Generation of a new protein purification matrix by loading ceramic hydroxyapatite with metal ions-demonstration with poly-histidine tagged green fluorescent protein

The gene encoding the green fluorescent protein (GFP) from the jellyfish Ae quorea victoria, was inserted under transcriptional control of the polyhedr in promoter of the Autographa californica nuclear polyhedrosis virus and ex pressed in the Spodoptera frugiperda insect cell line Sf9 during viral infe ction. The baculovirus transfervector pBlueBacHisB was used for constructin g the recombinant baculovirus, so that the green fluorescent protein could be tagged with a poly-histidine tail. This fusion protein was utilized as a marker for evaluating the properties of metal ion loaded ceramic hydroxyap atite as a matrix in protein purification. Ceramic hydroxyapatite loaded wi th Zn(II) was the best choice for purifying this poly-histidine tagged GFP, followed by Fe(III) of the metal ions tested. Ni(II) that is superior espe cially in many poly-histidine purification systems did not, when loaded to hydroxyapatite, have binding properties comparable to Zn(II) or Fe(III). El ution of polyhistidine tagged GFP was best performed with phosphate buffers or EDTA that could compete with the phosphate molecules in hydroxyapatite or complexly bind the metal ions, respectively. (C) 1999 Elsevier Science B .V. All rights reserved.