A REVIEW ON INTEIN MEDIATED RECOMBINANT PROTEIN PURIFICATION PROCESSES WITHOUT USE OF PROTEASE

Öz

In this study, researches on inteins and recombinant proteins to increase splitting activity of inteins that are utilised in recombinant protein purification processes without use of protease have been investigated. Researches on protein cleaving by use of inteins are aimed to make inteins and protein purification processes become more effective. The processes conducted in vivo ve in vitro, intein and target protein included polyclonal PCR products are cloned and the expressed plasmids are fused to cloned strains. Kinteics of activity of cleaving and activity of cleaving rates have been determined in different pH, temperature and filtration times.   In the researches, protein cleaving activity of the different microbial natural and engineered inteins have been investigated and cleaving activity of recombinant Nostoc punctiforme DnaE C-inteins were found to be very high. The studies have been focused on the C-inteins due to recepient ability of the amino acid additions.

Anahtar Kelimeler

• Intein,Recombinant protein purification,Cloning

YENİDEN BİRLEŞTİRİLMİŞ PROTEİNLERİN PROTEAZ KULLANILMADAN SAFLAŞTIRILMASINDA İNTEİN ARACILI AYIRMA SÜREÇLERİNİN İNCELENMESİ ÜZERİNE BİR DERLEME

Öz

Bu çalışmada, yapay proteinlerin proteaz kullanılmadan saflaştırılması süreçlerinde yararlanılan inteinlerin ayrılma aktivitelerinin artırılması için inteinler ve hedef proteinler üzerinde yapılan araştırmalar incelenmiştir. İntein kullanımı ile protein ayrışması üzerinde yapılan araştırmalar, intein ve protein saflaştırma süreçlerinin daha verimli hale getirilmesini amaçlamaktadır. In vivo ve in vitro olarak gerçekleşen süreçlerde, intein ve hedef protein içeren poliklonal PCR ürününün çoğaltılması ve ekspres plazmidlerin klonlanan suşlara taşınması işlemleri yapılmaktadır. İnteinin proteinden ayrılma aktivitesi ve ayrılma oranı sabiti kinetikleri, değişik pH, sıcaklık ve süzme sürelerinde belirlenmekte ve hedef proteinlere ilişkili olarak karşılaştırılmaları yapılmaktadır. Araştırmalarda değişik mikrobiyal doğal ve yapay inteinlerin protein ayrıştırma aktiviteleri incelenmiş ve yapay Nostoc punctiforme DnaE C-inteinlerinin ayrışma aktivitelerinin en yüksek olduğu belirlenmiştir. Çalışmalar amino asit eklenmesine daha yatkın olması nedeniyle C-inteinler üzerinde yoğunlaşmaktadır.

Anahtar Kelimeler

• İntein,Rekombinant protein saflaştırma,Klonlama,Kinetik

Kaynakça

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