Characterization of metal affinity of green fluorescent protein and its purification through salt promoted, immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) was investigated as a meth od of recovery for green fluorescent protein (GFPuv). It was found that in the absence of genetic modification to enhance metal affinity, GFPuv displa yed strong metal affinity to Cu(II) and Ni(II), and weak or negligible affi nity to Zn(II) and Co(II). Changes in the mobile phase NaCl concentration d uring Ni(II)-IMAC strongly affected purity and yield of GFPuv, with fine re solution under higher NaCl concentrations. Finally, IMAC via Cu(II) and Zn( II) with intervening diafiltration was used to recover GFPuv with high yiel d and purity. (C) 2001 Elsevier Science B.V. All rights reserved.