Automated, high-throughput platform for protein solubility screening using a split-GFP system.
Overproduction of soluble and stable proteins for functional and structural studies is a major bottleneck for structural genomics programs and traditional biochemistry laboratories. Many high-payoff proteins that are important in various biological processes are "difficult to handle" as protein reagents in their native form. We have recently made several advances in enabling biochemical technologies for improving protein stability (http://www.lanl.gov/projects/gfp/), allowing stratagems for efficient protein domain trapping, solubility-improving mutations, and finding protein folding partners. In particular split-GFP protein tags are a very powerful tool for detection of stable protein domains. Soluble, stable proteins tagged with the 15 amino acid GFP fragment (amino acids 216-228) can be detected in vivo and in vitro using the engineered GFP 1-10 "detector" fragment (amino acids 1-215). If the small tag is accessible, the detector fragment spontaneously binds resulting in fluorescence. Here, we describe our current and on-going efforts to move this process from the bench (manual sample manipulation) to an automated, high-throughput, liquid-handling platform. We discuss optimization and validation of bacterial culture growth, lysis protocols, protein extraction, and assays of soluble and insoluble protein in multiple 96 well plate format. The optimized liquid-handling protocol can be used for rapid determination of the optimal, compact domains from single ORFS, collections of ORFS, or cDNA libraries.
Listwan
Pawel
P
Bioscience Division, MS-M888, Los Alamos National Laboratory, Bikini Atoll Rd, SM30, Los Alamos, NM 87545, USA.
Terwilliger
Thomas C
TC
Waldo
Geoffrey S
GS
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012371:Robotics / Q000379:methods*; D012995:Solubility
D011506:Proteins; D049452:Green Fluorescent Proteins