Comparison of cell-based and cell-free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies. Proteins 2005 May 15;59(3):633-43
Date
03/25/2005Pubmed ID
15789406DOI
10.1002/prot.20436Scopus ID
2-s2.0-20944435522 (requires institutional sign-in at Scopus site) 56 CitationsAbstract
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.
Author List
Tyler RC, Aceti DJ, Bingman CA, Cornilescu CC, Fox BG, Frederick RO, Jeon WB, Lee MS, Newman CS, Peterson FC, Phillips GN Jr, Shahan MN, Singh S, Song J, Sreenath HK, Tyler EM, Ulrich EL, Vinarov DA, Vojtik FC, Volkman BF, Wrobel RL, Zhao Q, Markley JLAuthors
Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of WisconsinBrian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
ArabidopsisArabidopsis Proteins
Cell-Free System
Cloning, Molecular
Crystallography, X-Ray
Escherichia coli
Genome, Plant
Magnetic Resonance Spectroscopy
Seeds
Triticum
