Construction and characterization of versatile cloning vectors for efficient delivery of native foreign proteins to the periplasm of Escherichia coli

Plasmid
M G JoblingRandall K Holmes

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 ...Continue Reading

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Citations

Dec 6, 2000·Proceedings of the National Academy of Sciences of the United States of America·M G Jobling, R K Holmes
Dec 9, 2003·The Journal of Biological Chemistry·Amanda L HorstmanMeta J Kuehn
Jun 24, 2003·Infection and Immunity·Alejandra V E CapozzoMarina S Palermo
Jan 11, 2014·Nature Protocols·Daniel J WilliamsonW Bruce Turnbull
Mar 27, 2013·Applied Microbiology and Biotechnology·Kheng Oon LowRosli Md Illias
Nov 30, 2011·The Journal of Cell Biology·Carla P GuimaraesHidde L Ploegh
May 5, 2010·Protein Expression and Purification·Juliette K TinkerBritni M Arlian
Jan 5, 2010·Biotechnology and Applied Biochemistry·Yanina R SevastsyanovichJeffrey A Cole
Apr 25, 2000·The Journal of Biological Chemistry·A L Horstman, M J Kuehn
Aug 25, 2018·Scientific Reports·Bruce R Lichtenstein, Birte Höcker

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