Affiliation:
1. Department of Biology, Washington University, St. Louis, Missouri 63130.
Abstract
The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Reference79 articles.
1. Evidence for a coding pattern on the non-coding strand of the E. coli genome;Alff-Steinberger C.;Nucleic Acids Res.,1984
2. Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt;Banas J. A.;Infect. Immun.,1990
3. Barrett J. F. T. A. Barrett and R. Curtiss III. 1986. Biochemistry and genetics of dextranase from Streptococcus mutans (sic sobrinus) 6715 p. 205-215. In S. Hamada S. M. Michalek H. Kiyono L. Menaker and J. R. McGhee (ed.) Molecular microbiology and immunology of Streptococcus mutans. Elsevier Science Publishing Inc. New York.
4. Purification and partial characterization of the multicomponent dextranase complex of Streptococcus sobrinus and cloning of the dextranase gene;Barrett J. F.;Infect. Immun.,1987
5. Renaturation of dextranase activity from culture supernatant fluids of Streptococcus sobrinus after sodium dodecyl sulfate polyacrylamide gel electrophoresis;Barrett J. F.;Anal. Biochem.,1986
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