Hitrap talon crude

The ORFs of BoHSP90-A and BoHSP90-B were amplified from the cDNA and subcloned into the pET-32a vector using the EcoRI and XhoI restriction sites. The resulting plasmids pET-32a/BoHSP90-A and pET-32a/BoHSP90-B were transformed into the E. coli BL21™ (DE3) strain (Transgene, China) using the standard method. The plasmids from bacterial cells at each step were extracted using QIAGEN Plasmid Mini Kit (Qiagen, Hilden, Germany) and subjected to restriction enzyme digestion and sequencing analysis in order to verify successful cloning and the orientation of genes in the vector. The proteins were expressed in E. coli by inducing with 1 mM isopropyl β-D-thiogalactoside (IPTG). The rBoHSP90-A and rBoHSP90-B were expressed as His-fusion proteins and their expressions were monitored through SDS-PAGE. The uninduced plasmids pET-32a/BoHSP90-A and pET-32a/BoHSP90-B and IPTG induced empty vector (pET-32a) were used as negative controls (Figure 1).
The rBoHSP90-A and rBoHSP90-B were expressed in the insoluble-fraction of E. coli and were purified using HiTrap TALON crude (GE Healthcare, Sweden). The affinity chromatography was performed according to the manufacturer’s instructions. The quantities of recombinant proteins were measured by BCA protein assay kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions.

Plasmids encoding cDNAs for RSV subgroup A strain A2 F protein (wild-type post-fusion lacking the signal peptide and transmembrane domain), the SC-TM construct and the subgroup B strain 18537 protein construct (wild-type post-fusion F protein lacking the signal peptide and transmembrane domain) were synthesized (Genscript). The RSV B 18537 Ds-Cav1 (pre-fusion) construct was a gift from B. Graham (NIH). Plasmids were expanded in E. coli DH5α cells and DNA was purified using Qiagen Plasmid Maxiprep kits (Qiagen). For each litre of transfected culture, 1.3 mg of plasmid DNA was mixed with 2 mg of polyethylenimine in Opti-MEM I + GlutaMAX cell culture medium (Fisher). After 10 min, the DNA mixture was added to HEK293 cells at 1 × 10⁶ cells per ml. The culture supernatant was collected after 6 days and the protein was purified by a HiTrap Talon crude (GE Healthcare Life Sciences) column for RSV F protein variants and mutants. Expression and purification of mAbs 101F, motavizumab and D25 have been described previously^{14 (link)}. Commercial preparations of palivizumab (Synagis; Medimmune) were obtained from the pharmacy at Vanderbilt University Medical Center.