High-Level Production of MMLV Reverse Transcriptase Enzyme in Escherichia Coli

Yıl 2021, Cilt: 33 Sayı: 4, 549 - 555, 30.12.2021

Özlem Kaplan Rizvan İmamoğlu İsa Gökçe

https://doi.org/10.7240/jeps.877806

Cited By: 1

Öz

Reverse transcriptase (RT) of Moloney murine leukemia virus (MMLV) is the most widely used enzyme for cDNA synthesis and RNA amplification. In this study, we aimed to produce MMLV RT enzyme recombinantly due to its importance in molecular studies. In this context, the DNA fragment encoding the MMLV RT enzyme was cloned into pTOLT plasmid and expressed in E. coli BL21 (DE3) pLysE cells. Since the high-level expression of the protein caused the protein molecules to aggregate in the inclusion bodies, co-expression of MMLV RT and chaperone plasmids (pG-KJE8, pGro7, pKJE7, pGTf2, pTf16) was performed to obtain the MMLV RT protein in soluble form. Contrary to our expectations, because it could not be obtained in soluble form, the protein was recovered from the inclusion bodies using refolding process. Finally, the protein was purified by affinity chromatography and the activity of the protein was checked using RT-PCR technique.

Anahtar Kelimeler

Inclusion bodies, Moloney murine leukemia virus, Reverse transcriptase, cDNA synthesis

Destekleyen Kurum

Turkish Scientific and Technical Research Council (TUBITAK)

Proje Numarası

119Z581

Teşekkür

This study was supported by the Turkish Scientific and Technical Research Council (TUBITAK) under Grant number: 119Z581

High-Level Production of MMLV Reverse Transcriptase Enzyme in Escherichia Coli

Öz

Moloney murin lösemi virüs (MMLV) ters transkriptazı (RT), cDNA sentezi ve RNA amplifikasyonu için en yaygın kullanılan enzimdir. Bu çalışmada moleküler çalışmalardaki önemi nedeniyle MMLV RT enziminin rekombinant olarak üretilmesi amaçlanmıştır. Bu bağlamda, MMLV RT enzimini kodlayan DNA fragmanı pTOLT plazmidine klonlanmış ve E. coli BL21 (DE3) pLysE hücrelerinde eksprese edilmiştir. Proteinin yüksek düzeyde ekspresyonu, protein moleküllerinin inklüzyon cisimciklerinde toplanmasına neden olduğundan MMLV RT proteininin çözünür formda elde edilebilmesi için MMLV RT ve şaperon plazmidlerin (pG-KJE8, pGro7, pKJE7, pGTf2, pTf16) birlikte ekspresyonu gerçekleştirilmiştir. Beklentilerimizin aksine, protein çözünür formda elde edilemediği için, yeniden katlama prosesi kullanılarak inklüzyon cisimciklerinden geri kazanılmıştır. Son olarak protein, afinite kromatografisiyle saflaştırılmış ve proteinin aktivitesi RT-PCR tekniği kullanılarak kontrol edilmiştir.

Anahtar Kelimeler

İnklüzyon cisimcikleri, Moloney murin lösemi virüsü, Ters transkriptaz, cDNA sentezi

Proje Numarası

119Z581

Kaynakça

• Yasukawa, K., Nemoto, D. &Inouye, K. (2008). Comparison of the thermal stabilities of reverse transcriptases from avian myeloblastosis virus and Moloney murine leukaemia virus. J Biochem, 143, 261-268.
• Levin, J. G., Crouch, R. J., Post, K., Hu, S. C., Mckelvin, D., Zweig, M., Court, D. L. & Gerwin, B. I. (1988). Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity. J Virol, 62, 4376-4380.
• Konishi, A., Nemoto, D., Yasukawa, K. & Inouye, K. (2011). Comparison of the thermal stabilities of the alphabeta heterodimer and the alpha subunit of avian myeloblastosis virus reverse transcriptase. Biosci Biotechnol Biochem,75, 1618-1620.
• Rittie, L. & Perbal, B. (2008). Enzymes used in molecular biology: a useful guide. J Cell Commun Signal, 2, 25-45.
• Arnorsdottir, J., Helgadottir, S., Thorbjarnardottir, S. H., Eggertsson, G. & Kristjansson, M. M. (2007). Effect of selected Ser/Ala and Xaa/Pro mutations on the stability and catalytic properties of a cold adapted subtilisin-like serine proteinase. Biochim Biophys Acta, 1774, 749-55.
• Kusano, M., Yasukawa, K. & Inouye, K. (2010). Effects of the mutational combinations on the activity and stability of thermolysin. J Biotechnol, 147, 7-16.
• Das, D. & Georgiadis, M. M. (2001). A directed approach to improving the solubility of Moloney murine leukemia virus reverse transcriptase. Protein Sci, 10, 1936-41.
• Fei, X., Xuemei, M., Xiansong, W., 2012. Soluble Expression and Purification of Histidine-Tagged Moloney Murine Leukemia Virus Reverse Transcriptase by Ni-NTA Affinity Chromatography. Affinity Chromatography, 17, 357-368.
• Khosrowabadi, E., Takalloo, Z., Sajedi, R. H. & Khajeh, K. (2018). Improving The Soluble Expression Of Aequorin In Escherichia Coli Using The Chaperone-Based Approach By Co-Expression With Artemin. Prep Biochem Biotechnol, 48, 483-489.
• Sachsenhauser, V. & Bardwell, J. C. (2018). Directed Evolution To Improve Protein Folding In Vivo. Curr Opin Struct Biol, 48, 117-123.
• De Marco, A. (2007). Protocol For Preparing Proteins With Improved Solubility By Co-Expressing With Molecular Chaperones In Escherichia Coli. Nat Protoc, 2, 2632-9.
• Yao, D., Fan, J., Han, R., Xiao, J., Li, Q. & Xu, G. (2020). Enhancing soluble expression of sucrose phosphorylase in Escherichia coli by molecular chaperones. Protein Expr Purif, 169, 105571.
• Levy, R., Weiss, R., Chen, G., Iverson, B. L. & Georgiou, G. (2001). Production Of Correctly Folded Fab Antibody Fragment In The Cytoplasm Of Escherichia Coli Trxb Gor Mutants Via The Coexpression Of Molecular Chaperones. Protein Expr Purif, 23, 338-47.
• Ronez, F., Desroche, N., Arbault, P. & Guzzo, J. (2012). Co-Expression Of The Small Heat Shock Protein, Lo18, With Beta-Glucosidase In Escherichia Coli Improves Solubilization And Reveals Various Associations With Overproduced Heterologous Protein, Groel/Es. Biotechnol Lett, 34, 935-9.
• Shuo-Shuo, C., Xue-Zheng, L. & Ji-Hong, S. (2011). Effects Of Co-Expression Of Molecular Chaperones On Heterologous Soluble Expression Of The Cold-Active Lipase Lip-948. Protein Expr Purif, 77, 166-72.
• Jhamb, K. & Sahoo, D. K. (2012). Production Of Soluble Recombinant Proteins In Escherichia Coli: Effects Of Process Conditions And Chaperone Co-Expression On Cell Growth And Production Of Xylanase. Bioresour Technol, 123, 135-43.
• Voulgaridou, G. P., Mantso, T., Chlichlia, K., Panayiotidis, M. I. & Pappa, A. (2013). Efficient E. Coli Expression Strategies For Production Of Soluble Human Crystallin Aldh3a1. Plos One, 8, E56582.
• Yan, X., Hu, S., Guan, Y. X. & Yao, S. J. (2012). Coexpression Of Chaperonin Groel/Groes Markedly Enhanced Soluble And Functional Expression Of Recombinant Human Interferon-Gamma In Escherichia Coli. Appl Microbiol Biotechnol, 93, 1065-74.
• Sorensen, H. P. & Mortensen, K. K. (2005). Soluble Expression Of Recombinant Proteins In The Cytoplasm Of Escherichia Coli. Microb Cell Fact, 4, 1.
• Hartl, F. U. & Hayer-Hartl, M. (2002). Molecular Chaperones In The Cytosol: From Nascent Chain To Folded Protein. Science, 295, 1852-8.
• Folwarczna, J., Moravec, T., Plchova, H., Hoffmeisterova, H. & Cerovska, N. (2012). Efficient Expression Of Human Papillomavirus 16 E7 Oncoprotein Fused To C-Terminus Of Tobacco Mosaic Virus (Tmv) Coat Protein Using Molecular Chaperones In Escherichia Coli. Protein Expr Purif, 85, 152-7.
• Chen, Y., Xu, W. & Sun, Q. (2009). A Novel And Simple Method For High-Level Production Of Reverse Transcriptase From Moloney Murine Leukemia Virus (Mmlv-Rt) In Escherichia Coli. Biotechnol Lett, 31, 1051-7.
• Anderluh, G., Gokce, I. & Lakey, J. H. (2003). Expression Of Proteins Using The Third Domain Of The Escherichia Coli Periplasmic-Protein Tola As A Fusion Partner. Protein Expr Purif, 28, 173-81.
• Melissis, S. C., Papageorgiou, A. C., Labrou, N. E. & Clonis, Y. D. (2010). Purification Of M-Mlvh- Rt On A 9-Aminoethyladenine-(1,6-Diamine-Hexane)-Triazine Selected From A Combinatorial Library Of Dntp-Mimetic Ligands. J Chromatogr Sci, 48, 496-502.
• Kuduğ, H., Ataman, B,. İmamoğlu, R., Düzgün, D. & Gökçe, İ. (2019). Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization. International Advanced Researches and Engineering Journal, 3, 20-25.
• Kaplan, Ö., İmamoğlu, R., Şahingöz, İ. & Gökçe, İ. (2021). Recombinant production of Thermus aquaticus single-strand binding protein for usage as PCR enhancer. International Advanced Researches and Engineering Journal, 5, 42-46.
• Garcia-Fruitos, E., Martinez-Alonso, M., Gonzalez-Montalban, N., Valli, M., Mattanovich, D. & Villaverde, A. (2007). Divergent Genetic Control Of Protein Solubility And Conformational Quality In Escherichia Coli. J Mol Biol, 374, 195-205.
• Martinez-Alonso, M., Garcia-Fruitos, E. & Villaverde, A. (2008). Yield, Solubility And Conformational Quality Of Soluble Proteins Are Not Simultaneously Favored In Recombinant Escherichia Coli. Biotechnol Bioeng, 101, 1353-8.
• Martinez-Alonso, M., Vera, A. & Villaverde, A. (2007). Role Of The Chaperone Dnak In Protein Solubility And Conformational Quality In Inclusion Body-Forming Escherichia Coli Cells. Fems Microbiol Lett, 273, 187-95.
• Rinas, U., Hoffmann, F., Betiku, E., Estape, D. & Marten, S. (2007). Inclusion Body Anatomy And Functioning Of Chaperone-Mediated In Vivo Inclusion Body Disassembly During High-Level Recombinant Protein Production In Escherichia Coli. J Biotechnol, 127, 244-57.