Analysis of B-polyST mutants and

Copyright information:Taken from "Biochemical characterization of a polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases"Molecular Microbiology 2007;65(5):1258-1275.Published online Jan 2007PMCID:PMC2169525.© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd A. Single-point mutations were introduced into the D/E-D/E-G and HP motif of B-polyST. Wild-type and mutant enzymes were expressed in and soluble (s) and insoluble (i) fractions of the bacterial lysates were analysed for expression by SDS-PAGE and Western blotting using anti-T7-tag antibody. B. PolyST activity was determined in protein lysates with similar expression levels by the radiochemical assay. Relative activities were calculated compared with the wild-type enzyme (100%). C. Mutant strains of were generated by replacing the native polyST gene with mutant polySTs carrying the respective point mutations. Capsule expression was analysed with the polySia-specific antibody 735 by slide agglutination. D. Capsular polySia of mutant strains with residual capsule expression was quantified by ELISA using mab 735. Equal loading of the wells with B was controlled using mab P1.7 for detection of the meningococcal major outer membrane protein PorA. Each value represents the average of three independent determinations with the standard deviation indicated.