Izražanje in izolacija rekombinantne človeške kinaze CK1a

Mitkov, Kostadin (author), Gaber, Aljaž (mentor)

CK1a (kazeinska kinaza 1 a) je protein, ki ima več funkcij. Ima aktivnost Ser/Thr proteinske kinaze in pomembno regulativno vlogo pri številnih celičnih procesih, kot so procesiranje in popravljanje DNA, proliferacija, dinamika citoskeleta, vezikularni transport, apoptoza in diferenciacija celic. CK1a lahko tudi spremeni aktivnost ključnih proteinov, ki sodelujejo pri prenosu in integraciji signalov. V skladu s tem konceptom je CK1a ena od ključnih sestavin, ki tvorijo ß-katenin uničevalni kompleks in je tesno povezana z uravnavanjem in razgradnjo ß-katenina v signalni poti Wnt. CK1a fosforilira ß-katenin, ki ga nato skupaj z drugimi proteini, vključeni v uničevalni kompleks, pripravi za ubikvitinacijo in proteasomsko razgradnjo. Molekularni mehanizmi v zvezi z uničevalnim kompleksom ß-katenina so še vedno premalo raziskani in slabše razumljeni. Da bi preučili, kako CK1a interagira z ß-kateninom in drugimi proteini ß-katenin uničevalnega kompleksa ter kako ga fosforilira, je bil cilj naše raziskave pripraviti prečiščeno aktivno obliko proteina CK1a. Pripravljen je bil DNA konstrukt CK1a, ki sta mu na N-konec bila dodana fuzijska partnerja heksahistidinska oznaka (His6) in sfGFP. Konstrukt je bil vstavljen v tarčni vektor pET-32b(+), ki smo ga uporabili za transformacijo dveh bakterijskih kultur celic E. coli. Ena kultura je bila transformirana samo z rekombinantnim vektorjem, ki je vseboval pripravljen konstrukt, druga kultura pa je bila poleg rekombinantnega vektorja kotransformirana s plazmidom pRARE. Izražanje, izolacija in čiščenje proteina so bili izvedeni v skladu s postopkom, opisanim v literaturi. Glede na rezultate analize z NaDS-PAGE smo ugotovili, da je bil protein uspešno izražen le v bakterijski kulturi, ki je bila kotransformirana s plazmidom pRARE. Poleg tega je zelo verjetno, da so naš protein med postopkom izolacije razcepile endogene bakterijske proteaze. Te ugotovitve so omogočile vpogled v optimizacijo postopka za razvoj protokola za pripravo prečiščene aktivne oblike CK1a. Takšen protokol bi bil zelo uporaben za nadaljnje raziskave ß-katenin uničevalnega kompleksa in signalne poti Wnt.

• Language: Slovenian
• Keywords: CK1a, ß-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-149236
• COBISS.SI-ID: 167374851
• Publication date in RUL: 05.09.2023

Secondary language

• Language: English
• Title: Expression and isolation of recombinant human CK1a kinase

Abstract

CK1a (Casein kinase 1 a) is a multifunctional protein that has Ser/Thr protein kinase activity and plays major regulatory roles in many cellular processes such as DNA processing and repair, proliferation, cytoskeleton dynamics, vesicular trafficking, apoptosis, and cell differentiation. CK1a can also alter the activity of critical proteins involved in signal transduction and signal integration. In accordance with this concept, CK1a is one of the key components forming the ß-catenin destruction complex and is tightly connected to the regulation and degradation of ß-catenin in the Wnt signalling pathway. CK1a phosphorylates ß-catenin, which then subsequently, along with other proteins involved in the destruction complex, primes it for ubiquitination and proteasomal degradation. The molecular mechanisms regarding the ß-catenin destruction complex are still inadequately researched and poorly understood. In order to study how CK1a phosphorylates and interacts with ß-catenin and the other proteins within the ß-catenin destruction complex, our research aimed to prepare a purified active form of CK1a protein. A DNA construct of CK1a was prepared, with a hexa-histidine tag (His6) and sfGFP added to its N-terminus as fusion partners. The construct was inserted into the target vector pET-32b(+), which was then used to transform two cultures of E. coli cells. One culture was transformed only with the recombinant vector containing the prepared construct, while the other culture was cotransformed with the plasmid pRARE, in addition to the recombinant vector. Subsequently, expression, isolation, and purification of the protein were performed according to the procedure described in the article. According to the results obtained from the analysis with NaDS-PAGE, we concluded that the protein was successfully expressed only in the bacterial culture cotransformed with the pRARE plasmid. Furthermore, it is highly likely that our protein was cleaved during the isolation process by endogenous bacterial proteases. These findings provided insights into optimizing the procedure in order to develop a protocol for preparing a purified active form of CK1a. Such a protocol would be valuable for further research on the ß-catenin destruction complex and the Wnt signaling pathway.

• Keywords: CK1a, ß-catenin, destruction complex