Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase.

ABSTRACT:

Objective

Neisseria meningitidis is a Gram-negative bacterium that causes meningitis. N. meningitidis serogroup W (NmW) capsule polymerase synthesizes capsular polysaccharide of this serogroup. This enzyme could be a tool for meningococcal glycoconjugate vaccine development. Our long-term goal is to control activity of the NmW capsule polymerase for production of defined carbohydrates for vaccines. The enzyme lacks a simple, high-throughput activity assay. Here, we describe the use of high-throughput bioluminescence assays (CMP-Glo and UDP-Glo by Promega) to investigate NmW capsule polymerase activity. These assays detect free nucleotides produced during transfer of sugar from UDP-Galactose and CMP-Sialic Acid to an acceptor. Kinetic studies using NmW hydrolyzed polysaccharide (PS)

SUBMITTER: Sheikhi Moghaddam L 

PROVIDER: S-EPMC8600345 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature