Siyan (mavi) Fluoresan Protein Aquamarine’nin Biyoreaktörde Üretilmesi ve Saflaştırılması

Yıl 2020, Sayı: 18, 300 - 305, 15.04.2020

Hülya Kuduğ Ceylan , Rizvan İmamoğlu , İsa Gökçe

https://doi.org/10.31590/ejosat.660492

Öz

Aquamarine proteini siyan (mavi ) floresan protein ailesinin bir üyesi olup ve Aequorea victoria'dan elde edilen GFP (yeşil floresan protein) türevi floresan bir proteindir. Aquamarine benzeri floresan proteinler genetik mühendisliği teknikleri ile geliştirilmiş özellikleri sayesinde canlı hücrelerin biyolojik olarak görüntülenmesi ve hücre içerisinde lokalizasyon çalışmaları için sıklıkla kullanılmaktadır. Bu çalışmada Escherichia coli BL21(AI) hücreleri pROEX Aqua plasmid DNA’sı ile transforme edilmiş ve % 0.04 konsantrasyonda arabinoz ilavesi ile protein ekspresyonu indüklenmiştir. 3L kültür hacimli biyoreaktörde yüksek ekspresyon seviyesinde üretilen Histidin etiketli hedef protein Ni+2 afinite kromotografisi ile saflaştırılmıştır. Saflaştırılan protein konsantrasyonu bir litre bakteri kültürü için 80 mg olarak belirlenmiştir. Rekombinant Aquamarine’ne ait fotolüminesans özellikler florometre ile analiz edilmiştir. SDS-PAGE analizi rekombinant Aquamarine proteine ait tek bir bandın olduğunu ve saflaştırılan proteinin ileri çalışmalarda kullanılmak üzere yüksek verimde ve saflıkta (>%95) elde edildiğini göstermektedir.

Anahtar Kelimeler

Aquamarine, Siyan (mavi) Floresan Proteini, GFP, E. coli, Rekombinant protein

Destekleyen Kurum

Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (TÜBİTAK)

Proje Numarası

114Z956

Teşekkür

Bu çalışmanın gerçekleşmesi için verdiği mali destekten dolayı (Proje No: 114Z956) Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (TÜBİTAK)’na teşekkür ederiz.

Production of Cyan (blue) Fluorescent Protein Aquamarine in a Bioreactor and Purification

Öz

Aquamarine is a member of the cyan fluorescent protein (CFP) protein family and is a GFP (green fluorescent protein) derived fluorescent protein from Aequorea victoria. Aquamarine-like fluorescence proteins are often used for biological imaging and localization studies of living cells due to their properties developed by genetic engineering techniques. In this study, Escherichia coli BL21 (AI) cells were transformed with pROEX Aqua plasmid DNA and protein expression was induced by the addition of arabinose at a concentration of 0.04%. Histidine-labeled target protein produced at high expression levels in the bioreactor that has 3L culture volume was purified by Ni+2 affinity chromatography. About 80 mg of the purified protein was yielded per liter of bacterial culture. The photoluminescence properties of the Aquamarine were analyzed by fluorimeter. SDS-PAGE analysis shows a single band corresponding to the recombinant Aquamarine that was obtained high yield and purity (>95%) for further studies.

Anahtar Kelimeler

Aquamarine, Cyan Fluorescent Protein, GFP, E. coli, Recombinant Protein

Proje Numarası

114Z956

Kaynakça

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