Purification of gamma-crystallin from human lenses by acetone precipitation method.
OBJECTIVE
The aim of this study was to develop a new purification method for human lens gamma-crystallin by utilizing its unique property of remaining soluble during acetone precipitation of water soluble (WS) proteins.


METHODS
The WS protein fractions from lenses of donors of different ages were precipitated with 50% acetone (v/v) and the supernatant and precipitated protein fractions were collected following centrifugation. Among lens crystallins, gamma-crystallin remained soluble (recovered in the supernatant following centrifugation) while other crystallins were precipitated. To determine the recovery of maximal levels of gamma-crystallin as soluble protein during acetone precipitation, the WS proteins were precipitated under different conditions, and both supernatant and precipitated fractions were quantified for proteins and analyzed by size-exclusion chromatographic and Western blot methods. Based on these results, a three-step purification procedure for gamma-crystallin was developed which consisted of acetone precipitation followed by preparative isoelectric focusing (IEF) and size-exclusion HPLC of the soluble fraction.


RESULTS
During precipitation of WS proteins by 50% (v/v) acetone, only gamma-crystallin remained soluble. The identity of gamma-crystallin was based on its Mr of 20 kDa on SDS-PAGE, co-elution with lens homogenate gamma-crystallin during a size-exclusion Agarose chromatography, immunoreactivity with anti-gamma-crystallin antibody on a Western blot and an overlap of its partial N-terminal sequence with gammaC-crystallin. A three-step procedure, as described above, provided a highly purified preparation of gammaC-crystallin from the WS protein fraction. The three-step procedure was also utilized to recover a highly purified human lens recombinant gammaD-crystallin preparation from E. coli lysate.


CONCLUSIONS
The unique property of human lens gamma-crystallin of remaining soluble during acetone precipitation can be utilized to purify this crystallin by a three-step procedure. This procedure is also applicable in the purification of recombinant gammaD-crystallin from E. coli lysate.
Srivastava
O P
OP
Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham 35294, USA.
Srivastava
K
K
D000096:Acetone; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D011232:Chemical Precipitation; D002648:Child; D002675:Child, Preschool; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D007908:Lens, Crystalline / Q000737:chemistry*; D008875:Middle Aged; D008970:Molecular Weight; D012995:Solubility
D003459:Crystallins; D000096:Acetone