Role of primary structure and disulfide bond formation in beta-lactamase secretion

Abstract

Plasmid pBR322-encoded ,-lactamase was shown to contain a single disulfide bond, which caused the protein to migrate faster in sodium dodecyl sulfatepolyacrylamide gels than the fully reduced form. A similar difference in mobility of the in vitro synthesized precursor before and after reduction indicates that it also contained a disulfide bond. Formation of the disulfide bond in vivo, however, occurred concomitant with processing. In vivo accumulation of the precursor by inhibition of secretion did not allow disulfide bond formation to occur. This result is consistent with post-translational translocation of the precursor. Synthesis of a fragment of ,-lactamase lacking the carboxy terminus was obtained by insertion of a foreign DNA segment into the PstI site of bla. Processing and secretion of the protein did not appear to be greatly affected, indicating that the carboxy terminus is not required for secretion. Type TEM1 ,B-lactamase (EC 3.5.2.6) is encoded by the gene bla carried on many R-factor plasmids harbored by Escherichia coli. The nucleotide sequence of this gene has been determined (18), and the deduced amino acid sequence, along with that obtained by protein sequencing (1), shows that ,-lactamase is synthesized as a 31,516 Mr precursor having a 23residue amino-terminal signal sequence. The primary translation product contains three cysteinyl residues at positions 18, 75, and 121. Proteolytic processing of the signal sequence has been shown to occur after translation of the precursor (12, 13). The mature enzyme of 28,909 Mr (1, 18) is localized in the E. coli periplasm (13). The mechanism for secretion is complex, and several models have been proposed (3, 11, 21). In this study, we examined two specific features of the structure of P-lactamase in relation to signal sequence processing and secretion. We found that P-lactamase contains a disulfide bond and determined the temporal sequence for disulfide bond formation and proteolytic processing. The data indicate that in vivo disulfide bond formation and processing of the precursor occur simultaneously. This is consistent with a posttranslational change in environment from the cytoplasm to the periplasm. The second structural feature examined concerns the carboxy terminus of ,-lactamase. In contrast to an earlier report that an intact carboxy-terminal region of t Journal paper no. 9080 from the Purdue University Agricultural Experiment Station.

Formation of disulphide bonds during secretion of proteins through the periplasmic‐independent type I pathway

“…3B). This was confirmed further by examining the differences in the electrophoretic mobility of proteins in non-reducing SDS± PAGE, which are indicative of the formation of intramolecular disulphide bonds (Scheele and Jacoby, 1982;Pollitt and Zalkin, 1983;Bardwell et al, 1991). The mobility in non-reducing SDS±PAGE of the scFv 6AC3 polypeptide was lower when produced in the periplasm of the dsbA mutant strain (Fig.…”

Identification and characterization of a new disulfide isomerase-like protein (DsbD) in Escherichia coli.

“…Cultures of wild-type or dsbD mutant bacteria grown in M9 minimal medium as above were pulse-labeled with 100 tCi/ml [35S]methionine for 2 min and chased with a 1000fold excess of cold methionine. Further oxidation processes were quenched by dropping the pH and blocking the cysteinyl groups, as described by Pollitt and Zalkin (1983).…”

Disulphide bridge formation in the periplasm of Escherichia coli: β‐lactamase::human lgG3 hinge fusions as a model system

“…Considering the different views on the role of the C-terminus of p-lactamase during export (Koshland and Botstein, 1982;Pollitt and Zaikin, 1983), we directly fused the cDNA of the human lgG3 hinge region to the 3' end of the p-lactamase gene. Since two neighbouring p-lactamase moieties could possibly interfere with correct association in the hinge region into F(ab')2-like dimers.…”