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 Aequorea victoria, was inserted under transcriptional control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The baculovirus transfervector pBlueBacHisB was used for constructing 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 hydroxyapatite as a matrix in protein purification. Ceramic hydroxyapatite loaded with 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 especially in many poly-histidine purification systems did not, when loaded to hydroxyapatite, have binding properties comparable to Zn(II) or Fe(III). Elution of poly-histidine 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.
Nordström
T
T
Department of Biochemistry and Pharmacy, Abo Akademi University, Finland.
Senkas
A
A
Eriksson
S
S
Pöntynen
N
N
Nordström
E
E
Lindqvist
C
C
D000818:Animals; D002460:Cell Line; D002516:Ceramics; D002846:Chromatography, Affinity / Q000379:methods*; D017886:Durapatite; D005290:Ferric Compounds; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006639:Histidine; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008670:Metals; D009532:Nickel; D017924:Nucleopolyhedroviruses / Q000235:genetics; D000077183:Occlusion Body Matrix Proteins; D010455:Peptides; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins; D015032:Zinc
D005290:Ferric Compounds; D008164:Luminescent Proteins; D008670:Metals; D000077183:Occlusion Body Matrix Proteins; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D049452:Green Fluorescent Proteins; C033223:polyhistidine; D006639:Histidine; D009532:Nickel; D017886:Durapatite; D015032:Zinc