Op. Srivastava et K. Srivastava, PURIFICATION OF GAMMA-CRYSTALLIN FROM HUMAN LENSES BY ACETONE PRECIPITATION METHOD, Current eye research (Print), 17(11), 1998, pp. 1074-1081
Purpose. The aim of this study was to develop a new purification metho
d for human lens gamma-crystallin by utilizing its unique property of
remaining soluble during acetone precipitation of water soluble (WS) p
roteins. Methods. The WS protein fractions from lenses of donors of di
fferent ages were precipitated with 50% acetone (v/v) and the supernat
ant and precipitated protein fractions were collected following centri
fugation. Among lens crystallins, gamma-crystallin remained soluble (r
ecovered in the supernatant following centrifugation) while other crys
tallins 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 a
nd analyzed by size-exclusion chromatographic and Western blot methods
. Based on these results, a three-step purification procedure for gamm
a-crystallin was developed which consisted of acetone precipitation fo
llowed by preparative isoelectric focusing (IEF) and size-exclusion HP
LC of the soluble fraction. Results. During precipitation of WS protei
ns by 50% (v/v) acetone, only gamma-crystallin remained soluble. The i
dentity of gamma-crystallin was based on its Mr of 20 kDa on SDS-PAGE,
coelution with lens homogenate gamma-crystallin during a size-exclusi
on Agarose chromatography, immunoreactivity with anti-gamma-crystallin
antibody on a Western blot and an overlap of its partial N-terminal s
equence with gamma C-crystallin. A three-step procedure, as described
above, provided a highly purified preparation of gamma C-crystallin fr
om the WS protein fraction. The three-step procedure was also utilized
to recover a highly purified human lens recombinant gamma D-crystalli
n preparation from E. coli lysate. Conclusions. The unique property of
human lens gamma-crystallin of remaining soluble during acetone preci
pitation can be utilized to purify this crystallin by a three-step pro
cedure. This procedure is also applicable in the purification of recom
binant gamma D-crystallin from E. coli lysate.