PPIases also prevent aggregation of antibody fragments ( Feige et al., 2010). Kappa light chain variable domains (Vκ) contain two conserved prolines in the cis conformation AG-014699 supplier at positions L8 and L95 ( Bothmann and Pluckthun, 2000) unlike the frameworks of heavy chain variable (VH) and lambda light chain variable (Vλ) antibody domains which, based on evaluation of sequences in the PDB database, do not
contain any cis-prolines ( Horne and Young, 1995). Cis-trans isomerization at Pro-L95 is a rate limiting step in the folding of Vκ domains and is essential for VL/VH docking and therefore for native protein conformation ( Suominen et al., 1987, Knappik and Pluckthun, 1995, Forsberg LY2109761 cost et al., 1997 and Ramm and Pluckthun, 2000). Interestingly, co-expression of the periplasmic Escherichia coli PPIase, FkpA, resulted in a significant improvement in secretion into the bacterial periplasm of functional scFv fragments containing either Vκ chains, which contain cis prolines, or Vλ chains which do not contain cis-prolines, suggesting that it has both molecular chaperone and PPIase enzymatic activities ( Bothmann and Pluckthun, 2000). Employing FkpA deletion mutants and functional assays, Saul et al. (2004) established that the FkpA carboxy and amino terminal domains carry independent PPIase and chaperone activities,
respectively. Previously, Missiakas et al. (1996) demonstrated that FkpA mafosfamide can act as a “global folding catalyst” that limits the levels of unfolded proteins in the outer membrane and periplasm. Periplasmic overexpression of FkpA facilitates the expression of multiple heterologous proteins, including an E. coli maltose binding protein misfolding mutant ( Arie et al., 2001), single-chain antibodies and antibody fusions ( Arie et al., 2001, Zhang et al., 2003, Padiolleau-Lefevre et al., 2006 and Sonoda et al., 2010). Another molecular chaperone in the E. coli periplasm is the 17 kDa Skp protein which forms a trimer with a central cavity. This cavity allows Skp to engulf native polypeptide substrates and prevents their subsequent aggregation (
Walton et al., 2009). Co-expression of Skp with a poorly soluble single chain Ab resulted in its secretion into the E. coli periplasm as well as improved solubility and phage display of the antibody fragment and diminished the toxicity of the antibody for the host cells ( Hayhurst and Harris, 1999). As observed with FkpA, other groups have demonstrated that co-expression of scFvs with Skp increased their secretion in E. coli ( Sonoda et al., 2010). Previously, it also was shown that overexpression of Skp lacking its signal sequence significantly improved the yield of a correctly folded Fab produced by a trxBgor mutant E. coli strain that enables the production of disulphide bonds in the bacterial cytoplasm ( Levy et al.