Strep-Tactin Superflow Plus (10 ml)

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Preparation of NV replicase (NV3Dpol): Plasmid pVL3Dwt (GenBank: AB039782 [19]) harbouring NV3Dpol gene was kindly provided from BML Inc. PCR was performed with KOD-plus- DNA polymerase (TOYOBO) and the PCR products were purified with QIAquick PCR Purification Kit (QIAGEN). NV3Dpol gene was amplified from pVL3Dwt by PCR using prNV3Dstart(+) 5′-ATGGGAGGTGACGACAAGGGC-3′ and prNV3Dstop(-) 5′-TTATTCGACGCCATCTTCATTCACA-3′. NV3Dpol gene was modified with the coding region for Strep-tag II sequence (WSHPQFEK) [21] using prNV3Dstart(+) and prNV3D-strep(-) 5′-GCATCGACTCCTTACTTTTCAAACTGCGGATGGCTCCATTCGACGCCATCTTCATTC-3′ (the sequence showed in italics indicates stop codon and Strep-tag II coding sequence) by PCR. At the downstream of the stop-codon, KpnI restriction site was added using prNV3Dstart(+) and prStrep-kpn1(-) 5′-GGGGTACCTTACTTTTCAAACTGCGGATGGCTCC-3′ (the sequence showed in italics indicates KpnI cutting site) by PCR. Then the PCR product was digested with KpnI (TaKaRa) and integrated into the pTD1 expression vector (SHIMADZU Biotech) [23] according to the manufacture’s instruction. The recombinant plasmid was named pTD-NV3Dpol-strep. NV3Dpol-strep expression construct DNA was amplified using prTD161-179 (5′-GCAGATTGTACTGAGAGTG-3′) and prTD845-827 (5′-GGAAACAGCTATGACCATG-3′), and transcribed in vitro with RiboMAXTM Large Scale RNA production system-T7 (Promega). The transcript, named NV3Dpol-strep mRNA was purified with NICK column (GE Healthcare). NV3Dpol-strep mRNA was translated with Transdirect insect cell cell-free protein synthesis kit (SHIMADZU Biotech) [28] according to the manufacture’s instruction.
The translated product was purified with Strep-tactin Superflow plus (QIAGEN). The strep-tag II modified NV3Dpol was bound onto Strep-tactin Superflow plus resin, pre-equilibrated with binding buffer (50 mM Tris–HCl [pH 8.0], 300 mM NaCl). The bound protein was washed with the binding buffer and eluted with elution buffer (50 mM Tris–HCl [pH 8.0], 300 mM NaCl, and 2.5 mM desthiobiotin (Sigma)). The eluted protein was then enriched and buffer-exchanged with buffer A (25 mM Tris–HCl [pH 8.0], 100 mM NaCl, 5 mM MgCl2, and 1 mM β-mercaptoethanol) with Microcon YM-50 column (Millipore), and stored at -80°C. In the enrichment step using Microcon YM-50 column, bovine serum albumin (BSA) (TaKaRa) was used as a carrier protein to avoid the non-specific adsorption of NV3Dpol to the column. The amount of NV3Dpol-strep was quantified on sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) referring a calibration curve with BSA.

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