Molekulsko kloniranje, izražanje in izolacija rekombinantne človeške kinaze GSK3β

Kovačić, Marko (author), Gaber, Aljaž (mentor)

Glikogen sintaza kinaza 3 beta (GSK3β) je serin/treonin kinaza, ki ima vlogo pri številnih biokemijskih procesih in boleznih. Sestavljena je iz N-končne domene, bogate z β-trakovi, in C-končne α-vijačne domene. GSK3β kot del biomolekularnega kondenzata β-katenin uničevalnega kompleksa sodeluje pri pozitivni in negativni regulaciji signalne poti Wnt. V odsotnosti signaliziranja z ligandi Wnt GSK3β hiperfosforilira β-katenin in ga označi za ubikvitinacijo ter proteasomsko razgradnjo. V okviru diplomskega dela smo želeli v bakterijskih celicah E. coli seva BL21[DE3] izraziti in izolirati človeško rekombinantno GSK3β, da bi bolje razumeli biološko vlogo kinaze in njene interakcije s proteini β-katenin uničevalnega kompleksa. Zapis za GSK3β smo vnesli v vektor pET-32b(+) in ga na 5' koncu sklopili z nukleotidnim zaporedjem za heksahistidinsko oznako, ki mu je sledil zapis za sfGFP. Heksahistidinsko oznako in sfGFP je bilo mogoče odcepiti s proteazo TEV. V nadaljevanju smo izrazili celoten vključek in poskusili izolirati GSK3β z nikljevo afinitetno kromatografijo. Postopek izražanja in izolacije proteina se ni izkazal za optimalnega, saj so najverjetneje že med izražanjem proteina endogene proteaze cepile GSK3β. Ugotovili smo, da se je ob dodatku plazmida pRARE, ki zapisuje za nekatere tRNA, ki so v E. coli redkejše, GSK3β slabše izražala v bakterijskih celicah E. coli seva BL21[DE3]. Za uspešnejše izražanje in izolacijo proteina bi lahko poskusili izraziti protein z dodatkom inhibitorjev endogenih proteaz v E. coli.

• Language: Slovenian
• Keywords: GSK3β, β-katenin, signalna pot Wnt, β-katenin uničevalni kompleks
• 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-149342
• COBISS.SI-ID: 168339715
• Publication date in RUL: 06.09.2023

Secondary language

• Language: English
• Title: Molecular cloning, expression and isolation of recombinant human GSK3β

Abstract

Glycogen synthase kinase 3 beta (GSK3β) is a serine/threonine kinase that has an important role in several biochemical processes and diseases. It consists of an N-terminal domain rich in β-strands and a C-terminal α-helical domain. As part of the biomolecular condensate of the β-catenin destruction complex, GSK3β participates in both positive and negative regulation of the Wnt signalling pathway. In the absence of signaling with Wnt ligands GSK3β hyperphosphorylates β-catenin and marks it for ubiquitination and proteasomal degradation. As part of the diploma thesis, we wanted to express and isolate human recombinant GSK3β in order to better understand its role and interactions with the proteins of the β-catenin destruction complex. We wanted to express GSK3β in E. coli strain BL21[DE3]. The gene for GSK3β was inserted into the pET32b(+) vector and fused at the 5’ end with a sequence for the hexahistidine tag, followed by gene for sfGFP. The hexahistidine tag and sfGFP could be cleaved by TEV protease. In the following, we expressed the genes and tried to isolate GSK3β by nickel affinity chromatography. The process of protein expression and isolation did not turn out to be optimal, since endogenous proteases most likely cleaved GSK3β during expression. We found out that when pRARE plasmid, which has genes for some tRNAs that are rare in E. coli, was included in protocol, GSK3β was less expressed in E. coli strain BL21[DE3]. For more successful expression and isolation of the recombinant human GSK3β, we could try to express the protein with the addition of inhibitors of endogenous proteases in E. coli.

• Keywords: GSK3β, β-catenin, Wnt signalling pathway, β-catenin destruction complex