05% Surfactant P20 at pH 7.4 with 1 mg/ml BSA (Sigma)), and filtered through 0.22 μ multiscreen GV filter plates (Millipore). Filtered periplasmic extracts were injected over immobilized ligand for 3 min at 30 μl/min. Dissociation was followed for 10 min. The surface was regenerated following each analyte injection with 10 mM glycine at pH 1.7. Data was double referenced by subtracting the reference spot within the flow cell which was an activated and deactivated blank surface,

as well as subtracting out blank injections. Following referencing, the data were fit to a 1:1 dissociation model using Biacore 4000 evaluation software. To express Skp and FkpA in the E. coli cytoplasm, DNAs encoding these chaperones lacking their signal sequences and containing V5 and FLAG tags, respectively, were amplified by PCR from the Selleck BMS-936558 XL1-Blue E. coli chromosome. The gene products, designated

as cytSkp-V5 and cytFkpA-FLAG, were cloned into the l-arabinose-inducible expression vector, pAR3 ( Perez-Perez and Gutierrez, 1995) either separately ( Fig. 1a), or as a bicistronic gene sequence cyt[Skp + FkpA] encoding both cytFkpA and cytSkp http://www.selleckchem.com/products/Adriamycin.html ( Fig. 1b) for expression in the E. coli cytoplasm. Vectors were also constructed containing Skp and FkpA with their native signal sequences for expression in the E. coli periplasm ( Fig. 1a). Plasmids containing cytSkp-V5 and/or cytFkpA-FLAG, were transformed into E. coli TG1 cell cultures, grown to log phase, induced with l-arabinose, and periplasmic and cytoplasmic

extracts prepared. Western blot analysis using Inositol monophosphatase 1 anti-V5 and anti-FLAG tag antibodies verified that cytSkp and cytFkpA expressed on the same or separate plasmids were produced in the cytoplasm of TG1 cells ( Fig. 2, Lanes 3 and 5). Lower amounts of cytSkp and cytFkpA also were observed in an E. coli periplasmic extract ( Fig. 2, Lanes 2 and 4) which may be due to escape of the chaperones through the inner membrane during the generation of the extracts. The two bands that appear upon overexpression of cytSkp in E. coli ( Fig. 2b) could be attributed to an incomplete processing of Skp corresponding to the precursor and mature forms of Skp. Other scientists have previously demonstrated similar results when probing Skp using anti-Skp antisera ( Volokhina et al., 2011). We first tested the effect of co-expressing FkpA and Skp on secretion into the bacterial periplasm of Fabs containing kappa light chains. Initially, two human kappa Fabs, ING1 (anti-EpCAM) and XPA23 (anti-IL1β) and a murine anti-human insulin receptor kappa Fab, 83-7 (Soos et al., 1986) were expressed in TG1 cells in the presence or absence of cytoplasmically or periplasmically-expressed FkpA or Skp, either alone or in combination. The level of Fab in the periplasm capable of binding to EpCAM and IL1β was assessed by ELISA.