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

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.

Fig. 1

Maps of expression vectors used in this study. pVP16, pVP65, pVP68K and pVP91A are used for E. coli expression. pEU-His and pEU-His-FV are used for wheat germ cell-free translation

Fig. 2

Performance of Escherichia coli vectors pVP16, pVP68K and pVP65K in an X-ray crystallography production pipeline. Twenty-one ORFs were tested in three vectors for production of protein suitable for structure determination. “NA” is Not Attempted

Fig. 3

Performance of three Escherichia coli vectors in production of ¹⁵N-labeled proteins. Ten proteins from the control workgroup with molecular weights suitable for NMR structure determination were tracked for progress along the production pipeline described in Materials and Methods. Purified proteins were subjected to ¹H-¹⁵N NMR HSQC analyses. “NA” is Not Attempted

Fig. 4

Performance of pEU-His-FV in production of ¹⁵N-labeled proteins by wheat germ cell-free translation. Targets with high or medium ratings in Small-Scale Screening, as described in Materials and Methods, were advanced to large-scale production, purification, and ¹H-¹⁵N HSQC screening