Abstract
The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications.
Highlights
The end goal of structural genomics is the rapid generation of threedimensional structures obtained from atomic level resolution studies of pure proteins (Xiao et al, 2010; Elsliger et al, 2010; Watson et al, 2007; Bonanno et al, 2005)
To facilitate rapid purification of proteins for structural genomics, we have developed the Protein Maker, a high-throughput parallel liquid-chromatography system that is capable of purifying up to 24 protein targets in a single unattended run (Fig. 1)
The same individual can process up to 40 proteins per week with the Protein Maker using the same protocols for individual sample lysis and staggered-loop consecutive injections for single-line size-exclusion chromatography
Summary
The end goal of structural genomics is the rapid generation of threedimensional structures obtained from atomic level resolution studies of pure proteins (Xiao et al, 2010; Elsliger et al, 2010; Watson et al, 2007; Bonanno et al, 2005). Suitable for a research environment (Walls et al, 2011), most standard instrumentation does not allow parallel purification or testing to investigate a large number of purification conditions simultaneously. To address this critical need on a structural genomics scale, much research and development has gone into the creation of pipelines designed to deliver the necessary high-quality materials at a rapid pace (Kim et al, 2008; Cymborowski et al, 2010; Stols et al, 2002; Dieckman et al, 2002; Steen et al, 2006). The result is the purification of multiple targets with identical or individualized buffer systems, decreasing the time required for purification while potentially increasing protein yields through time-efficient buffer optimization
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