Binding Rate Screen - a high-throughput assay in soluble lysate for prioritizing protein expression constructs.
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.
Tian-Yu
Jiamin
J
Oncology, Novartis Institutes for Biomedical Research, Emeryville, CA 94608, USA. jiamin.yu@novartis.com
Licht
Stuart
S
Pardee
Gwynn
G
Bhat
Arun
A
Cao
Ying
Y
Gao
Wei
W
Sangalang
Emma
E
Zaror
Isabel
I
D057166:High-Throughput Screening Assays / Q000379:methods*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007118:Immunoassay; D007368:Interferometry; D008027:Light; D008163:Luminescent Measurements; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine