Percorrer por autor "Gonzalez, Alejandra Febus"
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- Cloning and preliminary functional analysis of zebrafish BiP, an ATPase-Dependent endoplasmic reticulum chaperonePublication . Gonzalez, Alejandra Febus; Melo, Eduardo José Xavier Rodrigues de Pinho eThe endoplasmic reticulum chaperone BiP (Binding immunoglobulin Protein) plays a fundamental role in protein homeostasis, mediated by its ATPase activity. To investigate the evolutionary conservation and functionality of BiP in vertebrates, we cloned the open reading frame of Danio rerio BiP (zebrafishBiP, Uniprot Q7SZD3) into a pTrcHis-A vector for heterologous expression in Escherichia coli. The open reading frame cloned excluded the N-terminal signal peptide and the highly acidic sequence of eight amino acids after the peptide signal to align expression with previously studied mammalian BiP from Mesocricetus auratus (golden hamster, Uniprot P07823). Following PCR amplification, enzymatic digestion, ligation, and bacterial transformation, colonies were screened via colony PCR and sequencing. One clone, B1, showed 99.73% identity with the Uniprot reference, as two missense mutations (Pro→Leu) were identified. Preliminary functional assays using the malachite green method confirmed ATPase activity of hamster BiP, paving the way to test if recombinant zfBiP is biochemically active. These results lay the foundation for comparative analyses of BiP functionality across species and its potential role in the endoplasmic reticulum proteostasis and protein disaggregation.