Priprava rekombinantne zunajcelične domene proteina EGFR in njegovega interakcijskega partnerja EGF

Latysheva, Daria (author), Lenarčič, Brigita (mentor)

EGFR je transmembranski glikoprotein, ki igra pomembno vlogo pri nastanku in razvoju raka. Pred kratkim so ugotovili, da zunajcelična domena proteina EpCAM (EpEX) deluje kot ligand EGFR. Interakcija med proteinoma je šibka, zato je za analizo interakcije potrebno uporabiti metodo za analizo šibkih in prehodnih interakcij, kot je na primer analiza agregacije kroglic (BAA). Za preučevanje interakcij med temi proteini z metodo BAA je potrebno pripraviti rekombinantni protein EGFR-Ex z FC-regijo, hkrati pa je potrebno način vezave EpEX na EGFR primerjati z interakcijo med EGFR in EGF, ki je znan ligand EGFR. V sklopu diplomskega dela smo se osredotočili na pripravo rekombinantnega proteina EGFR-Ex z FC-oznako in EGF. Za EGFR-Ex smo pripravili DNA konstrukt s FC-regijo, vendar ga nismo uspeli izraziti v bakulovirusnem sistemu. Za protein EGF smo pripravili DNA-konstrukt z inteinom Mxe GyrA in pomožnim proteinom SUMO, ki omogočata pravilno zvitje proteina EGF. Konstrukt EGF smo izrazili v bakterijah E. coli BL21[DE3].

• Language: Slovenian
• Keywords: EGFR, EGF, EpCAM
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2019
• PID: 20.500.12556/RUL-109856
• COBISS.SI-ID: 1538395331
• Publication date in RUL: 09.09.2019

Secondary language

• Language: English
• Title: Preparation of recombinant extracellular domain of EGFR and its interaction partner EGF

Abstract

EGFR is a transmembrane protein that plays a crucial role in cancer development. It has recently been discovered that the extracellular domain of another transmembrane protein EpCAM (EpEX) acts as an EGFR ligand. However, this interaction is weak and, therefore, needs to be examined with methods, suitable for analysing weak and/or transient interactions. One such method is Bead Aggregation Assay – BAA. In order to study this interaction with BAA a recombinant fusion protein EGFR-FC had to be prepared. Our goal was to compare the binding pattern of EpEX and EGFR with that of EGFR and EGF, which is a known ligand of EGFR. A DNA construct of EGFR-Ex with FC-fusion was prepared; however, expression in insect cells with baculovirus expression system was unsuccessful. EGF DNA construct was prepared in fusion with intein Mxe GyrA / SUMO system, which facilitates correct protein folding. EGF construct was expressed in E. coli BL21[DE3].

• Keywords: EGFR, EGF, EpCAM