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Comparison of cell‐based and cell‐free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies
Author(s) -
Tyler Robert C.,
Aceti David J.,
Bingman Craig A.,
Cornilescu Claudia C.,
Fox Brian G.,
Frederick Ronnie O.,
Jeon Won Bae,
Lee Min S.,
Newman Craig S.,
Peterson Francis C.,
Phillips George N.,
Shahan Mark N.,
Singh Shanteri,
Song Jikui,
Sreenath Hassan K.,
Tyler Ejan M.,
Ulrich Eldon L.,
Vinarov Dmitriy A.,
Vojtik Frank C.,
Volkman Brian F.,
Wrobel Russell L.,
Zhao Qin,
Markley John L.
Publication year - 2005
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20436
Subject(s) - arabidopsis thaliana , genome , computational biology , cell , biology , microbiology and biotechnology , genetics , gene , mutant
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 15 N‐labeled proteins to be analyzed by 1 H‐ 15 N 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 1 H‐ 15 N 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. Proteins 2005. © 2005 Wiley‐Liss, Inc.