A high-throughput screen for polysialyltransferase activity

Analytical Biochemistry
Timothy G KeysRita Gerardy-Schahn

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 truncation...Continue Reading

References

Jan 1, 1996·Annual Review of Microbiology·I S Roberts
Aug 15, 1997·Biochimica Et Biophysica Acta·A I Fernandes, G Gregoriadis
Mar 31, 1999·Proceedings of the National Academy of Sciences of the United States of America·M OstermeierS J Benkovic
Jun 1, 2000·Proceedings of the National Academy of Sciences of the United States of America·K A Datsenko, B L Wanner
Feb 24, 2001·Cellular and Molecular Life Sciences : CMLS·G GregoriadisB McCormack
Apr 9, 2001·International Journal of Pharmaceutics·A I Fernandes, G Gregoriadis
Jan 28, 2003·Current Opinion in Chemical Biology·Geoffrey S Waldo
Mar 15, 2003·Science·Hans E SchoemakerMarcel G Wubbolts
Jan 20, 2004·Nature Structural & Molecular Biology·Cecilia P C ChiuNatalie C J Strynadka
Jul 28, 2005·International Journal of Pharmaceutics·Gregory GregoriadisPeter Laing
Feb 18, 2006·Journal of Bacteriology·Ekaterina N Andreishcheva, Willie F Vann
Sep 9, 2006·Gene·Ekaterina N Andreishcheva, Willie F Vann
Jul 22, 2009·Nature Chemical Biology·Nicholas J Turner
Sep 30, 2009·Journal of Molecular Biology·Haeyoung JeongJihyun F Kim
Apr 20, 2011·Proceedings of the National Academy of Sciences of the United States of America·Theresa LindhoutWarren W Wakarchuk

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Citations

Oct 15, 2013·Analytical Biochemistry·Ching-Ching YuStephen G Withers
Apr 15, 2014·Nature Chemical Biology·Timothy G KeysRita Gerardy-Schahn
Feb 11, 2016·Biochemical Society Transactions·John B McArthur, Xi Chen
Apr 13, 2019·Glycobiology·Bettina JaneschWarren W Wakarchuk
Nov 5, 2017·Metabolic Engineering·Timothy G KeysMarkus Aebi
Oct 14, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Ryoma HombuSheldon Park

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