Publication protocol
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.
Full paper
Login or
join for free to view the full paper.
Videos
Check out videos that might be relevant for performing Protein tag Purification of Strep-tagged proteins using Strep-Tactin Superflow Plus (10 ml) from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.