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. 1994 Feb 15;298(Pt 1):189–195. doi: 10.1042/bj2980189

Engineering and overexpression of periplasmic forms of the penicillin-binding protein 3 of Escherichia coli.

C Fraipont 1, M Adam 1, M Nguyen-Distèche 1, W Keck 1, J Van Beeumen 1, J A Ayala 1, B Granier 1, H Hara 1, J M Ghuysen 1
PMCID: PMC1138000  PMID: 8129719

Abstract

Replacement of the 36 and 56 N-terminal amino acid residues of the 588-amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modified ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optimized by varying the copy number of the recombinant plasmids and the amount of LacI repressor, and export was facilitated by increasing the SecB content of the producing strain. The periplasmic PBP3s (yield 8 mg/l of culture) were purified to 70% protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-bound PBP3, they undergo processing by elimination of the C-terminal decapeptide I578-S588, they bind penicillin in a 1:1 molar ratio and they catalyse hydrolysis and aminolysis of acyclic thioesters that are analogues of penicillin. The membrane-anchor-free PBP3s have ragged N-termini. The G57-V577 PBP3, however, is less prone to proteolytic degradation than the F37-V577 PBP3.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adam M., Damblon C., Jamin M., Zorzi W., Dusart V., Galleni M., el Kharroubi A., Piras G., Spratt B. G., Keck W. Acyltransferase activities of the high-molecular-mass essential penicillin-binding proteins. Biochem J. 1991 Oct 15;279(Pt 2):601–604. doi: 10.1042/bj2790601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adam M., Damblon C., Plaitin B., Christiaens L., Frère J. M. Chromogenic depsipeptide substrates for beta-lactamases and penicillin-sensitive DD-peptidases. Biochem J. 1990 Sep 1;270(2):525–529. doi: 10.1042/bj2700525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baneyx F., Georgiou G. Construction and characterization of Escherichia coli strains deficient in multiple secreted proteases: protease III degrades high-molecular-weight substrates in vivo. J Bacteriol. 1991 Apr;173(8):2696–2703. doi: 10.1128/jb.173.8.2696-2703.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Belder J. B., Nguyen-Distèche M., Houba-Herin N., Ghuysen J. M., Maruyama I. N., Hara H., Hirota Y., Inouye M. Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12. Mol Microbiol. 1988 Jul;2(4):519–525. doi: 10.1111/j.1365-2958.1988.tb00058.x. [DOI] [PubMed] [Google Scholar]
  5. Bowler L. D., Spratt B. G. Membrane topology of penicillin-binding protein 3 of Escherichia coli. Mol Microbiol. 1989 Sep;3(9):1277–1286. doi: 10.1111/j.1365-2958.1989.tb00278.x. [DOI] [PubMed] [Google Scholar]
  6. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Broome-Smith J. K., Hedge P. J., Spratt B. G. Production of thiol-penicillin-binding protein 3 of Escherichia coli using a two primer method of site-directed mutagenesis. EMBO J. 1985 Jan;4(1):231–235. doi: 10.1002/j.1460-2075.1985.tb02340.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  10. Ghuysen J. M. Serine beta-lactamases and penicillin-binding proteins. Annu Rev Microbiol. 1991;45:37–67. doi: 10.1146/annurev.mi.45.100191.000345. [DOI] [PubMed] [Google Scholar]
  11. Goloubinoff P., Gatenby A. A., Lorimer G. H. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature. 1989 Jan 5;337(6202):44–47. doi: 10.1038/337044a0. [DOI] [PubMed] [Google Scholar]
  12. Hara H., Yamamoto Y., Higashitani A., Suzuki H., Nishimura Y. Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3. J Bacteriol. 1991 Aug;173(15):4799–4813. doi: 10.1128/jb.173.15.4799-4813.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Houba-Hérin N., Hara H., Inouye M., Hirota Y. Binding of penicillin to thiol-penicillin-binding protein 3 of Escherichia coli: identification of its active site. Mol Gen Genet. 1985;201(3):499–504. doi: 10.1007/BF00331346. [DOI] [PubMed] [Google Scholar]
  14. Kumamoto C. A., Beckwith J. Evidence for specificity at an early step in protein export in Escherichia coli. J Bacteriol. 1985 Jul;163(1):267–274. doi: 10.1128/jb.163.1.267-274.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kumamoto C. A. Molecular chaperones and protein translocation across the Escherichia coli inner membrane. Mol Microbiol. 1991 Jan;5(1):19–22. doi: 10.1111/j.1365-2958.1991.tb01821.x. [DOI] [PubMed] [Google Scholar]
  16. Kusukawa N., Yura T., Ueguchi C., Akiyama Y., Ito K. Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli. EMBO J. 1989 Nov;8(11):3517–3521. doi: 10.1002/j.1460-2075.1989.tb08517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  19. Linström E. B., Boman H. G., Steele B. B. Resistance of Escherichia coli to penicillins. VI. Purification and characterization of the chromosomally mediated penicillinase present in ampA-containing strains. J Bacteriol. 1970 Jan;101(1):218–231. doi: 10.1128/jb.101.1.218-231.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mottl H., Keck W. Purification of penicillin-binding protein 4 of Escherichia coli as a soluble protein by dye-affinity chromatography. Eur J Biochem. 1991 Sep 15;200(3):767–773. doi: 10.1111/j.1432-1033.1991.tb16243.x. [DOI] [PubMed] [Google Scholar]
  21. Mottl H., Keck W. Rapid screening of a large number of immobilized textile dyes for the purification of proteins: use of penicillin-binding protein 4 of Escherichia coli as a model enzyme. Protein Expr Purif. 1992 Oct;3(5):403–409. doi: 10.1016/s1046-5928(05)80042-7. [DOI] [PubMed] [Google Scholar]
  22. Nagasawa H., Sakagami Y., Suzuki A., Suzuki H., Hara H., Hirota Y. Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli. J Bacteriol. 1989 Nov;171(11):5890–5893. doi: 10.1128/jb.171.11.5890-5893.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nakamura M., Maruyama I. N., Soma M., Kato J., Suzuki H., Horota Y. On the process of cellular division in Escherichia coli: nucleotide sequence of the gene for penicillin-binding protein 3. Mol Gen Genet. 1983;191(1):1–9. doi: 10.1007/BF00330881. [DOI] [PubMed] [Google Scholar]
  24. Nanninga N. Cell division and peptidoglycan assembly in Escherichia coli. Mol Microbiol. 1991 Apr;5(4):791–795. doi: 10.1111/j.1365-2958.1991.tb00751.x. [DOI] [PubMed] [Google Scholar]
  25. Rentier-Delrue F., Swennen D., Martial J. pIN-III-ompA secretion vectors: modification of the ompA signal peptide sequence for easier insert cloning. Nucleic Acids Res. 1988 Sep 12;16(17):8726–8726. doi: 10.1093/nar/16.17.8726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stark M. J. Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli. Gene. 1987;51(2-3):255–267. doi: 10.1016/0378-1119(87)90314-3. [DOI] [PubMed] [Google Scholar]
  27. Verschoor E. J., Luirink J., De Waard S., De Graaf F. K., Oudega B. Cloning, expression and release of native and mutant cloacin DF13 immunity protein. Antonie Van Leeuwenhoek. 1989 Apr;55(4):325–340. doi: 10.1007/BF00398511. [DOI] [PubMed] [Google Scholar]
  28. Vicente M., Palacios P., Dopazo A., Garrido T., Pla J., Aldea M. On the chronology and topography of bacterial cell division. Res Microbiol. 1991 Feb-Apr;142(2-3):253–257. doi: 10.1016/0923-2508(91)90038-c. [DOI] [PubMed] [Google Scholar]
  29. Wickner S. H. Three Escherichia coli heat shock proteins are required for P1 plasmid DNA replication: formation of an active complex between E. coli DnaJ protein and the P1 initiator protein. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2690–2694. doi: 10.1073/pnas.87.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wickner W., Driessen A. J., Hartl F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. doi: 10.1146/annurev.bi.60.070191.000533. [DOI] [PubMed] [Google Scholar]
  31. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. van der Linden M. P., Mottl H., Keck W. Cytoplasmic high-level expression of a soluble, enzymatically active form of the Escherichia coli penicillin-binding protein 5 and purification by dye chromatography. Eur J Biochem. 1992 Feb 15;204(1):197–202. doi: 10.1111/j.1432-1033.1992.tb16624.x. [DOI] [PubMed] [Google Scholar]

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