(485ap) Cell-Free Protein Synthesis with the Addition of Molecular Chaperones

Several advances have allowed E. coli based cell-free protein synthesis (CFPS) to become a reliable and economical method of protein production. While production of simple, native proteins and some classes of complex proteins has often been high, production of certain classes of heterologous proteins such as antibodies, T-cell receptors, and some membrane receptors have typically still been relatively low. We have thus tried to mimic the folding environment of the eukaryotic endoplasmic reticulum (ER) by controlling the chemical conditions and through the addition of several different molecular chaperones. We show that introducing these modifications to cell-free protein synthesis can help improve the folding and production of non-native, secreted proteins such as antibody fragments and the extracellular domains of T-cell receptors and the surface protein CD-19.

Addition of the Hsp70-family ER chaperone BiP helps increase the soluble production of these complex targets. Furthermore, we have modified BiP to bind to E. coli ribosomes, thereby promoting cotranslational folding much like its native function in the ER and show that this modification leads to further production improvements. Finally, we demonstrate that cell extracts expressing the chaperonin GroEL can help discourage intermolecular disulfide bond formation for CD-19 in a way that BiP cannot.