The recent outpouring of human genome sequence and gene expression data has fundamentally changed drug discovery. The rate at which new molecular targets are being identified is increasing rapidly, and the ability to provide purified recombinant human proteins for high throughput screening will soon become a bottleneck in the identification of lead molecules. A key technical hurdle is the capability to rapidly screen multiple expression parameters to identify conditions for expression of properly folded human proteins in E. coli and baculovirus infected insect cells. Achieving this will require methods for rapidly quantifying the expression level of recombinant proteins in crude cell extracts. Accordingly, PanVera has undertaken development of a novel fluorescent method for quantifying affinity-tagged proteins; the extensively used polyhistidine motif (HisTag) will serve as a model system in Phase I. With the developments included in this Phase I proposal, this assay method will enable rapid and sensitive quantification of the absolute levels of any protein with a HisTag using a simple mix-and-read, multiwell format. The availability of such homogenous assays for quantification of diverse proteins will enhance the capabilities of individual investigators trying to isolate novel proteins and will accelerate industrial efforts to multiplex and automate protein expression methods.
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| Herrick, Dawn Z; Sterbling, Stephenie; Rasch, Katie A et al. (2006) Position of synaptotagmin I at the membrane interface: cooperative interactions of tandem C2 domains. Biochemistry 45:9668-74 |
