Production of human papilloma virus E6 and E7 oncoproteins in Escherichia coli

Abstract

Humani papiloma virus (HPV) spada u obitelj Papilomavirusa. U 1970‐ima dokazano je kako infekcija čovjeka ovim virusom kod žena može dovesti do razvoja raka vrata maternice. Za onkogenična svojstva HPV‐a odgovorni su E6 i E7 geni, odnosno njihovi proteini. Cilj ovog istraživanja bio je eksprimirati E6 i E7 gene tako da dobijemo nativne i topive proteine koji se mogu koristiti za daljnje istraživanje. Uvidom u slijed nukleotida genoma HPV 16 odabrane su početnice za umnažanje E6 i E7 gena. Pomoću vektorskog sustava prenešeni su u bakteriji E. coli gdje se odvijala ekspresija. U ovom radu smo, mijenjali uvjete ekspresije kako bi našli optimalne za dobivanje nativnih proteina. Iako je ekspresija bila uspješna, nijedan od promijenjenih parametara nije utjecao na topivost proteina. Kao alternativni pristup, proteine smo izolirali u denaturirajućim uvjetima.Human papilloma virus (HPV) belongs to Papillomavirus family. In the 1970s it has been demonstrated that human infection by this virus in women may lead to the development of cervical cancer. The oncogenic properties of HPV are E6 and E7 genes. The aim of this study was the expression of E6 and E7 genes in order to obtain native, productive proteins for further research. Based on the genome sequence of HPV type 16, specific primers for E6 and E7 amplification were designed. Using a vector system the amplicon were transferred into E. coli bacteria where they were expressed. In this study, we have changed the conditions of the expression in order to find the optimal condition. Although the expression was successful, the proteins remained unsoluble under every tested change. Therefore, the alternative approach that we applied was to extract the proteins in denaturating conditions