Izražanje in izolacija inhibitornega peptida za TACE, ki je zasnovan na osnovi zaporedja propeptidne regije

Povšin, Jure (author), Gaber, Aljaž (mentor)

Disintegrinska metaloproteaza TACE oziroma ADAM17 je encim, ki spada v skupino šedaz ter specifično cepi prekurzorski faktor tumorske nekroze alfa. Poleg tega ima encim pomembno vlogo tudi pri procesiranju celičnih adhezijskih proteinov, citokinskih receptorjev ter ligandov EGF receptorjev, ki so potrebni za spodbujanje imunskega odziva in regeneracije. Zunajcelično večdomensko regijo TACE sestavljajo: prodomena, katalitična domena, disintegrinska domena, obmembranska domena ter regija CANDIS. Prodomena ima inhibitorno vlogo in je bila osrednja točka našega raziskovalnega dela. Priprava nativno zvite in stabilne prodomene je bila kar nekaj časa otežena, dokler niso znanstveniki naredili dvojnega mutanta prodomene (TPD-DM) z mutacijama R58A in C184A. V okviru raziskovalnega dela smo želeli pripraviti dovolj veliko količino take prodomene, da bi jo lahko uporabili za pripravo specifične kolone za čiščenje encima TACE z afinitetno kromatografijo. S tem bi olajšali in pospešili sam postopek čiščenja encima TACE. Uspelo nam je pripraviti genska zapisa za dva konstrukta TPD-DM, ki sta imela na N-koncu dodano heksahistidinsko oznako za lažje čiščenje proteina, eden od konstruktov pa je imel še regijo za cepitev s proteazo TEV, ki je omogočala odstranitev heksahistidinske oznake. Po uspešnem izražanju in čiščenju proteinov s kolono za IMAC ter dializi, so se nam proteini neželeno oborili, kar nam je onemogočilo izolacijo proteinov za vezavo na kolono. Poskušali smo optimizirati elucijo, z zmanjšanjem koncentracije imidazola v elucijskem pufru, in dializo, da bi ohranili enako koncentracijo soli, kot je bila v elucijskem pufru, vendar brez večjih uspehov. Ugotovili pa smo, da odcep heksahistidinske oznake najverjetneje poveča obarjanje proteina.

• Language: Slovenian
• Keywords: TACE, prodomena, izražanje in čiščenje proteinov.
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-136217
• COBISS.SI-ID: 105482755
• Publication date in RUL: 20.04.2022

Secondary language

• Language: English
• Title: Expression and isolation of TACE inhibitory peptide, designed on the basis of the propeptide sequence

Abstract

The disintegrin metalloprotease TACE or ADAM17 is an enzyme that functions as a sheddase and specifically cleaves the precursor tumor necrosis factor alpha. The enzyme also plays an important role in processing cell adhesion proteins, cytokine receptors and ligands of EGF receptors, that are needed for the stimulation of the immune response and regeneration. The extracellular multidomain region of TACE consist of: a prodomain, a catalytic domain, a disintegrin-like domain, a membrane-proximal domain and a CANDIS region. The prodomain acts as an inhibitor of TACE and was the focal point of our research work. The preparation of a natively folded and stable prodomain was difficult for quite some time until scientists made a double mutant prodomain (TPD-DM) with R58A and C184A mutations.As part of our research work we tried to prepare a large enough amount of such prodomain to be used for the preparation of a column that is specific for the purification of the TACE enzyme using affinity chromatography. In this way we could facilitate and speed up the process of purification of the TACE enzyme. We were able to prepare the genetic material for two TPD-DM constructs that had a hexahistidine tag added to the N-terminus to facilitate protein purification, and one of the constructs also had a TEV protease cleavage region that enabled the removal of the hexahistidine tag. After successful expression and purification of proteins by IMAC column and dialysis, our proteins precipitated, making it impossible for us to isolate the proteins for binding to the TACE specific purification column. We tried to optimize the elution, by reducing the concentration of imidazole in the elution buffer, as well as dialysis, to maintain the same salt concentration in the dialysis buffer as it was in the elution buffer but without much success. However we found out that cleavage of the hexahistidine tag most likely increases protein precipitation.

• Keywords: TACE, prodomain, protein expression and purification.