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Chinwe O. Asomugha, Ratna Gupta, Om P. Srivastava; Biophysical And Chaperone Properties Of N-terminal Domain, Core Domain, And C-terminal Extension Of Human αA- And αB-crystallins. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4738.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the biophysical properties of the N-terminal domain, core domain and C-terminal extensions of human αA- and αB-crystallins and correlate these properties to their chaperone activity.
Wild type (WT) αA- and αB-crystallins, previously cloned in pET 100D TOPO vector, were used as templates to generate different constructs encoding specific regions (N-terminal domain [NT], core domain [CD], and C-terminal extension, [CT]). The specific regions amplified by PCR using plasmid DNA from WT αA and WT αB were: αA NT (residues 1-63), αA CD (residues 64-142), αA CT (residues 143-173), αB NT (residues 1-66), αB CD (residues 67-146), and αB CT (residues 147-175). Resultant blunt-end PCR products were ligated to a pET100 Directional TOPO vector. DNA sequencing confirmed the desired constructs. Positive clones were transformed into the BL-21 Star (DE3) expression cell line, and expression and solubility of the proteins were confirmed by SDS-PAGE and Western Blot analysis using monoclonal antibodies against a 6X His-tag epitope. Proteins were purified using Ni2+-affinity columns, under native or denaturing conditions, and used for biophysical and chaperone function analyses.
A total of six constructs were generated: αA NT, αA CD, αA CT, αB NT, αB CD, and αB CT. SDS-PAGE and Western blot analyses showed that αA CD and αB CD were present in both the soluble and insoluble fractions, whereas species with NT alone became insoluble and species with CT alone became soluble. Confirmed constructs showed alterations in biophysical properties and chaperone function compared to WT α-crystallins. αA NT and αB CT exhibited the most notable changes in secondary structural content. Also, αA NT and all constructs of αB-crystallin showed altered surface hydrophobicity compared to their respective WT α-crystallins.
Although the individual α-crystallin regions (i.e., N-terminal domain, core domain, and C-terminal extension) exhibited varied biophysical properties, each region alone retained some level of chaperone function.
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