Wheat germ cell-free expression system for protein production.

The Center for Eukaryotic Structural Genomics, in cooperation with Ehime University and CellFree Sciences, has developed a novel wheat germ cell-free technology for the production of eukaryotic proteins. Protein production and purification are robust and scalable for high-throughput applications. The protocols have been used to express and purify proteins from Arabidopsis thaliana, human, mouse, rat and zebra fish. This unit describes expression and purification protocols for both small-scale testing (microgram) and large-scale production (milligram) of N-His6- and N-GST-tagged proteins. The methods described in this unit can be used to produce both unlabeled and labeled proteins required for structure-based determinations by NMR spectroscopy or X-ray crystallography.

Comparison of cell-based and cell-free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies.

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N-labeled proteins to be analyzed by 1H-15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H-15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+/- (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-free platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination.