We designed six bispecific expression cassettes based on three CBM variants fused to either the N- or C- terminus of HVHP428 and characterized these fusion proteins. One format, consisting of an N-terminal CBM9 fusion (CBM9-VTB-HVHP428), was expressed and purified in soluble cell assay form from E. coli with only a small fraction prone to degradation. This bispecific pentamer was capable of binding to cellulose-based filters through the pentameric CBM and also retained its ability to agglutinate S. aureus cells through the pentameric sdAb. Furthermore, cellulose Inhibitors,Modulators,Libraries filters containing immobilized CBM9-VTB-HVHP428 were capable of detecting S. aureus cells to a limit of detection of 105 Inhibitors,Modulators,Libraries cfu/mL (p = 0.05) when used in a vacuum flow-through assay.Figure 1.Overview of fusion protein construction and characterization.
2.?Materials and Methods2.1. Cell Lines and PlasmidsAll bacterial cloning and expression was conducted in the pVT1 expression vector  using electrocompetent TG1 Escherichia Inhibitors,Modulators,Libraries coli from New England Biolabs (Mississauga, ON, Canada). For yeast cloning, constructs were assembled in the pPICZ��A vector and expression was conducted Inhibitors,Modulators,Libraries in Pichia pastoris strain X-33 that was purchased from Invitrogen (Mississauga, ON, Canada). S. aureus (ATCC12598) was purchased from American Type Culture Collection (Manassas, VA, USA).2.2. Cloning and Expression of sdAb-CBM FusionsThe human sdAb HVHP428 , which specifically recognizes S. aureus protein A, and three cellulose-binding modules: (i) CBM2a , (ii) CBM2a(m), a CBM2a mutant devoid of five putative N-glycosylation sites  and codon-optimized for P.
GSK-3 pastoris (JCH, unpublished data), and (iii) CBM9 , were used. DNA encoding CBM2a and CBM9 were gifts from Dr. C.A. Haynes, University of British Columbia. A total of fourteen constructs were assembled with various combinations and orientations of HVHP428, CBM, or both. Of these, ten were expressed in either E. coli or P. pastoris. Briefly, CBM and sdAb genes were amplified by PCR and flanked with DNA encoding linker sequences and restriction sites BbsI/ApaI or BsiWI/ApaI for N-terminal fusions and BspEI/BamHI for C-terminal fusions. PCR primers for gene amplification and introduction of restriction sites for ligation are summarized in Table 1. Three linkers designated L1, L2, and L3, which separate the CBM, VTB, and HVHP428 domains, were the same linkers as those used for a clone in a previously described bispecific pentamer .
Amplified insert DNA was digested with the corresponding restriction enzymes and ligated into a similarly digested pVT1 vector. One clone, HVHP428-VTB-CBM2a(m), KPT-330 solubility which contained a CBM devoid of the putative N-glycosylation site A-X-T/S (X = any amino acid except P), was subcloned into the pPICZ��A expression vector from pVT1 and transformed into chemically competent P.