Rapid purification of recombinant green fluorescent protein using the hydrophobic properties of an HPLC size-exclusion column.
The green fluorescent protein (GFP) of the jelly fish Aequoria victoria was cloned into an Escherichia coli cell line that is a methionine auxotroph. The recombinant GFP (rGFP) was isolated from the cells and purified using a simple procedure consisting of only two chromatographic steps: size-exclusion chromatography and ion-exchange HPLC. Due to the hydrophobic nature of the protein, the surface characteristics of the HPLC size column, and the high initial salt concentration, the rGFP sticks to the size column and is eluted by reducing the salt concentration. Due to this unique behavior the purification procedure can readily be scaled to handle larger quantities of rGFP.
Naval Research Laboratory, Washington, DC 20375, USA.
J R
JR
Deschamps
C E
CE
Miller
K B
KB
Ward
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D037401:Scyphozoa / Q000235:genetics
D017931:DNA Primers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins