Simple Cell-Free Extract for Expression of Disulfide Bonded Proteins without Addition of Exogenous Polymerase and Chaperones

Cell-free expression of proteins with multiple disulfide bonds using E. coli extract remains a challenge due to the increased cost of purchasing specialized extracts and the extra steps required to supplement a modified E. coli strain (KGK10) with exogenous polymerase (T7RNAP) and chaperones (DsbC). In this work, we present a facile method to produce extract from a readily available, commercial strain (T7 SHuffle®, New England Biosciences ::DsbC Δgor Δtrxb) that does not require supplementation of T7RNAP or chaperones. Using face centered cubic design of experiments (DoE) [1], we determined optimal times of IPTG induction and harvest using Luciferase from Gaussia princeps (Gluc) and super folder GFP (sfGFP) as reporter proteins for disulfide bond formation and overall expression efficiencies, respectively. Interestingly, the optimum extract conditions for disulfide formation (Gluc expression) were different than those for overall expression (sfGFP). These were found to be 252 min to IPTG induction and 344 min to harvest when growing 1 L of culture in a 2.5 L Tunair shake flask using a 20 mL overnight seed culture. To further optimize the expression system, we explored the effect of altering extract concentration. Using another designed experiment, we found that diluting the extract 3-fold, while also diluting iodoacetamide to 30mM, lead to a 141% increase in luminescence. We also explored the addition of exogenous T7 RNAP and DsbC but found both to have a negative impact on Gluc expression. Using the 3-fold diluted extract, we expressed 3 enzymes with disulfide bonds (hevamine, endochitinase A, and AppA) using mail-order, minimal DNA templates that were amplified with isothermal rolling circle amplification [2].

[1] J.L. Dopp, N.F. Reuel, Biochem. Eng. J. 138 (2018) 21–28. doi:10.1016/j.bej.2018.06.021.

[2] J.L. Dopp, S.M. Rothstein, T.J. Mansell, N.F. Reuel, Bioeng. 116 (2019) 667–676. doi:10.1002/bit.26912.