Ni nta sepharose resin

The full-length JEV-E glycoprotein (GP78 strain) gene was PCR amplified using primers JEV-E F (5′-CCGGGATCCATGGGCAATCGTGACTTC-3′) and JEV-E R (5′GCAAGCTTGATGTCAATGGCACATCCAGT-3). The 1280-bp product, corresponding to the full-length mature protein, was purified by gel extraction using a commercial kit (Qiagen, Germany) and was cloned into the pET28a vector (Qiagen, Germany) according to the manufacturer’s instructions. The ligated products were transformed into Escherichia coli BL21 (DE3) cells. Plasmids were isolated from 10 randomly selected clones (colonies appeared on agar plate were randomly picked up and grown overnight in 5 ml broth) and were tested for the presence of the insert by size determination on an agarose gel (1%) and PCR amplification of the target gene. Two clones positive for the insert in the correct orientation were subjected to double-pass sequencing to check for possible mismatches using the commercial services (Xcelris Labs, India) employing an automated sequencer. The expression of recombinant protein was induced for 6 hrs. at 25 °C in 250-ml LB medium culture containing 0.2 mM isopropyl-D-thiogalactopyranoside (IPTG). The over-expressed recombinant protein was purified to near-homogeneity by using Ni-NTA Sepharose resin (Qiagen, Germany) according to the manufacturer’s instructions.

Ni‐nitrilotriacetic acid resin (NTA) pull down assay was performed as previously described [31 (link)]. Briefly, cells were transfected with RH‐tagged plasmids and then lysed in lysis buffer. The protein samples were harvested and incubated with Ni‐NTA‐Sepharose resin (Qiagen, Dusseldorf, Germany) at 4 °C overnight, according to the manufacturer’s instructions. The resin was then continuously washed with washing buffer at room temperature. After washing, the RH‐tagged proteins were eluted in the elution buffer and subjected to WB.