Original title:
Příprava a exprese izoforem proteinu p53 pomocí GATEWAY expresního systému
Translated title:
Preparation and expression of p53 protein isoforms using the GATEWAY expression system
Authors:
Wikarská, Monika ; Hrstka, Miroslav (referee) ; Brázda, Václav (advisor) Document type: Master’s theses
Year:
2019
Language:
slo Publisher:
Vysoké učení technické v Brně. Fakulta chemická Abstract:
[slo][eng]
Produktom tumor-supresorového génu TP53 je proteín p53 a vďaka dvom promótorom a alternatívnemu zostrihu aj jeho 11 izoforiem skrátených z N- a/alebo C- terminálneho konca. Izoformy p53 sa nachádzajú v zdravom aj nádorovom tkanive a vo vzťahu k diagnostike, prognóze a liečbe rakoviny sú intenzívne študované. V tejto práci boli prostredníctvom LR reakcie Gateway systému klonovania pripravené expresné vektory kódujúce sekvencie izoforiem p53 určené na produkciu proteínov baktériami E.coli kmeňa BL-21. Na ich izoláciu afinitnou chromatografiou boli za rovnakým promótorom kódované spolu s dvoma fúznymi proteínmi, glutatión-S-transferázou a polyhistidínovou kotvou. Správnosť inzertov po homológnej rekombinácii bola overená Sangerovou sekvenáciou. Výsledky sekvenácie potvrdili úspešné naklonovanie izoforiem p53, 133p53, 160p53, p53 a 160p53 do expresného vektoru pDEST15-N6xHis-GST-GW-DEST. Proteín 160p53 bol exprimovaný v bunkách BL-21 a izolovaný prostredníctvom oboch značiek. Izoláciou prostredníctvom polyhistidínovej kotvy bola získaná vyššia koncentrácia proteínu ako izoláciou sprostredkovanou interakciou glutatión-S-transferázy.
The TP53 gene can express protein p53 and 11 another isoform proteins N- and/or C-terminally truncated by using two promoters and alternative splicing. The p53 isoforms are found in both healthy and tumorous tissues, and are intensively studied in relation to cancer diagnosis, prognosis and treatment. In this work, the p53 isoforms were subcloned into expression vectors by LR reaction adapted from Gateway cloning system. The expression vectors were designed for protein production by bacteria E. coli strain BL-21. The constructs containing p53 isoforms were encoded together with two fusion proteins, glutathione-S-transferase and polyhistidine tag under the control of the same promotor for the affinity chromatography protein isolation. All the clones underwent Sanger sequencing for verification after homologous recombination. Sequencing confirmed the accuracy of the subcloned isoforms p53, 133p53, 160p53, p53 and 160p53 into an expression vector pDEST15-N6xHis-GST-GW-DEST. Protein 160p53 was expressed in BL-21 and isolated using both HIS and GST tag interacion. Isolation using HIS tag yielded in a higher protein concentration then the isolation mediated by the interaction of the glutathione-S-transferase.
Keywords:
gateway cloning; isoforms p53; protein p53
Institution: Brno University of Technology
(web)
Document availability information: Fulltext is available in the Brno University of Technology Digital Library. Original record: http://hdl.handle.net/11012/177453