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

Aceti, David J.Bingman, Craig A.Wrobel, Russell L.Frederick, Ronnie O.Makino, Shin-ichiNichols, Karl W.Sahu, Sarata C.Bergeman, Lai F.Blommel, Paul G.Cornilescu, Claudia C.Gromek, Katarzyna A.Seder, Kory D.Hwang, SoyoonPrimm, John G.Sabat, GrzegorzVojtik, Frank C.Volkman, Brian F.Zolnai, ZsoltPhillips, George N.Jr.Markley, John L.Fox, Brian G.2016-08-302016-08-302015Aceti, David J., Bingman, Craig A., Wrobel, Russell L., et al.. "Expression platforms for producing eukaryotic proteins: a comparison ofﾠE. coliﾠcell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies." <i>Journal of Structural and Functional Genomics,</i> 16, no. 2 (2015) Springer: 67-80. http://dx.doi.org/10.1007/s10969-015-9198-1.https://hdl.handle.net/1911/91364Vectors 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.engThis is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Springer.Expression platforms for producing eukaryotic proteins: a comparison ofﾠE. coliﾠcell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategiesJournal articlehttp://dx.doi.org/10.1007/s10969-015-9198-1