Alignments BI 2536 research buy of multiple protein sequences to view areas of conservation amongst A domains were performed using Clustal W http://www.ebi.ac.uk/ Generation of 3D-models for FnBPB (N23) types I-VII Theoretical models of the structure of
region A (N23) types I-VII were obtained by submitting the amino acid sequences for this segment of each protein to the Phyre service of the 3D-PSSM website http://www.sbg.bio.ic.ac.uk/phyre/. This web-based tool models the structure of these sequences based structure of the equivalent domains of the S. aureus clumping factor ClfA. All structures were viewed using the pyMOL viewing software. Expression of recombinant FnBPB A domain proteins Primers were designed to amplify DNA encoding residues 162-480 (N23 sub-domain) of FnBPB isotype I from strain 8325-4 by PCR. The primers included BamHI and SmaI restriction sites to facilitate cloning into the multiple cloning site of the N-terminal six-histidine tag expression
vector pQE30 (Qiagen) and incorporated a 3′ stop codon. The equivalent N23 regions of FnBPB isotypes types II-VII were PCR-amplified from strains N315, MSSA476, P1, 2, 3077 and 233, respectively. The PCR products were cloned separately into pQE30 and transformed into E. coli cells for protein production. Each construct was verified by sequencing (GATC Biotech AG, Germany) and proteins were purified by TSA HDAC Ni2+ chelate chromatography . Concentrations were determined using the BCA Protein Assay Kit (Pierce). Proteins were dialysed against PBS for 24 h at 4°C, aliquoted and stored at -70°C. Direct binding of recombinant FnBPB A domain proteins to immobilized elastin, fibrinogen
and fibronectin Human aortic elastin (Elastin Products Company; 50 μg/ml) was coated onto microtiter wells for 18 hr under UV light. Wells coated with human fibrinogen (Calbiochem; 10 μg/ml), and fibronectin (Calbiochem; 10 μg/ml) were placed at 4°C overnight. All plates were blocked with 5% skimmed milk in phosphate Cyclin-dependent kinase 3 buffered saline (PBS) for 2 hr at 37°C. Following three washes with PBS containing 0.05% v/v Tween 20 (PBST) various concentrations of purified rFnBPB N23 constructs in PBS were added and incubated at 37°C for 2 hr. After three washes with PBST, bound protein was detected by incubation with a 1:500 dilution of monoclonal antibody 7E8 that recognizes the N-terminal hexahistidine fusion tag. After 1 h incubation with shaking at room temperature, the wells were washed three times with PBST followed by 100 μl per well of goat-anti-mouse IgG antibodies conjugated to horseradish-peroxidase (HRP, Dako; Denmark) A-1210477 cost diluted 1:2000. After incubation for 1 h at room temperature, wells were washed three times with PBST, and bound HRP-conjugated antibodies were detected with 10 μg per well of 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma) in 0.05 M phosphate-citrate buffer containing 0.006% (v/v) hydrogen peroxide.