Interestingly, soluble expression of a 6XHis-Halo-PKA fusion protein, while expressing at higher levels than the initial construct (Fig. ), did not elevate soluble protein levels as hoped. As a result, we further speculated that the presence of an N-terminal 6XHis tag might also be deleterious to protein expression. Indeed, subsequent expression tests of a fusion containing a C-terminal 6XHis tag (Halo-PKA-6XHis) rather than an N-terminal 6XHis tag exhibited improved overall protein expression significantly over the 6XHis-MBP-PKA-Halo and 6XHis-Halo-PKA fusions (Fig. ). After identifying this lead construct (Halo-PKA-6XHis), we then sought to further optimize its expression in E. coli. Initial attempts at an osmolyte-based approach did not result in enhanced soluble protein expression (Supporting Figure ). However, these experiments identified a significant amount of leaky expression in the uninduced culture, with even more fusion protein present in the uninduced culture than the induced cultures. We hypothesized that leaky expression in overnight starter cultures was detrimental to larger scale protein expression, and that inhibition of this expression would improve soluble expression of the construct. We subsequently investigated the impacts of incorporating dextrose (0.2% w/v) into overnight E. coli starter cultures; this greatly improved soluble protein expression in the expanded cultures that followed. We also investigated the impacts of time and temperature on soluble protein expression, as summarized in Supporting Figure . Whereas previously we had experienced significant problems with expression and purification of our Halo-PKA fusions (Supporting Figure ), these optimizations resulted in a reliable expression procedure that could be readily purified in appreciable quantities (Supporting Figure ; 4 -5 mg of purified Halo-PKA protein recovered following IMAC and FPLC purification steps) from 500 mL expression cultures.