Construction, expression and characterization of TEV protease mutants engineered for improved solubility

Abstract

In recent years, the highly sequence specific tobacco etch virus protease (TEVp) has emerged as one of the most popular and widely used reagents for removal of fusion tags from target proteins. Its use, however, has been hampered due to relatively poor solubility and inefficient expression in E. coli. Although a lot of progress has been made, there is still need for new and improved TEVp variants. Recently, two different gain-of-function TEVp mutants were described; one containing the substitutions L56V/S135G, which conferred improved solubility and activity in vitro, while the other mutant, containing the substitutions T17S/N68D/I77V, was claimed to yield more soluble protease than the wild-type (wt) protease upon overexpression in E. coli. Here, we analyzed if the L56V/S135G substitutions could promote increased solubility also in vivo, as that would be beneficial to TEVp production and had never been investigated before. We also intended to create a novel, and hopefully superior, TEVp variant with all five mutations combined (T17S/L56V/N68D/S135G/I77V) in a single protease molecule. This variant and the two parental TEVp variants as well as the wt protease, were all expressed in E. coli and characterized with respect to the expression levels, solubility and activity using several different techniques; among them, a newly developed fluorescence-assisted whole-cell assay that directly reports on the apparent protease activity in vivo. Our results show that the L56V/S135G substitutions improve the solubility not only in vitro but also in vivo, which did hold true for the activity as well. Disappointingly, the protease variant containing all five substitutions (T17S/L56V/N68D/S135G/I77V) did not show the best performance, which instead the L56V/S135G variant did. In contrast to an earlier report, we show that the substitutions T17S/N68D/I77V, did not improve the TEVp solubility. In fact, they reduced the activity, and even appeared to have a slightly negative effect on solubility, of all protease constructs in which they were present. Thus, the best current and most promising TEVp variant for future protease engineering efforts, towards improved expression properties and enhanced catalytic efficiency, are those containing the L56V/S135G substitutions.QC 2011051