RESUMO
tau-Crystallin is a taxon-restricted crystallin found in eye lenses of reptiles and a few avian species but presumably absent in mammals. The level of tau-crystallin in the lens varies among different species. In the crocodile lens, it is the least abundant crystallin and is present in trace amounts. We present a method for cloning, overexpression, and purification of crocodilian tau-crystallin utilizing a combination of gel filtration and ion-exchange chromatography yielding an extremely purified protein. The protein gets profusely expressed resulting in a fairly high yield and exists as a monomeric entity of 47.5 kDa molecular mass. The recombinant tau-crystallin exists in a properly folded native state as probed by circular dichroism and fluorescence spectroscopy and exhibits enolase activity.
Assuntos
Bioquímica/métodos , tau-Cristalinas/biossíntese , tau-Cristalinas/química , tau-Cristalinas/isolamento & purificação , Jacarés e Crocodilos , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Dicroísmo Circular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Fosfopiruvato Hidratase/metabolismo , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coloração pela Prata , Espectrometria de FluorescênciaRESUMO
tau-Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete tau-crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the a-enolase gene from non-lenticular tissues. Quantitatively, the tau-crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile tau-crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5'- and 3'-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete tau-crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5'- and 3'-ends respectively. Further, it was found to be identical to its putative counterpart enzyme a-enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of tau-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.
