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

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dc.citation.firstpage67en_US
dc.citation.issueNumber2en_US
dc.citation.journalTitleJournal of Structural and Functional Genomicsen_US
dc.citation.lastpage80en_US
dc.citation.volumeNumber16en_US
dc.contributor.authorAceti, David J.en_US
dc.contributor.authorBingman, Craig A.en_US
dc.contributor.authorWrobel, Russell L.en_US
dc.contributor.authorFrederick, Ronnie O.en_US
dc.contributor.authorMakino, Shin-ichien_US
dc.contributor.authorNichols, Karl W.en_US
dc.contributor.authorSahu, Sarata C.en_US
dc.contributor.authorBergeman, Lai F.en_US
dc.contributor.authorBlommel, Paul G.en_US
dc.contributor.authorCornilescu, Claudia C.en_US
dc.contributor.authorGromek, Katarzyna A.en_US
dc.contributor.authorSeder, Kory D.en_US
dc.contributor.authorHwang, Soyoonen_US
dc.contributor.authorPrimm, John G.en_US
dc.contributor.authorSabat, Grzegorzen_US
dc.contributor.authorVojtik, Frank C.en_US
dc.contributor.authorVolkman, Brian F.en_US
dc.contributor.authorZolnai, Zsolten_US
dc.contributor.authorPhillips, George N.Jr.en_US
dc.contributor.authorMarkley, John L.en_US
dc.contributor.authorFox, Brian G.en_US
dc.date.accessioned2016-08-30T20:50:15Zen_US
dc.date.available2016-08-30T20:50:15Zen_US
dc.date.issued2015en_US
dc.description.abstractVectors 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.en_US
dc.identifier.citationAceti, 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.en_US
dc.identifier.doihttp://dx.doi.org/10.1007/s10969-015-9198-1en_US
dc.identifier.urihttps://hdl.handle.net/1911/91364en_US
dc.language.isoengen_US
dc.publisherSpringeren_US
dc.rightsThis is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Springer.en_US
dc.titleExpression platforms for producing eukaryotic proteins: a comparison ofﾠE. coliﾠcell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategiesen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpost-printen_US
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