title: A high-throughput screen for polysialyltransferase activity.
abstract: Polysialic acid is common to humans and a few bacterial pathogens and it holds great potential for the development of new therapeutic reagents. Currently, the bacterial polysialyltransferases (polySTs) are the only source of polysialic acid for research and biotechnological purposes either directly, by enzymatic polysialylation of therapeutic proteins, or indirectly, by harvest of polysialic acid from bacterial fermentation. Further engineering and optimization of these enzymes is hindered by the lack of high-throughput screening methodologies for polysialyltransferase activity. Here we report the development of an efficient in vivo activity screen for bacterial polySTs. The screen exploits complementation of a dormant capsule export complex in the expression strain, Escherichia coli BL21-Gold(DE3). This strain was metabolically engineered to synthesize CMP-Neu5Ac, the donor sugar for the polysialylation reaction. Using the new strain, a colony blotting procedure that enables the routine testing of more than 10(4) polyST genes was developed. To test the usefulness of the methodology, we screened a library of N-terminally truncated polySTs derived from the Neisseria meningitidis serogroup B (NmB)-polyST. We identified truncations that remove a putative membrane interaction domain, resulting in soluble and active enzymes.
authors.affiliation: Department of Biochemistry, Institute for Cellular Chemistry, Hannover Medical School, Hannover 30625, Germany.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Keys
authors.forename: Monika
authors.initials: M
authors.lastname: Berger
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
mesh_terms: D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D003568:Cytidine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D004926:Escherichia coli; D057166:High-Throughput Screening Assays; D006801:Humans; D060847:Metabolic Engineering; D008969:Molecular Sequence Data; D038541:Neisseria meningitidis, Serogroup B / Q000201:enzymology / Q000235:genetics; D007652:Oxo-Acid-Lyases / Q000235:genetics; D012794:Sialic Acids / Q000096:biosynthesis / Q000737:chemistry*; D012799:Sialyltransferases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D012794:Sialic Acids; C561601:cytidine-5'-monophosphosialic acid; C021319:polysialic acid; D012799:Sialyltransferases; C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; C024519:N-acyl-D-glucosamine 2-epimerase; D003568:Cytidine Monophosphate