Gst agarose beads

Human cDNA for PKM2, PKM1, and VDAC3 were separately cloned into a pET28a (+) and pGEX-4T-1 vector with an N-terminal His-tag and N-terminal GST-tag and purified from the Escherichia coli strain BL21 (Invitrogen) using Ni–agarose beads and GST agarose beads (Qiagen) as described previously^{7 (link)}. PKL was purchased from Sino Biological Inc. in China.

The GST-tag pull-down method was used to verify the interaction between BmRRS1 fusion protein and BV-eGFP. The constructed PGEX-4T-1-BmRRS1 was transformed into Escherichia coli BL21, and different final concentrations of IPTG (0,0.2 mM, 0.4 mM, 0.8 mM, 1 mM) were designed to induce the expression of BmRRS1 protein at 37 °C and 16 °C. The optimal final concentration and temperature were screened and the protein solubility was analyzed by ultrasonic crushing. The bacteria induced by IPTG were collected and the BmRRS1 protein was purified by pGEX-4T-1 prokaryotic expression vector and GST agarose beads (QIAGEN, Germany). The protein concentration was obtained by SDS-PAGE, BSA quantification and Image J 1.8.0 software analysis. BmRRS1 (300 ng) and BV-eGFP (viral titer 1 × 10⁸ pfu/mL) were incubated for 24 h at 4 °C on a shaker, followed by the addition of agarose beads to bind with the BmRRS1 fusion protein for 4 h at 4 °C. After washing three times with TNET buffer, proteins bound to GST agarose beads were eluted with elution buffer and detected by Western blotting. Fusion proteins were separated by SDS-PAGE and analyzed with rabbit anti-VP39 and mouse anti-GST. The pGEX-4T-1 vector was induced and purified according to the above method, and the purified GST protein was used as a negative control.