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C. O. Asomugha, R. Gupta, O. P. Srivastava; Solubility Properties of N-Terminal Domain, Core Domain, and C-Terminal Extension of Human A- and B-Crystallins. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4612.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the solubility properties of the N-terminal domain, core domain and C-terminal extension of human αA- and αB-crystallins and correlate to their biophysical properties and 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 [named as NT], core domain [named as CD], and C-terminal extension, [named as 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 pET100 Directional TOPO vector. DNA sequencing was carried out to confirm the desired constructs. Positive clones were transformed into the expression cell line BL-21 Star (DE3), and expression and solubility of the proteins were confirmed by SDS-PAGE and by Western Blot analysis using monoclonal antibodies against a 6X His-tag epitope.
A total of six constructs were generated: αA NT, αA CD, αA CT, αB NT, αB CD, and αB CT. DNA sequencing confirmed all but one of the desired constructs of αA-crystallin and all constructs of αB-crystallin. Analysis of the expressed proteins by SDS-PAGE and Western blot methods showed that αA CD and αB CD were present in both the soluble and insoluble fractions. However, αA CD was present mostly in the insoluble fraction, unlike αB CD which was present mostly in the soluble fraction.
Determination of the solubility properties of different regions of αA- and αB- crystallins is important for understanding their relative importance to the chaperone function of these crystallins. Based on the above results, the core region of αA-crystallin is relatively more insoluble than the core region of αB-crystallin. This property might affect the chaperone activity of both αA- and αB-crystallins.
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