Priprava heterodimera ektodomen proteinov EpCAM in Trop2

Maučec, Ana (author), Pavšič, Miha (mentor)

EpCAM in Trop2 sta homologna transmembranska proteina, ki sta vpletena v procese diferenciacije in proliferacije, ter prispevata k celični adheziji, njuna vloga v celičnih procesih pa še ni v celoti pojasnjena. V medicini ju pogosto uporabljajo kot biološka označevalca in tarči zdravljenj karcinomov, saj sta ravno pri tej vrsti raka pogosto prekomerno izražena. Homodimerna paraloga sta si zelo podobna po zaporedju in zvitju in sta včasih prisotna na površini istih epitelijskih celic. Opisanih je bilo kar nekaj primerov paralognih homodimernih proteinov, ki med seboj lahko tvorijo tudi heterodimerne komplekse. Simulacije molekulske dinamike predvidene strukture heterodimera so nakazale na možnost nastanka takšnega kompleksa, interakcije med podenotama pa naj bi bile močne. Te izsledke smo želeli eksperimentalno ovrednotiti in pripraviti heterodimer in vitro. V ta namen smo pripravili rekombinantni ekspresijski vektor pFastBac Dual z zapisoma za zunajcelični del neglikozilirane mutante EpCAM s heksahistidinsko oznako in delno neglikozilirane mutante Trop2 s StrepII oznako. Rekombinantna proteina smo proizvedli v insektni celični liniji Sf9. Po izražanju smo v dveh zaporednih korakih izvedli izolacijo potencialnega heterodimera: najprej z nikljevo afinitetno kromatografijo in nato še z afinitetno kromatografijo Strep-Tactin. Po drugi stopnji izolacije v vezani frakciji, kjer smo v primeru nastanka pričakovali heterodimer, le-tega nismo zaznali. Njegova vezava na nosilec Strep-Tactin bi lahko bila otežena zaradi steričnih ovir ali nedostopnosti StrepII oznake, vendar po NaDS-PAGE analizi v nevezani frakciji nismo zaznali prisotnosti Trop2, torej to ni bil razlog. Po detekciji T2EX-StrepII s Strep-Tactinom-HRP v mediju in homogenizatu insektnih celic po izražanju, smo ocenili, da je raven izražanja T2EX-StrepII približno 120-krat nižja od ravni izražanja ngEpEX-His. V primeru, da heterodimer nastane, bi verjetno nastal v premajhnih količinah, da bi ga z našim pristopom lahko zaznali.

• Language: Slovenian
• Keywords: EpCAM, Trop2, heterodimer, paraloga, afinitetne oznake
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-149574
• COBISS.SI-ID: 169309699
• Publication date in RUL: 07.09.2023

Secondary language

• Language: English
• Title: Preparation of EpCAM and Trop2 ectodomain heterodimer

Abstract

EpCAM and Trop2 are homologous transmembrane proteins that are involved in cell-proliferation as well as differentiation and contribute to adhesion, though their role in cellular processes is not yet fully known. In medicine, they are often used as biomarkers and targets of carcinoma treatments, as they are often overexpressed in this type of cancer. Homodimeric paralogues are remarkably similar in their amino-acid sequences and fold and can be expressed on the surface of the same epithelial cells. There have been quite a number of paralogous homodimeric proteins that can form heterodimeric complexes reported to date. Molecular dynamics simulations of the predicted structure of the heterodimer have pointed at the possibility of such a complex existing and interactions between subunits are predicted to be strong. Our aim was to experimentally evaluate those findings and prepare the heterodimer in vitro. For that purpose, we prepared a recombinant pFastBac Dual expression vector containing the extracellular parts of a non-glycosylated mutant of EpCAM with a hexahistidine tag and partly non-glycosylated mutant of Trop2 with a StrepII tag. Recombinant proteins were produced in the insect cell line Sf9. Following expression, a two-step isolation of the potential heterodimer was performed: first with nickel and then with Strep-Tactin affinity chromatography. After the second isolation step, we expected the potential heterodimer to be in the bound fraction, yet none was detected. We proposed that binding to the Strep-Tactin resin could be obstructed due to steric hindrances and StrepII tag inaccessibility. That was however not the case as no Trop2 was detected in the unbound fraction after SDS-PAGE analysis. After detection of T2EX-StrepII with Strep-Tactin-HRP in the medium and homogenisate of insect cells after expression, we estimated that the expression level of T2EX-StrepII is approximately 120 times lower than that of ngEpEX-His. In the case of heterodimer existing, there would probably not be enough of it formed to be detected using our approach.

• Keywords: EpCAM, Trop2, heterodimer, paralogues, affinity tags