Journal
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
Volume 59, Issue 3, Pages 633-643Publisher
WILEY
DOI: 10.1002/prot.20436
Keywords
NIH Protein Structure Initiative; structural genomics; X-ray crystallography; NMR spectroscopy; screening; protein folding; protein solubility
Categories
Funding
- NIGMS NIH HHS [1 P50 GM64598] Funding Source: Medline
Ask authors/readers for more resources
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 N-15-labeled proteins to be analyzed by H-1-N-15 NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-firee 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, NAIR sample too dilute. Those targets that reached the goal of analysis by H-1-N-15 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-firee 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. (c) 2005 Wiley-Liss, Inc.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available