8) demonstrate that co-expression of cytFkpA during phage panning allowed selection of a greater number of sequence-unique clones with slower dissociation rates (kd values lower than 10− 3 1/s are represented by columns higher than the horizontal dashed line in Fig. 8). This effect was most pronounced for anti-kinase candidates from the scFv phage display kappa-only library where 8 of 26 clones selected via panning in the presence of cytFkpA had kd values lower than 10− 3 1/s, as compared
to none panned without cytFkpA. In studies reported here, we demonstrate that cytoplasmic expression of the periplasmic chaperone, peptidyl prolyl cis-trans isomerase, FkpA, improved the secretion of functional antibody fragments into the bacterial periplasm. The benefit of the cytoplasmically-expressed chaperone exceeded that of native FkpA expressed in the periplasm. We also report an improvement in periplasmic expression of functional Fab for all the tested mTOR inhibitor antibody fragments containing kappa light chains co-expressed with cytoplasmic FkpA. By comparison, cytoplasmic
expression of FkpA did not benefit secretion of antibody Trametinib fragments containing lambda light chains to the same degree. The results observed with antibody fragments containing kappa versus lambda light chains suggest that the PPIase activity of cytFkpA may play a major role in the enhancement of functional antibody fragment secretion into the periplasm since only kappa light chains carry cis-prolines. Pluckthun et al. have shown that co-expression of full-length FkpA in the periplasm can also improve the yields of scFv fragments without cis-prolines (i.e. lambda light chains), establishing the usefulness of the FkpA molecular chaperone domain ( Ramm and Pluckthun, 2000). Our results show that the molecular chaperone activity of cytSkp by itself is unable to improve secretion of functional ING1 and XPA23 Fab fragments, suggesting that the cis-trans peptidyl prolyl isomerase activity of cytFkpA in the E. coli cytoplasm is instrumental for these kappa antibody fragments prior to their transport into the oxidizing environment of the periplasm
where formation G protein-coupled receptor kinase of intra-chain and interchain disulphide bonds takes place. Thus, the cytoplasmic localization of the PPIase activity of cytFkpA could explain our results. In our studies, we employed leader peptides that enable periplasmic localization of antibody fragments through the Sec secretion pathway. The Sec route allows transmembrane transport of proteins into the periplasm in a loosely folded or unfolded, translocationally competent state or insertion into the cytoplasmic membrane through the SecYEG apparatus ( Natale et al., 2008, Dalal and Duong, 2011 and Zalucki et al., 2011). We can speculate that cytFkpA isomerizes key prolines of kappa light chains prior to the periplasmic export, and by doing so removes a folding bottleneck following the translocation of the heterologously expressed polypeptides into the periplasm.