Figure 5
Purification of recombinant PiCV-Cap opt protein using on-column cleavage by thrombin and SP cation exchange chromatography. (A) The GST tag of recombinant GST-PiCV-Capopt protein was removed by the on-column cleavage method as described in Material and Method and the residue rCapopt were eluted and collected for the next purified procedure. The results of the purification were examined by SDS-PAGE. Lane M, pre-stained protein marker; lane 1, total protein-expressed cell lysate; lane 2, flow-through, lane 3, GST affinity column washing; lane 4, Tag-free rCapopt elute collected after on-column cleavage; lane 5, first column washing after on-column cleavage; lane 6, second column washing after on-column cleavage; lane 7 GST tag elute after on-column cleavage. (B) The rCapopt eluted protein was further purified by SP cation exchange chromatography and the purification result assessed by SDS-PAGE. Lane M, pre-strained protein marker; lane 1, Input fraction of rCapopt elute collected from a previous on-column cleavage purified step; lane 2 flow-through; lane 3, SP cation exchange column washing; lane 4, eluted fraction of purified rCapopt. (C) Identity of the rCapopt protein was determined by MALDI-TOF. The red-labeled marker represents actual amino acid matches and there is 27% protein sequence coverage.