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Journals / Türk Biyokimya Dergisi / 2015 / Cilt: 40 - Sayı: 4

Sumo füzyon partneri kullanarak hCA IX izoenziminin ekspresyonu ve antitümör ilaçların etkilerinin incelenmesi

Expression of hCA IX isoenzyme by using sumo fusion partner and examining the effects of antitumor drugs

Murat ŞENTÜRK
Emrah YERLİKAYA
Ramazan DEMİRDAĞ
Ömer İrfan KÜFREVİOĞLU
Orhan ERDOĞAN
Pages
334–342
Publication Date
2022-11-04
DOI
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AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

In vitro inhibition studies on the recombinant hCA IX enzyme can shed light on the development of anticancer drugs for cancers overexpressing CA IX.

📌 3 Key Takeaways

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Objective & Hypothesis:Objective: In this study, investigating the effects of inhibition of the enzyme activity of some antitumor drugs and the Cancer-Related Human Carbonic Anhydrase IX (hCA IX) isoenzyme expressing as a SUMO fusion protein in an Escherichia coli expression system were aimed.Methods: hCA IX isoenzyme was expressed using SUMO fusion technology.
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Key Findings & Data:IC50 values of drugs showing the inhibitory effect were calculated.
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Conclusion & Impact:In vitro inhibition studies on the recombinant hCA IX enzyme can shed light on the development of anticancer drugs for cancers overexpressing CA IX.
🔬Design:Research Article
📍Scope:Comprehensive Sample
⚙️Variables:Domain Parameters

Abstract

Objective: In this study, investigating the effects of inhibition of the enzyme activity of some antitumor drugs and the Cancer-Related Human Carbonic Anhydrase IX (hCA IX) isoenzyme expressing as a SUMO fusion protein in an Escherichia coli expression system were aimed.Methods: hCA IX isoenzyme was expressed using SUMO fusion technology. The fusion protein was expressed in a totally soluble form and the expression was verified by SDS-PAGE analysis. Affinity chromatography was used in the purification processes. The effects of certain antitumor drugs on enzyme activity were investigated in vitro conditions by using esterase activity. IC50 values of drugs showing the inhibitory effect were calculated. Inhibition types and Ki values for antitumor drugs, which inhibit the enzyme, were determined by separately plotting Lineweaver-Burk plots.Results: The molecular weight of the fusion protein was approximately 85kDa. The optimal induction concentration of IPTG and the growth temperature were found to be 1.0mM and 30oC. The fusion protein was purified at approximately 3.07-fold with a yield of 92.58%, and a specific activity of 43707EU/mg proteins by nickel nitrilo-triacetic acid resin chromatography Conclusion: Our work is extremely important because CA IX plays a clinical role as a biomarker in cancer diagnosis and the use of specific inhibitors of the CA IX enzyme will be useful in the fight against cancer. In vitro inhibition studies on the recombinant hCA IX enzyme can shed light on the development of anticancer drugs for cancers overexpressing CA IX.

Özet

Amaç: Bu çalışmada kanser ilişkili İnsan Karbonik Anhidraz IX (hCA IX) izoenziminin Escherichia coli ekspresyon sistemi içinde bir SUMO füzyon proteini olarak ekspre edilmesi ve bazı antitümör ilaçların enzim aktivitesi üzerine inhibisyon etkilerinin incelenmesi amaçlanmıştır.Metod: hCA IX izoenzimi SUMO füzyon teknolojisi kullanılarak ekspre edildi. Füzyon protein tümüyle çözünür bir formda ekspre edildi ve ekspresyon SDS-PAGE analizi ile doğrulandı. Saflaştırma işlemlerinde afinite kromatografisi kullanıldı. Enzim aktivitesi üzerine bazı antitümör ilaçların etkileri in vitro şartlarda esteraz aktivitesi metodu kullanılarak incelendi. İnhibitör etkisi gösteren ilaçların IC50 değerleri hesaplandı. Enzimi ihhibe eden antitümör ilaçlar için Ki değerleri ve inhibisyon tipleri Lineweaver-Burk grafikleri ayrı ayrı çizilerek belirlendi.Bulgular: Füzyon proteinin molekül ağırlığı yaklaşık olarak 85 kDa'du. Optimal IPTG indükleme konsantrasyonu ve büyüme sıcaklığı 1,0 mM ve 30oC olarak bulundu. Füzyon protein nikel-nitrilotriasetik asit (Ni-NTA) afinite kromatografisi kullanılarak yaklaşık olarak 43707 EUxmg-1 spesifik aktiviteyle; %92,58 verimle; 3,07 saflaştırma katsayısıyla saflaştırıldı.Sonuç: CA IX enzimi, kanser tanısında belirteç olarak klinik rol oynadığından ve CA IX enziminin spesifik inhibitörlerinin kullanımı kansere karşı mücadelede yararlı olacağından dolayı çalışmamız önem arzetmektedir. Rekombinant hCA IX enzimi üzerine yapılacak olan in vitro inhibisyon çalışmaları CA IX'un aşırı ekspresyon gösterdiği kanserler için antikanser ilaçlarının geliştirilmesine ışık tutabilir.

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Cite This Article

ŞENTÜRK, M., YERLİKAYA, E., DEMİRDAĞ, R., KÜFREVİOĞLU, Ö. İ., ERDOĞAN, O. (2022). Sumo füzyon partneri kullanarak hCA IX izoenziminin ekspresyonu ve antitümör ilaçların etkilerinin incelenmesi. Türk Biyokimya Dergisi, Cilt: 40 - Sayı: 4, 334–342. https://ajindex.com/articles/621101

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