Drosophila expression system

AgBR1 and NeSt1 were previously cloned in-frame into the pMT-Bip-V5-His tag vector (Invitrogen) [21 ] and recombinant proteins expressed and purified using the Drosophila Expression System (Invitrogen). AgBR1 and NeSt1 recombinant proteins were purified from the supernatant by TALON metal affinity resin (Clontech) and eluted with 150 mM imidazole. The eluted samples were filtered through a 0.22-μm filter and concentrated with a 10-kDa concentrator (Sigma-Aldrich) by centrifugation at 4°C and washed and dialyzed in PBS. Recombinant protein purities were assessed by SDS-PAGE, and then quantified using the Pierce BCA Protein Assay kit (Thermo Scientific). To generate rabbit sera against recombinant proteins, rabbits were immunized subcutaneously with 100 μg of recombinant proteins in complete Freund’s adjuvant and boosted three times at 2 week-intervals with 50 μg of recombinant proteins in incomplete Freund’s adjuvant. Rabbits were euthanized 2 weeks after the final boost and serum was collected by cardiac puncture. Reactivity to recombinant proteins was verified by immunoblot.

Total RNA was isolated from the salivary glands of fed I. scapularis ticks using Trizol (Life Technologies, Inc) and cDNA was synthesized according to the manufacture’s protocol (iScript cDNA synthesis kit, Bio-RAD). Gene-specific primers were used to amplify the salp14 and the amplicon was cloned into pMT-Bip-V5-HisA vector. To validate the clones, the recombinant DNA was sequenced at the Keck sequencing facility, Yale University. Recombinant Salp 14 protein was generated using the Drosophila expression system according to the manufacturer’s protocol (Invitrogen, CA) and as already described for tick salivary proteins [20 (link), 43 (link), 44 (link)]. The purity of the recombinant protein was assessed by SDS-PAGE using 4–20% gradient precast gels (Biorad, CA) and quantified using the BCA protein assay kit (Thermo Scientific, MA).

Recombinant mPF4 or hPBP was prepared as described(28 (link)). The mPF4 and hPBP were isolated from BL21DE30 pLysS bacterial cells. These proteins were further purified by fast protein liquid chromatography using a Resource RPC FPLC column (Amersham Pharmacia Biotech). Purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. An immunoblot after electrotransfer to polyvinylidenedifluoride was used to confirm the identity of the proteins.
Human PF4 was generated as previously described from S2 insect cells(29 (link)). Briefly, cDNA encoding hPF4 was cloned into the plasmid pMT/BiP/V5-His A (Invitrogen) for expression in Drosophila Expression System (Invitrogen). Expression of hPF4 was induced by adding copper sulfate to 0.5 mM. Induced cells were incubated in Insect-Xpress (Lonza Walkersville) media for 3–5 more days; supernatant collected, sodium azide (0.02% final concentration) and ethylenediaminetetraacetic acid (EDTA, 2.5 mM final concentration) added, and the media filtered through an Express Plus 0.22-μm filter (Millipore). The hPF4 was purified from the media on a heparin HiTrap column on an ATKA Prime (GE Healthcare) at 4°C in 10 mM Tris, 1 mM EDTA, pH 8.0 buffer as described(29 (link)).

Human fibrinogen depleted of plasminogen, von Willebrand factor and fibronectin (FIB 3), human plasmin, human thrombin, and HRP-conjugated sheep anti-human fibrinogen antibodies were purchased from Enzyme Research Laboratories. The soluble form of human VLDL receptor, sVLDLR, was prepared using the Drosophila Expression System (Invitrogen) as previously described [23 (link)]. The recombinant fibrin(ogen) (Bβ1–66)₂ and (β15-66)₂ fragments were produced in E. coli and purified as we described earlier [23 (link)]. Human receptor-associated protein (RAP) was expressed in E. coli and purified as described in [24 (link)]. Anti-VLDL receptor monoclonal antibodies mAb 5F3 and mAb 1H10 were purified from hybridoma supernatants by affinity chromatography on Protein A-Sepharose (Sigma-Aldrich) as we described earlier [21 (link)]. Goat secondary anti-mouse polyclonal antibodies conjugated with HRP and HRP substrate SureBlue TMB were from KPL. Calcein AM fluorescent dye and phorbol 12-myristate 13-acetate (PMA) were obtained from BD Biosciences and Promega, respectively, and Gly-Pro-Arg-Pro peptide and N-formyl-Met-Leu-Phe (fMLP) were from Sigma-Aldrich.