EXPRESSION OF BIOLOGICALLY ACTIVE PROTEINS IN A BACTERIAL CELL-FREE SYNTHESIS SYSTEM USING CELL EXTRACTS WITH ELEVATED LEVELS OF EXOGENOUS CHAPERONES

摘要

The present disclosure describes methods and systems for improving expression of properly folded biologically active proteins of interest in cell-free synthetic systems. The methods and systems utilize bacterial cell-free extracts with an active oxidative phosphorylation system and include exogenous protein chaperones. Exogenous protein chaperones can be expressed by bacteria used to prepare cell-free extracts. The exogenous protein chaperone may be a protein disulfide isomerase and/or a peptidyl-prolyl cis-trans isomerase. The inventors have found that the combination of protein disulfide isomerase and peptidyl-prolyl cis-trans isomerase results in a synergistic increase in the amount of properly folded biologically active protein of interest.