Academic
Publications
Automated, high-throughput platform for protein solubility screening using a split-GFP system

Automated, high-throughput platform for protein solubility screening using a split-GFP system,10.1007/s10969-008-9049-4,Journal of Structural and Func

Automated, high-throughput platform for protein solubility screening using a split-GFP system   (Citations: 6)
BibTex | RIS | RefWorks Download
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.
Journal: Journal of Structural and Functional Genomics , vol. 10, no. 1, pp. 47-55, 2009
Cumulative Annual
View Publication
The following links allow you to view full publications. These links are maintained by other sources not affiliated with Microsoft Academic Search.
    • ...We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]...
    • ...The split-GFP technology developed in this laboratory [12‐16] has recently been used to develop an automated, HTP solubility screening assay, allowing us to process and screen thousands of protein constructs for solubility in a few days [1]...
    • ...Our integrated, high-throughput robotic system has previously been described [1]...
    • ...For the quantification of the soluble protein fraction, a set of eight different concentrations of the soluble GFP S11 tagged control protein, sulfite reductase, was obtained by serial dilution and used to generate a calibration curve as previously described [1]...

    Pawel Listwanet al. The optimization of in vitro high-throughput chemical lysis of Escheri...

Sort by: