Studies of RET and its binding partners ephrin-A2 and ephrin-A5b

Eph receptor tirozin kinaze i njegovi ligandi, efrini imaju važnu ulogu u različitim biološkim funkcijama. Trenutna istraživanja usmjerena su na ulogu efrin-A reverznog signala u razvoju neurona, budući da se pretpostavlja da je posredovan RET-transmembranskim tirozin kinaza ko-receptorom. Cilj istraživanja bio je klonirati, eksprimirati i pročistiti ekstracelularne domene efrin-A2 i efrin-A5b proteina iz zebrice (Danio rerio). Kloniranje korištenjem restrikcijskih enzima NotI i XbaI je provedeno u pk503.9-FLAG-8xHis-Thr vektoru izvedenom iz pFastBac-1. Bakulovirus-sustav ekspresije pokazao se uspješnijim za efrin-A5b nego za efrin-A2. Proteini su pročišćeni iz medija afinitetnom kromatografijom i gel filtracijom. Budući da je dobivena veća koncentracija čistog efrin-A5b, korišten je za studije vezivanja s RET. Rezultati “pull-down” testa nisu jasni i daljnja istraživanja su nužna kako bi se potvrdilo specifično vezivanje RET.Eph receptor tyrosine kinases and their ligands, the ephrins perform an important function in different biological functions. Recent work focused on the role of ephrin-A reverse signaling in neuronal development as it has been hypothesized to be mediated by RET-a transmembrane tyrosine kinase co-receptor. The goal of this project was to clone, express and purify the extracelullar domains of ephrin-A2 and ephrin-A5b proteins from zebrafish (Danio rerio). Cloning was done into the expression vector pFastBac-1 derivative pk503.9-FLAG-8xHis-Thr using restriction enzymes NotI and XbaI. Baculovirus-expression system showed to be more successful for ephrin-A5b than for ephrin-A2. Proteins were purified from the media by affinity chromatography and gel-filtration. Since the higher yield of pure ephrin-A5b was obtained, it was used for binding studies with RET. Results of „pull-down“ assay are not conclusive and further analysis are required in order to confirm specific binding of RET