Probond purification system

MBP, SLPA, and LacZ cDNA were introduced into the Champion™ pET Directional TOPO^® Expression Kit (Invitrogen, Carlsbad, CA, USA). BL21 Star™ (DE3) E. coli was transformed with the recombinant vectors, grown, and then their expression was induced by the addition of 1 mM IPTG into growth media according to manufacturer instructions. The recombinant His(6X)-tagged proteins were subsequently purified utilizing the ProBond™ Purification System (Invitrogen). The eluted protein was concentrated by PES, 3K MWCO (ThermoFisher Scientific, Waltham, MA, USA). Final concentration of protein was measured using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific).

The viral titer of the recombinant flagellin-Cap baculovirus was assayed and expressed as plaque forming unit (PFU) according to the standard protocol. Sf9 cells were infected with the recombinant virus followed by assaying protein expression at different harvest time intervals. The optimized harvesting time was 72 h postinfection. For protein production, Sf9 cells were infected with the recombinant baculovirus at a MOI of 1 PFU and harvested at the indicated time point. The expressed fusion protein was purified under denaturing conditions using the ProBond Purification System (Invitrogen, USA) in accordance with the manufacturer’s protocol. Briefly, the recombinant baculovirus-infected Sf9 cells for 72 h after infection were collected and centrifuged at 3,000 × g for 35 min. The cell lysates were resuspended in 8 ml of 6.0 M guanidine hydrochloride (pH 8.0) and applied to a ProBond Nickel-Chelating Resin with a His-tag for purification of recombinant fusion proteins. Prior to inoculation into mice, the purified proteins under denaturing conditions were further dialyzed against 0.5 M NaCl in 50 mM phosphaste buffered solution (PBS) (pH 7.4) with a step-wise reduction of urea concentrations. As a control, Sf9 cells were infected with a recombinant Cap baculovirus (35) and the purified recombinant Cap protein was included in animal experiments.

To obtain recombinant proteins NS2-Nt and NS2-Ct, recombinant baculoviruses (rBac) Bac-NS2-Nt and Bac-NS2-Ct were generated using the Bac-to-Bac system (Invitrogen, Barcelona, Spain), following the supplier protocols. Briefly, plasmid pSC11-NS2 containing the sequence coding for NS2 protein of BTV serotype 4 was used to amplify the sequence of NS2-Nt and NS2-Ct with specific primers (Table 2). The PCR products were then cloned into plasmid pFastBac1 (Invitrogen, Barcelona, Spain) digested with BamHI and NotI to obtain the transfer vectors HTA-NS2-Nt and HTA-NS2-Ct that were used to generate the recombinant baculoviruses that express the NS2-Nt or NS2-Ct BTV proteins in insect high five cells (H5), named Bac-NS2-Nt and Bac-NS2-Ct. These proteins were purified using the ProBond Purification System (Invitrogen, Barcelona, Spain) following the procedure indicated by the manufacturer for protein purification under native (NS2-Ct) or hybrid native-denaturing (NS2-Nt) conditions.

Another insect cell line High Five was used to produce large amount of recombinant proteins. High Five cells were cultured in Express Five serum-free medium (Invitrogen, Grand Island, NY, USA) supplemented with 5% FBS, 10 μg/ml gentamicin and 18 mM L-glutamine, and incubated in a non-humidified incubator at 28 °C. Optimal conditions for protein production were empirically established. Capillary western blot was used to detect recombinant R16/17 protein. Briefly, cell lysates with different MOIs were harvested at different time points. The expression of recombinant R16/17 protein was detected with a mouse anti-6xHis tag antibody (1:10, ab18184, Abcam) by ProteinSimple WES capillary western blot system according to a previously published method (Beekman et al. 2014 (link)). α-tubulin (mouse anti-α-tubulin antibody, 1:20, T5168, Sigma-Aldrich) was used as the loading control.
To scale up protein production, P2 viral stock was added to High Five cells in T175 flasks at a multiplicity of infection (MOI) of 20. Cells were harvested 3 days after infection, and the recombinant proteins were purified from cell lysates with the Probond purification system (Invitrogen, Grand Island, NY, USA). The identities of recombinant proteins were confirmed by western blot using anti-R17 and anti-His tag antibodies.

The Protean 3D program (DNASTAR, Madison, WI, USA) was used to predict that an 81-amino acid segment of HPgV-2 NS4A/4B (derived from GenBank accession number KT427414.1) would be localized to the cytoplasm. This region was chosen for expression in the pMAL-C5X vector, which, under the control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter, allowed for the addition of a maltose-binding protein (MBP) tag at the amino terminus and a 6-histidine tag at the carboxyl terminus (Genscript, Piscataway, NJ, USA). The construct was expressed in Escherichia coli strain BL21(DE3). Following IPTG induction for 4 h at 37°C, cells were lysed, and soluble protein was purified using the ProBond purification system (Invitrogen, Grand Island, NY, USA). Western blotting of the purified protein was performed using a WesternBreeze chromogenic kit (Invitrogen), and purified protein was detected using an anti-His antibody (Invitrogen). Protein was visualized using 5-bromo-4-chloro-3-indolylphosphate (BCIP)/nitroblue tetrazolium (NBT) staining (Novex by Life Technologies) and a Bio-Rad Gel Doc EZ imager, using Image Lab v4.0 software.