Binding Rate Screen - a high-throughput assay in soluble lysate for prioritizing protein expression constructs

Analytical Biochemistry
Jiamin Tian-YuIsabel Zaror

Abstract

Identification of constructs suitable for the recombinant protein production pipeline is a bottleneck for structural genomics efforts, as most methods require purified proteins and/or are labor-intensive. Here, we present a novel high-throughput approach, Binding Rate Screen, that can alleviate this bottleneck by screening expression constructs in crude soluble lysate. This functional screen utilizes the frequently employed hexahistidine (His(6)) tag as a reporter, and measures its binding rate to an affinity matrix as a metric to reflect aggregation, concentration, and purifiability of the target protein. The constructs with the highest binding rates also exhibit high expression of soluble monomeric protein as judged by analytical size-exclusion chromatography. Constructs expressing variations of the target protein can be prioritized on a time scale of minutes, which is at least 10-100 times faster than any other technologies currently available.

References

Sep 25, 2001·Analytical Biochemistry·R K Knaust, P Nordlund
Jul 21, 2004·Journal of Structural and Functional Genomics·G E FolkersR Kaptein
Dec 8, 2004·Nature Biotechnology·Stéphanie CabantousGeoffrey S Waldo
Jun 1, 2005·Trends in Biotechnology·David S Waugh
Oct 8, 2005·Journal of Structural and Functional Genomics·Frank J SugarMichael W W Adams
Nov 30, 2006·Drug Discovery Today·Matthew A Cooper
Apr 18, 2007·Journal of Biomolecular Screening·Tara M MezzasalmaMatthew J Todd

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