FIG. 1.

Expression of His-BGLF4 in E. coli BL21(DE3) and generation of BGLF4-specific antiserum. (A) Total cell lysate from bacteria carrying vector control (pRSETA) or pSJC1 (His-BGLF4). Lysate is shown uninduced (lanes 1 and 3) and after induction with IPTG (lanes 2 and 4) and was displayed on a 10% PAGE gel and stained with Coomassie blue. The predicted molecular mass of BGLF4 is approximately 52 kDa, as indicated by the arrowhead. (B) After lysis with buffer (lane 2; total lysate), the cell lysate was fractionated into soluble proteins (lane 3) and pellet. Most of the BGLF4 product appeared in insoluble form in E. coli (lane 4). V, vector control. (C) The recombinant BGLF4 protein was dissolved in 8 M urea–5 mM imidazole–100 mM NaCl and purified using a nickel column. After being washed with binding buffer (lane 2) and 100 mM imidazole buffer (lane 3), the BGLF4 protein was eluted with buffer containing 300 mM imidazole (lane 4). F, flowthrough. (D) Western immunoblotting demonstrates the specificity of rabbit anti-BGLF4 antiserum. Total cell lysates of E. coli carrying pSJC1 (lane 1) or pRSETA (lane 2) and purified BGLF4 (lane 3) were displayed on an SDS–10% PAGE gel, transferred onto a membrane, and probed with rabbit anti-BGLF4 antiserum as described in Materials and Methods. M, molecular weight markers.