Purification And Characterization Of An Alpha-1,6 Glucan-Binding Protein From Streptococcus Sobrinus
Date of Award
Doctor of Philosophy (Ph.D.)
Microbiology and Immunology
The glucan-binding protein, GBP#1, produced by the bacterium Streptococcus sobrinus 6715-49 has been purified, physicochemically characterized and its binding to glucans has been described. Affinity and gel permeation chromatography yielded an homogenous protein preparation as judged by SDS-PAGE and isoelectric focussing. A monoclonal antibody raised against the purified protein did not recognise other bacterial proteins. Isoelectric focussing indicated a pI of 4.6. Fluorimetry revealed a spectrum dominated by a tryptophan-like fluorescence at 340 nanometers. The amino acid composition was enriched for the acidic residues aspartic and glutamic. A single band corresponding to a molecular size of 7.5k was seen on SDS-PAGE, in close agreement with the weight calculated from the amino acid composition. Nondenaturing PAGE and gel permeation chromatography indicated sizes of 13k and 15k respectively. These data are consistent with the native form of GBP#1 being a homodimer. The protein interacts with alpha-1,6 glucose homopolymers but not with heteropolymers or other linkage patterns. The association constant is 3.3 x 10('7) M('-1) ((DELTA)G('o) = -42 kJ/mol) for Dextran T2000, as determined by affinity electrophoresis, and is 7.1 x 10('3) M('-1) ((DELTA)G('o) = -22 kJ/mol) for isomaltohexaose, as determined by micropartition assay. There is a weak pH optimum at 7 and no strict requirement for divalent cations. The protein has one binding site that accommodates isomaltosaccharides up to DP8 in size. The size and affinity of the site are similar to the groove-sited dextran-binding antibody QUPC 52. GBP#1 has the same affinity for both Dextran T2000 and the glucan product of the S. sobrinus glucosyltransferase S4 enzyme. These data suggest that GBP#1 is an homodimer with a denatured size of 8k and a single binding site accommodating up to eight glucose residues. The protein binds readily to both exogenous and endogenous alpha-1,6 linked glucans.
Landale, Edwin Conde, "Purification And Characterization Of An Alpha-1,6 Glucan-Binding Protein From Streptococcus Sobrinus" (1987). Dissertations from ProQuest. 1648.