both a unique EcoRI restriction site right at the beginning of the signal codons of the beta-lactamase (bla) gene and a unique BstEII site just at the end of the bla signal codons. Although the signal peptide encoded by the new plasmid differs from the wild type (pBR322) by 2 amino acid residues (Ser 2 to Arg 2 and Ala 23 to Gly 23), the synthesis, transport, and processing of the beta-lactamase remain unchanged in Escherichia coli. Two deletion mutants, in which the bla signal codons have been almost completely excised, have also been constructed. Bacteria containing either of these plasmids produce, but do not secrete, an active beta-lactamase. Last, the bla signal codons have been precisely joined to the cDNA version of the triose phosphate isomerase (tpi) gene from chicken. Expression of this fusion gene in E. coli gives a hybrid protein that is neither secreted into the periplasm nor proteolytically processed. This result supports the view that there are characteristics of the mature protein that are necessary for the secretion across the inner membrane of E. coli.