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| Improved Rhodopsin purification during the purification of peripherin/RDS |
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作者：Fansheng… 文章来源：Shen Zhen Eye Hospital 518001 点击数：776 更新时间：2006/6/28 15:34:01 
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| Purpose: To get more purified rhodopsin and peripherin/RDS for the study of phototransduction and morphogenesis of photoreceptor cells.
Methods: The bovine rod outer segment (ROS) was prepared from 60 bovine retina collected from freshly slaughtered cattle by sucrose flotation and subsequent sucrose density gradient. The bovine ROS membrane protein was achieved by washing rod outer segment membrane pellet with GTP and urea and centrifuging with 360kg for 40 min in a Beckman 60Ti rotor. Then the supernatant was loaded to a two consecutive hydroxylapatide (HA) columns and were eluted with 50%, 70% and 100% step gradients of 300 mM NaH2PO4 and a Mono Q column chromatography in dark (or under dim red light). The purified rhodopsin peripheirn/RDS were examnated with anti-Rhodopsin and anti-peripherin/RDS antibodies. The purity of rhodopsin was also examnated with spectral ratio A280/A500.
Results: Elution with 150 mM NaH2PO4 in the 1st HA column forms a big and sharp peak on the trace chart. The first fraction of the peak has highly homogeneity of rhodopsin without minor band that appeared in the other fractions in the Coomassie blue stained gel. The Western blot was probed by anti-peripherin/rds and did not show the labeling. Spectral ratio of A280/A500 of the first fraction of the peak was 1.5.
The SDS-PAGE analysis of 110-130 mM NaCl elution of peripherin/RDS from Mono Q column with both silver staining and immnoblots probed with anti-opsin and anti-peripherin/RDS showed apparent homogeneity of peripherin/RDS with no rhodopsin presence and no anti-opsin labeling. The absorbance of 500 nm shows greatly reducing from 50% step gradient to 70% step gradient.
Conclusions: Rhodopsin purification can be improved by HA column with 50% step gradient of 300 mM NaH2PO4, the ratio of A280/A500 of the first fraction containing rhodopsin was 1.52 which is lower than the best ratio of 1.65-1.75 reported before; and the fraction excluded peripherin/RDS and other minor proteins.
Peripherin/RDS can be homogeneiticaly purified by two consecutive HA columns with step gradients of 300 mM NaH2PO4 followed by Mono Q column chromatography.
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