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A high-throughput screen for polysialyltransferase activity.

Timothy G Keys¹, Monika Berger, Rita Gerardy-Schahn

¹Department of Biochemistry, Institute for Cellular Chemistry, Hannover Medical School, Hannover 30625, Germany.

Analytical Biochemistry

|May 15, 2012

Summary

This summary is machine-generated.

Researchers developed a new high-throughput screening method to study bacterial polysialyltransferases (polySTs). This method enables efficient testing of polyST genes, facilitating the development of novel therapeutic reagents and improving enzyme engineering for polysialic acid production.

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Area of Science:

Biochemistry
Molecular Biology
Biotechnology

Background:

Polysialic acid (PSA) is a crucial biomaterial found in humans and pathogens, with significant therapeutic potential.
Bacterial polysialyltransferases (polySTs) are key enzymes for PSA production, used directly for protein modification or indirectly via bacterial fermentation.
Current limitations in enzyme engineering stem from the absence of high-throughput screening methods for polyST activity.

Purpose of the Study:

To develop an efficient in vivo activity screen for bacterial polySTs.
To enable high-throughput screening of polyST genes for improved enzyme engineering.
To identify soluble and active polyST variants through functional screening.

Main Methods:

Engineered Escherichia coli BL21-Gold(DE3) to synthesize CMP-Neu5Ac, the essential sugar donor for polysialylation.
Developed a colony blotting procedure for in vivo screening of polyST activity.
Screened a library of N-terminally truncated Neisseria meningitidis serogroup B (NmB)-polyST variants.

Main Results:

Successfully established an efficient in vivo screening system for bacterial polySTs.
Developed a method capable of testing over 10^4 polyST genes routinely.
Identified specific N-terminal truncations in NmB-polyST that yield soluble and active enzymes by removing a putative membrane interaction domain.

Conclusions:

The developed screening methodology significantly advances the study and engineering of bacterial polySTs.
This tool facilitates the discovery of novel polyST variants with enhanced properties for biotechnological applications.
The identification of soluble NmB-polyST variants opens new avenues for therapeutic reagent development using polysialic acid.