Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies

Autor: Claudia C. Cornilescu, Zsolt Zolnai, Brian F. Volkman, George N. Phillips, Karl W. Nichols, Sarata C. Sahu, Craig A. Bingman, Ronnie O. Frederick, Frank C. Vojtik, John L. Markley, Russell L. Wrobel, Shin-ichi Makino, Paul G. Blommel, Grzegorz Sabat, Soyoon Sarah Hwang, David J. Aceti, John G. Primm, Lai F. Bergeman, Katarzyna A. Gromek, Kory D. Seder, Brian G. Fox
Rok vydání: 2015
Předmět:
Zdroj: Journal of Structural and Functional Genomics. 16:67-80
ISSN: 1570-0267
1345-711X
Popis: Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.
Databáze: OpenAIRE
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