Hülya KUDUĞ CEYLAN, Rizvan İMAMOĞLU, İsa GÖKÇE

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

Aquamarine proteini siyan (mavi) floresan protein ailesinin bir üyesi olup ve Aequorea victoria'dan elde edilen GFP (yeşil floresanprotein) türevi floresan bir proteindir. Aquamarine benzeri floresan proteinler genetik mühendisliği teknikleri ile geliştirilmiş özelliklerisayesinde 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 konsantrasyondaarabinoz ilavesi ile protein ekspresyonu indüklenmiştir. 3L kültür hacimli biyoreaktörde yüksek ekspresyon seviyesinde üretilen Histidinetiketli hedef protein Ni+2 afinite kromotografisi ile saflaştırılmıştır. Saflaştırılan protein konsantrasyonu bir litre bakteri kültürü için 80mg olarak belirlenmiştir. Rekombinant Aquamarine’ne ait fotolüminesans özellikler florometre ile analiz edilmiştir. SDS-PAGE analizirekombinant Aquamarine proteine ait tek bir bandın olduğunu ve saflaştırılan proteinin ileri çalışmalarda kullanılmak üzere yüksekverimde ve saflıkta (>%95) elde edildiğini göstermektedir.

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

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.

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Alvarez, L., Levin, C. H., Merola, F., Bizouarn, T., Pasquier, H., Baciou, L., Rusconi, F., Erard, M. (2010). Are the fluorescent properties of the cyan fluorescent protein sensitive to conditions of oxidative stress? Photochem Photobiol, 86(1), 55–61.
Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A., Tsien, R. Y., (2002). A monomeric red fluorescent protein. Proceedings of the National Academy of Sciences, 99(12), 7877–7882.
Choi, J. H., Keum, K. C., Lee, S. Y. (2006). Production of recombinant proteins by high cell density culture of Escherichia coli. Chemical Engineering Science, 61(3), 876–885.
Chudakov, D. M., Lukyanov, S., Lukyanov, K. A. (2005). Fluorescent proteins as a toolkit for in vivo imaging. Trends in Biotechnology, 23(12), 605–613.
Erard, M., Fredj, A., Pasquier, H., Beltolngar, D. B., Bousmah, Y., Derrien, V., Merola, F. (2013). Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging. Molecular BioSystems, 9(2), 258–267.
Goedhart, J., Von Stetten, D., Noirclerc-Savoye, M., Lelimousin, M., Joosen, L., Hink, M. A., Royant, A. (2012). Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nature Communications, 3, 751.
Heim, R., Cubitt, A., Tsien, R. (1995). Improved green fluorescence. Nature, 373(6516), 663-664.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680- 685.
Mérola, F., Fredj, A., Betolngar, D. B., Ziegler, C., Erard, M., Pasquier, H. (2014). Newly engineered cyan fluorescent proteins with enhanced performances for live cell FRET imaging. Biotechnology Journal, 9(2), 180–191.
Park, S. W., Kang, S., Yoon, T. S. (2016). Crystal structure of the cyan fluorescent protein Cerulean-S175G. Acta Crystallographica Section:F Structural Biology Communications, 72, 516–522.
Pascual, A., García, I., Ballesta, S., Perea, E. J. (1999). Uptake and intracellular activity of moxifloxacin in human neutrophils and tissue-cultured epithelial cells. Antimicrobial Agents and Chemotherapy, 43(1), 12–15.
Phillips, G. J. (2001). Green fluorescent protein--a bright idea for the study of bacterial protein localization. FEMS Microbiology Letters, 204(1), 9–18.
Rizzo, M. A., Granada, B., Piston, D. W. (2004). An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnology, 20, 445-449.
Rosano, G. L., Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology, 5, 1–17.
Sawano, A., Miyawaki, A. (2000). Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Research, 28(16), e78.
Shaner N. C., Steinbach, P. A., Tsien, R. Y. (2005). A guide to choosing fluorescent proteins. Nature Methods, 2(12), 905-909.
Shemiakina, I. I., Ermakova, G. V., Cranfill, P. J., Baird, M. A., Evans, R. A., Souslova, E. A, Staroverov, D. B., Gorokhovatsky, A. Y., Putintseva, E. V., Gorodnicheva, T. V., Chepurnykh, T. V, Strukova, L., Lukyanov, S., Zaraisky, A. G., Davidson, M. W., Chudakov, D. M., Shcherbo, D. (2012). A monomeric red fluorescent protein with low cytotoxicity. Nature Communications, 3(1), 1204.
Tsien, R. Y. (1998). The Green Fluorescent Protein. Annual Review of Biochemistry, 67(1), 509–544
Verkhusha, V. V., Lukyanov, K. A. (2004). The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nature Biotechnology, 22(3), 289–296.
Wilcox, T., Hirshkowitz, A. (2015). The effect of color priming on infant brain and behavior. NIH Public Access, 85(1), 302-313.