One-Pot E. coli Cell-Free Extract for in vitro Expression of Disulfide Bonded Proteins

Abstract

In vitro expression of proteins from E. coli extract is a useful method for prototyping and production of cytotoxic or unnatural products. However, proteins that have multiple disulfide bonds require custom extract that, to date, requires careful addition of exogenous isomerase enzymes or the use of expensive commercial kits. This cost and complexity currently limit access to some groups who wish to rapidly prototype proteins with disulfide bonds. Herein, we present a simple solution that does not require addition of supplemental enzymes. We use a commercially available SHuffle T7 Express lysY strain of E. coli that expresses both T7 RNAP and DsbC isomerase enzymes. We experimentally determine optimal growth conditions (IPTG induction and harvest times) to balance overall productivity and efficiency of disulfide bond formation using a luciferase (from Gaussia princeps) that contains five disulfide bonds as our reporter protein. We also demonstrate the ability for rapid prototyping by screening the activity of four luciferase candidates against ten luciferin analogues. To display the broad applicability of the extract, three other enzymes containing [≥]3 disulfide bonds (hevamine, endochitinase A, and periplasmic AppA) were also expressed from minimal genetic templates that had undergone rolling circle amplification and confirmed via activity assays.