Transcript, structural, and histologic characterization of a novel EF-Hand protein and its isoform specific to the Ambulacraria clade

Abstract

Transcriptomic databases have become one of the main sources for protein discovery. In our studies of transcripts from normal echinoderms, we have identified several transcripts that have attracted our attention. One of these is a previously unidentified transcript (<em>Orpin</em>) that appeared to be upregulated during intestinal regeneration. In Chapter 2, using bioinformatics tools we: (1) identified a second <em>Orpin</em> sequence (2) describe their motifs and domains, and perform phylogenetic analyses that suggest that <em>Orpins</em> might comprise a novel subfamily of EF-hand containing proteins specific to the Ambulacraria clade. Semiquantitative RT-PCR analyses revealed that <em>Orpin</em> mRNAs are expressed in various tissues but no significant differential expression was found in regenerating tissues.<br /> <br /> In Chapter 3 we expressed and purified genetically modified versions of <em>Orpin</em> to further characterize this EF-hand protein. We developed two protocols:<br /> <br /> The first protocol consisted in the production of His-OBSm, which is His tagged with two additional genetic modifications: the deletion of the signal peptide encoding sequence and the addition of a 25 residues peptide from the pET200 plasmid vector. These modifications made possible the purification of a soluble recombinant version of <em>Orpin B</em>.<br /> <br /> The second protocol was developed to produce a soluble recombinant <em>Orpin</em> that best resembled the original protein. This protocol consisted of the expression of a <em>GST-Orpin</em> lacking the signal peptide region. For this protocol, we tested different parameters that affect protein expression, including, additives, host cells, growth media and supplementation, and protein extraction methods. The best parameters were identified and used to obtain a soluble <em>Orpin</em> form. We propose that the developed strategies can be used to increase the soluble expression and purification of other EF-hand proteins that are difficult to express by standard methods.<br /> <br /> In chapter 4 we produced antibodies against His-OBSm and used these to identify the cells expressing the protein in <em>H. glaberrima</em> tissues. Antibody specificity was tested through Western Blots.<br /> <br /> Finally, we investigated the effect of the Wnt signaling pathway on <em>Orpin</em> mRNA expression in regenerating gut explants and found an apparent decrease expression of an<em> Orpin B</em> isoform following Wnt/B-catenin pathway activation.