A high-throughput screen for polysialyltransferase activity

作者：Keys Timothy G; Berger Monika; Gerardy Schahn Rita^*

来源：Analytical Biochemistry, 2012, 427(1): 60-68.

DOI：10.1016/j.ab.2012.04.033

• Polysialyltransferase
• Polysialic acid
• High-throughput screen
• Library screening
• Truncation library
• Colony blot

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.

• 出版日期2012-8-1