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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. J Struct Funct Genomics 2015 Jun;16(2):67-80

Date

04/10/2015

Pubmed ID

25854603

Pubmed Central ID

PMC4430420

DOI

10.1007/s10969-015-9198-1

Scopus ID

2-s2.0-84937761852   6 Citations

Abstract

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.

Author List

Aceti DJ, Bingman CA, Wrobel RL, Frederick RO, Makino S, Nichols KW, Sahu SC, Bergeman LF, Blommel PG, Cornilescu CC, Gromek KA, Seder KD, Hwang S, Primm JG, Sabat G, Vojtik FC, Volkman BF, Zolnai Z, Phillips GN Jr, Markley JL, Fox BG

Author

Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Cell-Free System
Cloning, Molecular
Escherichia coli
Eukaryota
Gene Expression
Genetic Vectors
Germ Cells
Protein Biosynthesis
Proteins
Triticum
jenkins-FCD Prod-444 eb4ebd1a08581aba961d3befd3b851a3c3ec6b46