title: Construction and characterization of versatile cloning vectors for efficient delivery of native foreign proteins to the periplasm of Escherichia coli.
abstract: Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones in Escherichia coli inhibited growth and resulted in low yields of cholera toxin (CT). We found that production of wild type CT or its B subunit (CT-B) as a periplasmic protein was toxic for E. coli, but by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit of E. coli heat-labile enterotoxin LTIIb we succeeded for the first time in producing CT holotoxin in high yield in E. coli. Based on these findings, we designed and constructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the periplasm of E. coli. We confirmed the usefulness of these vectors by producing two other secreted recombinant proteins. First, using phoA from E. coli, we demonstrated that alkaline phosphatase activity was 17-fold greater when the LTIIb-B signal sequence was used than when the native leader for alkaline phosphatase was used. Second, using the pspA gene that encodes pneumococcal surface protein A from Streptococcus pneumoniae, we produced a 299-residue amino-terminal fragment of PspA in E. coli in large amounts as a soluble periplasmic protein and showed that it was immunoreactive in Western blots with antibodies against native PspA. The vectors described here will be useful for further studies on structure-function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm in E. coli.
authors.affiliation: Department of Microbiology, University of Colorado Health Sciences Center, Denver 80262, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: Jobling
authors.forename: L M
authors.initials: LM
authors.lastname: Palmer
authors.forename: J L
authors.initials: JL
authors.lastname: Erbe
authors.forename: R K
authors.initials: RK
authors.lastname: Holmes
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D002772:Cholera Toxin / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D003593:Cytoplasm; D004269:DNA, Bacterial; D004768:Enterotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013296:Streptococcus pneumoniae / Q000235:genetics
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C051592:pneumococcal surface protein A; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli; D000469:Alkaline Phosphatase