O P Srivastava1, K Srivastava. 1. Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham 35294, USA.
Abstract
PURPOSE: 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.
PURPOSE: 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.
Authors: K Devraj; R Geguchadze; M E Klinger; W M Freeman; A Mokashi; R A Hawkins; I A Simpson Journal: J Neurosci Methods Date: 2009-07-23 Impact factor: 2.390
Authors: Harvey E Johnston; Kranthikumar Yadav; Joanna M Kirkpatrick; George S Biggs; David Oxley; Holger B Kramer; Rahul S Samant Journal: Anal Chem Date: 2022-07-18 Impact factor: 8.008