A plate-based high-throughput activity assay for polysialyltransferase from Neisseria meningitidis

作者：Yu Ching Ching; Hill Tara; Kwan David H; Chen Hong Ming; Lin Chun Cheng; Wakarchuk Warren; Withers Stephen G^*

来源：Analytical Biochemistry, 2014, 444: 67-74.

DOI：10.1016/j.ab.2013.09.030

• Polysialyltransferase
• Polysialic acid
• GFP
• Endosialidase
• High-throughput screening
• Click chemistry

Polysialyltransferases (PSTs) assemble polysialic acid (PSA) and have been implicated in many biological processes. For example, certain bacteria such as neuroinvasive Neisseria meningitidis decorate themselves in a PSA capsule to evade the innate immune system. Identifying inhibitors of PSTs therefore represents an attractive therapeutic goal and herein we describe a high-throughput, robust, and sensitive microtiterplate-based activity assay for PST from N. meningitidis. A trisialyl lactoside (GT3) serving as the acceptor substrate was immobilized on a 384-well plate by click chemistry. Incubation with PST and CMP-sialic acid for 30 min resulted in polysialylation. The immobilized PSA was then directly detected using a green fluorescent protein (GFP)-fused PSA-binding protein consisting of the catalytically inactive double mutant of an endosialidase (GFP-EndoNF DM). We report very good agreement between kinetic and inhibition parameters obtained with our on-plate assay versus our in-solution validation assay. In addition we prove our assay is robust and reliable with a Z%26apos; score of 0.79. All aspects of our assay are easily scalable owing to optimization trials that allowed immobilization of acceptor substrates prepared from crude reaction mixtures and the use of cell lysates. This assay methodology enables large-scale PST inhibitor screens and can be harnessed for directed evolution screens.

• 出版日期2014-1-1
• 单位清华大学