One-step purification of chimeric green fluorescent protein providing metal-binding avidity and protease recognition sequence.
Gene fusion technique was successfully applied as a potential approach to create a metal-binding site to assist one-step purification of green fluorescent protein (GFP). The chimeric GFP carrying hexapolyhistidine (H6GFPuv) was purified to homogeneous protein via the Immobilized Metal Affinity Chromatography charged with zinc ions. Removal of metal tagger could readily be performed by using enterokinase enzyme. Engineering of the hexahistidine and enterokinase cleavage sites (DDDDK) onto the chimeric protein did not significantly affect the fluorescent property and the binding avidity to Burkholderia pseudomallei protease of a chimeric protease-binding GFP (H6PBGFPuv). This concludes that engineering of repetitive histidine regions onto interested target protein along with the enterokinase cleavage sites will ease the complication of protein purification.
Prachayasittikul
Virapong
V
Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand.
Isarankura Na Ayudhya
Chartchalerm
C
Piacham
Theeraphon
T
Kiatfuengfoo
Rachada
R
D001665:Binding Sites; D016957:Burkholderia pseudomallei / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000378:metabolism; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D008670:Metals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D010447:Peptide Hydrolases; D004765:Enteropeptidase; D015032:Zinc