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J.M. Chaves, C. Asomugha, K. Srivastava, R. Gupta, O.P. Srivastava; Characterization of Truncated/Deamidated Mutants of Human Alpha A and Alpha B Crystallins . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2003.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize truncated/deamidated human αA and αB mutant proteins for structural and functional analyses.
αA–wild type (WT) and αB–WT, previously cloned in pDIRECT, and deamidated αA and αB generated using QUIK change XL system, were used as a template to generate truncated or truncated/deamindated mutants. PCR–directed truncation of N–terminal domain (residue number 1–63) or C–terminal domain (residue number 140–173) was performed using plasmid DNA of WT–αA, αA N101D, αA N123D, or αA N101/123D and specific primers. Similarly, PCR–directed truncation of N–terminal domain (residue number 1–67) or C–terminal domain (residue number 144–175) was performed using plasmid DNA from WT–αB, αB N78D, αB N146D, or αB N78/146D and specific primers. Resultant blunt end PCR products from individual clones were ligated to pET100 Directional TOPO vector. Restriction analyses of the isolated plasmid DNA were done to screen for positive clones. DNA sequencing was carried out to confirm the desired mutations, deletions, and orientations. Positive clones were transformed into the expression cell line BL–21 (DE3), and the expression of mutant proteins was confirmed by SDS–PAGE and MALDI–TOF analyses.
Restriction and DNA sequencing analyses confirmed the desired deletions in WT and deamidated αA and αB crystallins. Analysis of the expressed proteins by SDS–PAGE showed truncations in WT and deamidated αA and αB crystallin species. A total of eight αA deamidated/deleted mutants were generated: WT–αA–NT (N–terminally truncated), αA N101D–NT, αA N123D–NT, αA N101/123D–NT, WT–αA–CT (C–terminally truncated), αA N101D–CT, αA N123D–CT, and αA N101/123D–CT. Similarly, eight αB deamidated/deleted mutants were generated: WT–αB–NT, αB N78D–NT, αB N146D–NT, αB N78/146D–NT, WT–αB–CT, αB N78D–CT, αB N146D–CT, and αB N78/146D–CT. On SDS–PAGE analysis, each of the mutant proteins showed the desired molecular weights. Specific truncations in the mutant proteins were confirmed by MALDI–TOF analysis.
Mutant proteins of human αA and αB crystallins (see above) with both deamidation and either N– or C–terminal truncations were generated by PCR–based deletional mutagenesis. These will be used for studying relative significance of deamidation and/or truncation in structure and function analyses.
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