Abstract
Pullulanase secretion in Escherichia coli depends on the expression of a MalT-regulated operon called pulC. Characterization of the first two genes of this operon showed that they encode, respectively, a 31,000-Da protein (PulC) and a 70,600-Da protein (PulD) which has a putative signal peptide and that these two proteins are required for pullulanase secretion. The analysis of alkaline phosphatase hybrid proteins generated by TnphoA mutagenesis of pulC and pulD showed that both PulC and PulD contain export signals which can direct the alkaline phosphatase segment of the hybrids across the inner membrane. A representative PulC-PhoA hybrid protein fractionated mainly with the inner membrane upon isopycnic sucrose gradient centrifugation of membrane vesicles. This, together with sequencing data, suggests that PulC is an inner membrane protein. Antibodies raised against a purified PulD-PhoA hybrid protein were used to show that PulD was enriched in low density outer membrane vesicles.
Highlights
To cite this version: Christpohe d’Enfert, Isabelle Reyss, Cécile Wandersman, Anthony P
Characterization of the first two genes of this operon showed that they encode, respectively, a 31,000-Da protein (PulC) and a 70,600-Da protein (PulD) which has a putative signal peptide and that these two proteins arerequired for pullulanase secretion
The analysis of alkaline phosphatase hybrid proteins generated by TnphoA mutagenesis of pulC and pulD showed that both PulC and PulD contain export signals which can direct the alkaline phosphatase segment of the hybrids across the inner membrane
Summary
Commercial alkaline phosphatase (Sigma) were precipitated with ammonium sulfate (45% saturated) for 30 min at room temperature and centrifuged for 10 min at 10,000 X g. The approximate immunoglobulin concentration was determined by SDS-PAGE analysis. It was applied to the alkaline phosphatase affinity column at 4 “C at a flow rate of 10 ml/h. The bound PulD-PhoA85 hybrid protein was eluted with 0.2 M glycine HC1 (pH 2.2) containing 0.15 M NaCl. 1-ml fractions were initially mixed with 0.1 mlof 1 M Tris-HC1, pH 9.0, for alkaline phosphatase assays. Fractions corresponding to the nearly homogeneous PulD-PhoA85 hybrid protein (as determined by SDSPAGE analysis and immunodetection with an anti-PhoA serum)were pooled, dialyzed against 10 mM Tris-HC1, pH 8, and lyophilized. A rabbit was immunized three times with approximately 100 pgof hybrid protein
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
