Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli (original) (raw)

Abstract

A gene bank of Sau3A1-generated Streptococcus pneumoniae type 23 DNA fragments was constructed in Escherichia coli K-12 with the low-copy-number cosmid vector pOU61cos. Clone lysates were screened by immunoblotting using a mouse antiserum raised against a crude pneumococcal hyaluronidase preparation. One immunoreactive clone was isolated, and it produced high level of hyaluronidase activity. This clone contained a recombinant cosmid (designated pJCP800) with an approximately 35-kb DNA insert, and the putative hyaluronidase coding sequence was subcloned into pBluescript SK as a 3.8-kb PstI-ClaI fragment (designated pJCP802). The complete nucleotide sequence of this insert was determined. The region included an open reading frame sufficient to encode a polypeptide with an M(r) of 107,751. An active hyaluronidase with an M(r) of approximately 89,000 was purified to homogeneity from E. coli DH5 alpha(pJCP802). N-terminal amino acid sequence analysis of the purified protein suggested that translation initiation was occurring primarily at a TTG codon within the major open reading frame. However, immunoblot analysis using antiserum raised against the purified 89-kDa hyaluronidase indicated that E. coli DH5 alpha(pJCP802) also expressed the 107-kDa form of the enzyme. This antiserum labelled a 107-kDa protein in partially purified hyaluronidase preparations from S. pneumoniae. The hyaluronidase activity in this pneumococcal extract was also neutralized by the antiserum.

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Selected References

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  1. Berry A. M., Lock R. A., Hansman D., Paton J. C. Contribution of autolysin to virulence of Streptococcus pneumoniae. Infect Immun. 1989 Aug;57(8):2324–2330. doi: 10.1128/iai.57.8.2324-2330.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berry A. M., Yother J., Briles D. E., Hansman D., Paton J. C. Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae. Infect Immun. 1989 Jul;57(7):2037–2042. doi: 10.1128/iai.57.7.2037-2042.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boulnois G. J. Pneumococcal proteins and the pathogenesis of disease caused by Streptococcus pneumoniae. J Gen Microbiol. 1992 Feb;138(2):249–259. doi: 10.1099/00221287-138-2-249. [DOI] [PubMed] [Google Scholar]
  4. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  6. Gray B. M., Converse G. M., 3rd, Dillon H. C., Jr Epidemiologic studies of Streptococcus pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life. J Infect Dis. 1980 Dec;142(6):923–933. doi: 10.1093/infdis/142.6.923. [DOI] [PubMed] [Google Scholar]
  7. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  8. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  9. Hynes W. L., Ferretti J. J. Sequence analysis and expression in Escherichia coli of the hyaluronidase gene of Streptococcus pyogenes bacteriophage H4489A. Infect Immun. 1989 Feb;57(2):533–539. doi: 10.1128/iai.57.2.533-539.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jobling M. G., Holmes R. K. Construction of vectors with the p15a replicon, kanamycin resistance, inducible lacZ alpha and pUC18 or pUC19 multiple cloning sites. Nucleic Acids Res. 1990 Sep 11;18(17):5315–5316. doi: 10.1093/nar/18.17.5315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Knott V., Rees D. J., Cheng Z., Brownlee G. G. Randomly picked cosmid clones overlap the pyrB and oriC gap in the physical map of the E. coli chromosome. Nucleic Acids Res. 1988 Mar 25;16(6):2601–2612. doi: 10.1093/nar/16.6.2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lock R. A., Paton J. C., Hansman D. Purification and immunological characterization of neuraminidase produced by Streptococcus pneumoniae. Microb Pathog. 1988 Jan;4(1):33–43. doi: 10.1016/0882-4010(88)90046-0. [DOI] [PubMed] [Google Scholar]
  14. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  15. Musher D. M. Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. Clin Infect Dis. 1992 Apr;14(4):801–807. doi: 10.1093/clinids/14.4.801. [DOI] [PubMed] [Google Scholar]
  16. Paton J. C., Berry A. M., Lock R. A., Hansman D., Manning P. A. Cloning and expression in Escherichia coli of the Streptococcus pneumoniae gene encoding pneumolysin. Infect Immun. 1986 Oct;54(1):50–55. doi: 10.1128/iai.54.1.50-55.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Paton J. C., Lock R. A., Hansman D. J. Effect of immunization with pneumolysin on survival time of mice challenged with Streptococcus pneumoniae. Infect Immun. 1983 May;40(2):548–552. doi: 10.1128/iai.40.2.548-552.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pritchard D. G., Lin B. Group B streptococcal neuraminidase is actually a hyaluronidase. Infect Immun. 1993 Aug;61(8):3234–3239. doi: 10.1128/iai.61.8.3234-3239.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  20. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Williams K. L., Gooley A. A., Haynes P. A., Batley M., Curtin J. H., Stuart M. C., Champion A. C., Sheumack D. D., Redmond J. W. Analytical biotechnology: applications for downstream processing. Aust J Biotechnol. 1991 Apr;5(2):96–100. [PubMed] [Google Scholar]