As observed by ELISA (Fig. 4), expression of the CF1 kappa Fab benefited to a lesser extent (1.7 to 2-fold) from expression of cytFkpA. A tricistronic vector (Fig. 1b) was developed

for co-expressing the ING1 Fd and light chains in the periplasm along with cytFkpA under control of the lac promoter. Western blot analysis confirmed that most of the cytFkpA was expressed in the cytoplasm (data not shown). Accumulation of total and functional Fabs in the periplasm, assessed by expression and target ELISAs, was improved when co-expressed MI-773 molecular weight with cytFkpA ( Fig. 6a), thus establishing the usefulness of incorporating cytFkpA along with Fd and light chains as a tricistronic unit in the expression vector. We also confirmed by SPR that total periplasmic ING1 Fab was increased by co-expressing with cytFkpA from a single vector in the E. coli cytoplasm ( Fig. 6b). Yields of periplasmic soluble Fab ranged from 0.4 to 2.45 μg/ml without cytFkpA

and 3.5–14.2 μg/ml in the presence of cytFkpA. Since co-expression of cytFkpA enhances expression in the E. coli periplasm of functional Fabs with kappa (and some lambda) light chains, we examined the effects of co-expressing cytFkpA on selection of antigen-specific Fab or scFv fragments from naïve phage display libraries. Three rounds of phage panning were performed with biotinylated target (kinase) using a large kappa scFv library ( Schwimmer et al., 2013). Following the third round of panning, Z-VAD-FMK order clones were picked for evaluation of scFv expression in the periplasm. Periplasmic extracts were also tested for binding to kinase. Panning was performed with or without expression of cytFkpA from a separate arabinose-inducible vector (pAR3) containing a p15A origin of replication

which is compatible with the library phagemid vector that carries tuclazepam the lac promoter and harbors the ColE1 origin of replication. Ninety three output clones were selected after the third round of phage panning performed with or without cytFkpA expression. While scFv clones selected from panning campaigns without cytFkpA were induced only with IPTG, clones selected from panning with cytFkpA also were induced with l-arabinose to allow cytFkpA expression. The amount of functional scFv in the bacterial periplasmic extracts in the absence and presence of cytFkpA was assessed by ELISA. Overexpression of cytFkpA significantly increased both the frequency and expression levels of sequence-unique clones obtained by panning with a scFv phage display library containing kappa light chains (Table 2). Only 10% of the output clones selected from panning without cytFkpA were sequence-unique and antigen-specific, with an ELISA signal (OD450) greater than 3-fold over the background, compared to 48% of clones selected when cytFkpA was co-expressed. Thus, the diversity of the selected kinase-binding clones, as defined by the number of sequence-unique clones and their expression levels, was greatly improved in the presence of cytFkpA.