Araştırma Makalesi
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Molecular Modification of Escherichia coli Expression Plasmid pTolT by Point Mutation Method

Yıl 2019, Sayı: 17, 1 - 8, 31.12.2019

Öz

Bacteria are among the most preferred hosts for their easy culture conditions, simple structure and allowing genetic manipulations for recombinant protein production. Escherichia coli is the most widely used prokaryotic host in many studies. In addition to all these advantages, the high amount production of foreign proteins can cause inclusion bodies or toxic effects in Escherichia coli. The pTolT expression vector developed to overcome these problems allows the production of heterologous proteins with the Escherichia coli periplasmic region protein TolAIII. However, cysteines in positions 31 and 56 of the TolAIII influence the activity of the recombinant protein via unwanted disulfide bridges with other proteins produced by this system. The aim of this study was to remove cysteines in TolAIII protein and replace serine amino acids. The in vitro point mutation technique is an advanced technique that is frequently used for modifying vector DNA sequences, protein structure and function studies, and genetic research to regulate gene cloning and expression strategies. In this technique, polymerase chain reaction (PCR) by mutagenic primers produces many new copies of the template plasmid DNA. In the present study, C31S and C56S mutations in the TolAIII gene sequence have been performed by QuikChange site-directed mutagenesis method. Following the mutation confirmed by DNA sequence analysis results, the obtained new plasmid was called pTolT Delta. Thus, it will be easier for the recombinant proteins to be produced with the new expression system developed by this study to acquire their natural three-dimensional conformation.

Kaynakça

  • Mansfeld, J., Vriend, G., Dijkstra, B. W., Veltman, O. R., Van den Burg, B., Venema, G. & Eijsink, V. G. (1997). Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond. Journal of Biological Chemistry, 272(17), 11152-11156.
  • Bulaj, G. (2005). Formation of disulfide bonds in proteins and peptides. Biotechnology advances, 23(1), 87-92.
  • Ulusu, Y., Şentürk, S. B., Kuduğ, H., & Gökçe, İ. (2016). Expression, purification, and characterization of bovine chymosin enzyme using an inducible pTOLT system. Preparative Biochemistry and Biotechnology, 46(6), 596-601.
  • LaVallie, E. R., & McCoy, J. M. (1995). Gene fusion expression systems in Escherichia coli. Current opinion in biotechnology, 6(5), 501-506.
  • Makrides, S. C. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Mol. Biol. Rev., 60(3), 512-538.
  • Crowe, J., Dobeli, H., Gentz, R., Hochuli, E., Stiiber, D., & Henco, K. (1994). 6xffis-ni-nta chromatography as a superior technique in recombinant protein expression/purification. In Protocols for gene analysis (pp. 371-387). Humana Press.
  • Derman, A. I., Prinz, W. A., Belin, D., & Beckwith, J. (1993). Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science, 262(5140), 1744-1747.
  • Stewart, E. J., Åslund, F., & Beckwith, J. (1998). Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. The EMBO journal, 17(19), 5543-5550.
  • Levengood, S. K., Beyer, W. F., & Webster, R. E. (1991). TolA: a membrane protein involved in colicin uptake contains an extended helical region. Proceedings of the National Academy of Sciences, 88(14), 5939-5943.
  • Lazzaroni, J. C., Germon, P., Ray, M. C., & Vianney, A. (1999). The Tol proteins of Escherichia coli and their involvement in the uptake of biomolecules and outer membrane stability. FEMS microbiology letters, 177(2), 191-197.
  • Lubkowski, J., Hennecke, F., Plückthun, A., & Wlodawer, A. (1999). Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA. Structure, 7(6), 711-722.
  • Anderluh, G., Gökçe, I., & Lakey, J. H. (2003). Expression of proteins using the third domain of the Escherichia coli periplasmic-protein TolA as a fusion partner. Protein expression and purification, 28(1), 173-181.
  • Gokce, I., Anderluh, G., & Lakey, J. H. (2008). U.S. Patent No. 7,348,408. Washington, DC: U.S. Patent and Trademark Office.
  • Khoo, K., & Norton, R. S. (2011). Role of disulfide bonds in peptide and protein conformation. In Amino Acids, Peptides and Proteins in Organic Chemistry: Analysis and Function of Amino Acids and Peptides (pp. 395-417). Wiley-VCH Verlag GmbH & Co. KGaA.
  • Mallick, P., Boutz, D. R., Eisenberg, D., & Yeates, T. O. (2002). Genomic evidence that the intracellular proteins of archaeal microbes contain disulfide bonds. Proceedings of the National Academy of Sciences, 99(15), 9679-9684.

Escherichia coli Ekspresyon Plazmiti pTolT’nin Nokta Mutasyonu Yöntemiyle Moleküler Modifikasyonu

Yıl 2019, Sayı: 17, 1 - 8, 31.12.2019

Öz

Bakteriler rekombinant protein üretimi için kolay kültür edilmeleri, basit yapıları ve genetik manipülasyonlara açık olmaları nedeniyle en çok tercih edilen konakçılardandır. Özellikle Escherichia coli en yaygın olarak kullanılan prokaryotik konakçı olarak pek çok araştırmada karşımıza çıkmaktadır. Tüm bu avantajlarının yanında, yabancı proteinlerin yüksek miktardaki üretimleri, Escherichia coli’de inklüzyon cisimciklerinin oluşmasına veya toksik etkilere neden olabilmektedir. Bu problemlerin aşılması amacıyla geliştirilen pTolT ekspresyon vektörü heterolog proteinlerin Escherichia coli periplazmik bölge proteini TolAIII ile birlikte üretilmesine imkân sağlamaktadır. Bununla birlikte TolAIII proteininde 31 ve 56. pozisyonda bulunan sisteinler bu sistemle üretilen diğer proteinler ile istenmeyen disülfit köprüleri kurarak rekombinant proteinin aktivitesi üzerine etki etmektedir. Bu çalışmanın amacı TolAIII proteininde bulunan sisteinlerin kaldırılarak yerine serin amino asitlerinin getirilmesidir. In vitro nokta mutasyonu tekniği, gen klonlama ve ekspresyon stratejilerinin düzenlenmesi amacıyla vektör DNA dizilerinin modifiye edilmesinde, protein yapı ve fonksiyon çalışmalarında ve genetik araştırmalarda sıklıkla kullanılan gelişmiş bir tekniktir. Bu teknikte mutajenik primerler vasıtasıyla polimeraz zincir reaksiyonu (PZR) işlemi ile kalıp plazmid DNA’nın birçok yeni kopyasını meydana getirilir. Sunulan çalışmada QuikChange site-directed mutagenesis metoduyla TolAIII gen dizisinde C31S ve C56S mutasyonları gerçekleştirilmiştir. DNA sekans analizi sonuçları ile teyit edilen mutasyonun ardından elde edilen yeni plazmid pTolT Delta olarak adlandırılmıştır. Böylece bu çalışma ile geliştirilen yeni ekspresyon sistemi ile üretilecek rekombinant proteinlerin doğal üç boyutlu konformasyonlarını kazanmaları daha kolay olacaktır.

Kaynakça

  • Mansfeld, J., Vriend, G., Dijkstra, B. W., Veltman, O. R., Van den Burg, B., Venema, G. & Eijsink, V. G. (1997). Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond. Journal of Biological Chemistry, 272(17), 11152-11156.
  • Bulaj, G. (2005). Formation of disulfide bonds in proteins and peptides. Biotechnology advances, 23(1), 87-92.
  • Ulusu, Y., Şentürk, S. B., Kuduğ, H., & Gökçe, İ. (2016). Expression, purification, and characterization of bovine chymosin enzyme using an inducible pTOLT system. Preparative Biochemistry and Biotechnology, 46(6), 596-601.
  • LaVallie, E. R., & McCoy, J. M. (1995). Gene fusion expression systems in Escherichia coli. Current opinion in biotechnology, 6(5), 501-506.
  • Makrides, S. C. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Mol. Biol. Rev., 60(3), 512-538.
  • Crowe, J., Dobeli, H., Gentz, R., Hochuli, E., Stiiber, D., & Henco, K. (1994). 6xffis-ni-nta chromatography as a superior technique in recombinant protein expression/purification. In Protocols for gene analysis (pp. 371-387). Humana Press.
  • Derman, A. I., Prinz, W. A., Belin, D., & Beckwith, J. (1993). Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science, 262(5140), 1744-1747.
  • Stewart, E. J., Åslund, F., & Beckwith, J. (1998). Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. The EMBO journal, 17(19), 5543-5550.
  • Levengood, S. K., Beyer, W. F., & Webster, R. E. (1991). TolA: a membrane protein involved in colicin uptake contains an extended helical region. Proceedings of the National Academy of Sciences, 88(14), 5939-5943.
  • Lazzaroni, J. C., Germon, P., Ray, M. C., & Vianney, A. (1999). The Tol proteins of Escherichia coli and their involvement in the uptake of biomolecules and outer membrane stability. FEMS microbiology letters, 177(2), 191-197.
  • Lubkowski, J., Hennecke, F., Plückthun, A., & Wlodawer, A. (1999). Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA. Structure, 7(6), 711-722.
  • Anderluh, G., Gökçe, I., & Lakey, J. H. (2003). Expression of proteins using the third domain of the Escherichia coli periplasmic-protein TolA as a fusion partner. Protein expression and purification, 28(1), 173-181.
  • Gokce, I., Anderluh, G., & Lakey, J. H. (2008). U.S. Patent No. 7,348,408. Washington, DC: U.S. Patent and Trademark Office.
  • Khoo, K., & Norton, R. S. (2011). Role of disulfide bonds in peptide and protein conformation. In Amino Acids, Peptides and Proteins in Organic Chemistry: Analysis and Function of Amino Acids and Peptides (pp. 395-417). Wiley-VCH Verlag GmbH & Co. KGaA.
  • Mallick, P., Boutz, D. R., Eisenberg, D., & Yeates, T. O. (2002). Genomic evidence that the intracellular proteins of archaeal microbes contain disulfide bonds. Proceedings of the National Academy of Sciences, 99(15), 9679-9684.
Toplam 15 adet kaynakça vardır.

Ayrıntılar

Birincil Dil Türkçe
Konular Mühendislik
Bölüm Makaleler
Yazarlar

Yakup Ulusu 0000-0002-8755-2822

Yayımlanma Tarihi 31 Aralık 2019
Yayımlandığı Sayı Yıl 2019 Sayı: 17

Kaynak Göster

APA Ulusu, Y. (2019). Escherichia coli Ekspresyon Plazmiti pTolT’nin Nokta Mutasyonu Yöntemiyle Moleküler Modifikasyonu. Avrupa Bilim Ve Teknoloji Dergisi(17), 1-8.