PURIFICATION OF GAMMA-CRYSTALLIN FROM HUMAN LENSES BY ACETONE PRECIPITATION METHOD

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.