Rapid purification of recombinant betaB2-crystallin using hydrophobic interaction chromatography.
BetaB2-crystallin, the major subunit of beta-crystallins, is difficult to purify either from lens homogenate or from betaH-or betaL-crystallins. It has been prepared by heterologous expression in Escherichia coli. Most often, the methods used for purifying a recombinant globular protein employ the combination of ion-exchange with gel filtration chromatography. In the case of betaB2-crystallin too, different approaches have been used to obtain the purified protein, majority of which use a combination of ion-exchange and gel filtration chromatography. We present a new approach to purify betaB2-crystallin using hydrophobic interaction chromatography. In this method, the protein is bound to the hydrophobic matrix in the presence of high concentration of a non-chaotropic salt and eluted by decreasing the salt concentration. The method that we have used for the purification of this globular protein has definite advantages over the earlier methods in its simplicity and efficiency. The most noted advantage of this procedure is the rapid purification with a relatively purified product and a comparatively high yield (>20 mg/L of culture). Over all, the present protocol provides a rapid, efficient and simplified procedure for the preparation of betaB2-crystallin in large yield, sufficient for structural and functional studies.
Jobby
M K
MK
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India.
Sharma
Yogendra
Y
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D007908:Lens, Crystalline / Q000737:chemistry; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D038206:beta-Crystallin B Chain / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D038206:beta-Crystallin B Chain; C113634:beta-crystallin B2