Effects of pH on Purification of GFPuv/Cytochrome c-552 Fusion Protein

Fusion gene of GFPuv and Cytochrome c-552 was inserted into the pTrcHis B vector and transferred to E. coli. A fusion protein of GFPuv and Cytochrome c-552 was expressed in BL21. This fusion protein was composed of a His-tag for purification using an immobilized metal affinity chromatography(IMAC). IMAC constitutes a rather facile means of unravelling the principles of recognition and, in particular, of identifying the counterligands on the protein surface, which interact with the ligated and immobilized metal ions. Histidine when present on the surface of a protein molecule under a favorable solvent condition, may serve as electron donors in coordination with the immobilized chelates of some transition metal ions$(Ni^{2+})$.

SDS-PAGE 결과 elution pH에 따라 분리되는 band pattern은 비슷하게 2band의 양상을 보이지만, FI값을 비교하여 보았을 때 다른 pH보다 8.0에서 가장 높은 수준을 보였으므로 GFPuv/cytochrome c-552 fusion Protein의 분리 ${\cdot}$ 정제에 가장 적합한 pH를 8.0으로 정하였다.