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J. M. Chaves, R. Gupta, K. Srivastava, O. P. Srivastava; Interaction of Human A-Crystallin With Phakinin and Filensin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4095. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the interaction of αA-crystallin with two intermediate filament [IF] proteins (phakinin and filensin).
Phakinin, filensin, and vimentin, cloned in PT7 and pBAD-hys MycA vectors (provided by Dr. Paul Fitzgerald of University of California-Davis), respectively, were expressed in BL-21 AI cells using 0.1% arabinose. Phakinin, filensin and vimentin were purified using a DEAE-cellulose column followed by a hydroxyapatite column under denaturing conditions. If needed, these were further purified using a size-exclusion Sephadex G-75 column under denaturing conditions. Wild type (WT) αA, and its N (residue number 1-63)- or C (residue number 140-173)-terminally truncated mutants (named αA-NT and αA-CT, respectively), cloned in pET 100 TOPO vector were expressed in BL-21 (DE3) cells using 1% IPTG, and purified using a Ni2+-affinity column. The interaction between WT-αA, αA-NT, or αA-CT and phakinin and/or filensin was determined by either electron microscopic method (by negative staining using 2% uranyl acetate) or by sedimentation analysis (Centrifugation at 80,000xg for 30 minutes at 20ºC followed by SDS-PAGE analysis of pellet and supernatant).
Following ultracentrifugation of individual proteins (phakinin, filensin, vimentin, WT-αA, αA-NT and αA-CT), the SDS-PAGE analyses showed that αA-species remained in the supernatant and phakinin, filensin, and vimentin were mostly found in the pellet. On ultracentrifugation of a mixture of WT-αA, αA-NT, or αA-CT (at varying ratios) with filensin and/or phakinin (also at varying ratios), WT-αA and αA-CT were found in the pellet with phakinin and/or filensin. However, αA-NT generally did not sediment with phakinin and filensin. The EM studies showed filament formation in mixtures of WT-αA or αA-CT with phakinin and filensin. Again, relatively fewer filaments were seen in the mixture of αA-NT with phakinin and filensin.
The N-terminally truncated αA-crystallin formed relatively lesser number and shorter filamentous structures with phakinin and filensin, whereas such structures were seen in abundance in mixtures of WT-αA or αA-CT with phakinin and filensin.
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