Priprava plazmida za izražanje fuzijskega proteina sestavljenega iz GFP in proteaze iz Vibrio cholerae

Babnik, Ana (author), Gunčar, Gregor (mentor)

Domena cisteinske proteaze (CPD) je del večfunkcijskega samoprocesirajočega toksina, ki vsebujejo ponavljajoče motive (MARTX) iz Vibrio cholerae. Pri vstopu MARTX toksina v citosol evkariontske celice se zaradi prisotnosti InsP6 aktivira CPD. Ta ima avtoproteolitično aktivnost, saj cepi toksin na samostojne domene, ki povzročijo kovalentno povezovanje aktina, kar vodi v depolarizacijo aktinskega citoskeleta. V diplomskem delu smo z metodo kloniranja s sestavljanjem in vivo pripravili plazmid z zapisom za rekombinantni protein, sestavljen iz GFP, CPD in heksahistidinske oznake. Bakterije Escherichia coli DH5α smo transformirali z vektorjem pET22b, ki je vseboval zapis za naš fuzijski protein. Z metodo PCR na osnovi ene kolonije smo preverili, katere kolonije so ustrezne za izolacijo plazmidne DNA. Nukleotidno zaporedje plazmida je bilo določeno z metodo po Sangerju in potrjeno s poravnavo zaporedij.

• Language: Slovenian
• Keywords: domena cisteinske proteaze, zeleni fluorescenčni protein, kloniranje s sestavljanjem in vivo, rekombinantni protein
• 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-137932
• COBISS.SI-ID: 116811523
• Publication date in RUL: 06.07.2022

Secondary language

• Language: English
• Title: Preparation of plasmid for expression of fusion protein composed of GFP and protease from Vibrio cholerae

Abstract

Cysteine protease domain (CPD) is a domain of multifunctional autoprocessing repeats-in-toxin (MARTX) toxin from Vibrio cholerae. Upon entry of MARTX in eukaryotic cytosol CPD is activated by InsP6. Autoproteolytic processing of toxin by CPD causes covalent actin cross-linking that leads to actin depolarization and cell rounding. We prepared a plasmid for expression of a recombinant fusion protein composed of GFP, CPD and hexahisitdine tag using in vivo assembly cloning method. pET22b vector with our recombinant fusion protein was transformed into Escherichia coli DH5α. Presence of vector with the right construct in bacterial colonies was verified by colony PCR. Plasmid DNA was isolated from suitable colonies and the sequence was determined by Sanger sequencing. We confirmed the sequence of the recombinant protein with sequence alignment.

• Keywords: cysteine protease domain, green fluorescent protein, in vivo assembly cloning, recombinant protein