End‐to‐End Semi‐automated Mid‐scale Protein Screening Platform for Drug Discovery Research

Abstract

AbstractThe drug discovery landscape is ever‐evolving and constantly demands revolutionary technology advancements in protein expression and production laboratories. We have built a higher‐throughput mid‐scale semi‐automated protein expression and screening platform to accelerate drug discovery research. The workflow described here enables comprehensive expression and purification screening assessment of challenging or difficult‐to‐express recombinant proteins in a fast and efficient manner by delivering small but sufficient amounts of high‐quality proteins. The platform has been implemented for a wide range of applications that include identification of optimal constructs and chaperones for poorly expressing proteins, assessment of co‐expression partners for expressing stable multiprotein complexes, and suitable buffer/additive screening for insoluble or aggregation‐prone proteins. The approach allows parallel expression, purification, and characterization of 24 different samples using co‐infection or a polycistronic approach in insect cells and enables parallel testing of multiple parameters to improve protein yields. The strategy has been successfully adopted for screening intracellular and secreted proteins in Escherichia coli, mammalian transient expression, and baculovirus expression vector systems. Proteins purified from this platform are used for several structural and functional screens, such as negative staining, biochemical activity assays, mass spectrometry, surface plasmon resonance, and DNA‐encoded chemical library screens. In this article, for simplicity, we have focused on detailed expression and purification screening of intracellular protein complexes from insect cells. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Baculovirus generation via homologous recombinationSupport Protocol 1: Anti‐glycoprotein 64 antibody assayBasic Protocol 2: Generation of insect cell biomass expressing target protein(s)Basic Protocol 3: Mid‐scale affinity purificationSupport Protocol 2: Automated method for affinity purification on Hamilton STARBasic Protocol 4: Size exclusion chromatographySupport Protocol 3: Chromeleon 7 operation on Vanquish Duo