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

Year 2017, Volume: 6 Issue: 2, 95 - 102, 31.08.2017

Yakup Ermurat

https://doi.org/10.18036/aubtdc.267017

Abstract

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.

Keywords

Intein, Recombinant protein purification, Cloning

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

Abstract

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.

Keywords

İntein, Rekombinant protein saflaştırma, Klonlama, Kinetik

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