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B. Qu, Y. Liu, A. Prescott, R. Dahm, R. Quinaln; Evolutionary Development of Beaded Filament Proteins. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4076. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the evolutionary development of beaded filament proteins.
Sequences were mined from the available genome databases, alignments and evolutionary distances determined using online tools. The expression constructs (pET23) for beaded filament proteins from Zebrafish were constructed by a PCR, sequencing and subcloning strategy. This vector system allows the inducible expression of the cloned cDNAs in /Escherishia coli/ by the addition of IPTG. Protein expression was monitored by SDS-PAGE. Like other intermediate filament proteinsexpressed beaded filament proteins form inclusion bodies in the bacteria. These were purified by previously published protocols and purified to homogeneity by anion exchange chromatography. The functional integrity of these recombinantly expressed proteins was analysed by in vitro assembly protocols, culminating in the analysis of filament structure by electron microscopy using negatively stained samples. To facilitate cell biological studies, a peptide specific antibody wasgenerated to Bfsp2 and used for immunofluoresence microscopy.
Both Bfsp1 and 2 have distinctive sequence features that are preserved across species. Interestingly the existance of a tail domain is common to many fish species. The proteins appear to show the expectimmunostaining pattern in sections from Zebrafish lenses and therefore we examined the in vitro assembly properties of Bfsp2. The data revealthat despite the differences in protein structure the homologies are sufficient to preserve many of the features required for assembly.
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