Year 2019, Volume 3 , Issue 1, Pages 20 - 25 2019-04-15

Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization

Hülya Kuduğ [1] , Bahadır Ataman [2] , Rizvan İmamoğlu [3] , Duygu Düzgün [4] , İsa Gökçe [5]


New and improved genetic engineered variants of fluorescent proteins (FPs) have become useful tools for bioimaging in biomedical researches. Red fluorescent proteins (RFPs) first derived from the sea anemone Discosoma show high performance in vivo labeling and imaging. mCherry is a member of RFPs which has very high photostability, resistant to photo bleaching and rapid maturation. These advantages ensure that mCherry can be successfully fused to many proteins and widely used for quantitative imaging techniques. In this study, the constructed recombinant plasmid pBADCherry was expressed in Escherichia coli BL21(AI) then culture conditions, inducer concentration and induction time were optimized. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that 5 hours induction at 0.04% of arabinose concentration was optimal for the highest mCherry yield. The expression of hexa histidine-tagged (6xHis) recombinant mCherry was induced by arabinose and purification performed using nickel (Ni2+) affinity chromatography. High throughput expression of 81 mg fluorescent protein from a liter of E. coli culture carried out in bioreactor.

Bioreactor, E.coli, Fluorescent Proteins, mCherry, pBAD
  • 1. Shimomura, O., The discovery of aequorin and green fluorescent protein. Journal of Microscopy, 2005. 217: p. 3–15.
  • 2. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C., Green fluorescent protein as a marker for gene expression. Science, 1994. 263(5148): p. 802–805.
  • 3. Chudakov, D.M., Matz, M.V., Lunyakov, S., Lunyakov, K.A., Fluorescent proteins and their applications in imaging living cells and tissues. Physiological Reviews, 2010. 90(3): p.1103–1163.
  • 4. Shaner, N.C., Patterson, G.H., Davidson, M.W., Advances in fluorescent protein technology. Journal of Cell Science, 2007. 120: p. 4247–4260.
  • 5. Haddock, S.H., Mastoianni, N., Christianson, L.M., A photoactivatable green-fluorescent protein from the phylu. Ctenophora. Proceedings Biological Sciences, 2010. 277: p. 1155–1160.
  • 6. Chudakov, D.M., Belousov, V.V., Zaraisky, A.G., Novoselov, V.V., Staroverov, D.B., Zorov, D.B., Lukyanov, S., Lukyanov, K.A., Kindling fluorescent proteins for precise in vivo photolabeling. Nature Biotechnology, 2003. 21(2): p. 191–194.
  • 7. 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., A monomeric red fluorescent protein with low cytotoxicity. Nature Communications, 2012. 3(1): p. 1204.
  • 8. Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A., Tsien, R.Y., A monomeric red fluorescent protein. Proceedings of the National. Academy of Sciences, 2002. 99(12): p. 7877–7882.
  • 9. Shen, Y., Lai, T., Campbell, R.E., Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications. Neurophotonics, 2015. 2(3): p. 031203.
  • 10. Bin, W., Yan, C., Joachim, D.M., Fluorescence fluctuation spectroscopy of mCherry in living cells. Biophysical Journal, 2009. 96(6): p. 2391–2404.
  • 11. Shaner, N.C., Campbell R.E., Steinbach P.A., Giepmans B.N., Palmer A.E., Tsien R.Y., Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology, 2004. 22(12): p. 1567–1572.
  • 12. Ransom, E.M., Ellermeier, C.D., Weiss, D.S., Use of mCherry red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile. Applied and Environmental Microbiology, 2015. 81(5): p. 1652–1660.
  • 13. Lee, You-Jin, Jung K.-H., Modulation of the tendency towards inclusion body formation of recombinant protein by the addition of glucose in the araBAD promoter system of Escherichia coli. Journal of Microbiology and Biotechnology, 2007. 17(11): p. 1898–1903.
  • 14. Guzman, L.-M., Belin, D., Carson, M.J., Beckwith, J., Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of Bacteriology, 1995. 177(14), p. 4121-4130.
  • 15. Newman, J.R., Fuqua C., Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene, 1999. 227: p. 197-203.
  • 16. Patkar, A., Vijayasankaran, N., Urry, D.W., Srienc F., Flow cytometer as a useful tool for process development: Rapid evaluation of expression systems. Journal of Bacteriology, 2002. 93(3): p. 217-229.
  • 17. Boström, M., Markland, K., Sandén, A.M., Hedhammar, M., Hober, S., Larsson, G., Effect of substrate feed rate on recombinant protein secretion, degradation and inclusion body formation in Escherichia coli. Applied Microbiology and Biotechnology, 2005. 68(1): p. 82-90.
  • 18. Sandén, A.M., Boström, M., Markland, K., Larsson, G., Solubility and proteolysis of the Zb-MalE and Zb-MalE31 proteins during overproduction in Escherichia coli. Biotechnology and Bioengineering, 2005. 90(2): p. 239-247.
  • 19. Greefield, L., Boone, T., Wilcox, G., DNA sequence of the araBAD promoter in Escherichia coli B/r. Proceedings of the National Academy of Sciences, 1978. 75(10): p. 4724-4728.
  • 20. Siegele, D.A., Hu, J.C., Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proceedings of the National Academy of Sciences, 1997. 94(15): p. 8168–8172.
  • 21. Khlebnikov, A., Risa, Ø, Skaug, T., Carrier, T., Keasling, J.D., Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture. Journal of Bacteriology, 2007. 182(24): p. 7029-7034.
  • 22. Chae H.J., Delisa M.P., Cha H.J., Weigand W.A., Rao G., Bentley W.E., Framework for online optimization of recombinant protein expression in high-cell-density Escherichia coli cultures using GFP-fusion monitoring. Biotechnology and Bioengineering, 2000. 69(3): p. 275-285.
  • 23. Lu, C., Albano, C.R., Bentley W.E., Rao, G., Differential rates of gene expression monitored by green fluorescent protein. Biotechnology and Bioengineering, 2002. 79(4): p. 429-437.
  • 24. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970. 227(5259): p. 680-685.
  • 25. Sarabipour, S., King, C., Hristova, K., Uninduced high-yield bacterial expression of fluorescent proteins. Analytical Biochemistry, 2017. 449(1): p. 155–157.
  • 26. Demain, A.L., Vaishnav, P., Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances, 2009. 27(3): p. 297-306.
  • 27. Shaner, N.C., Steinbach, P.A., Tsien, R.Y., A guide to choosing fluorescent proteins. Nature Methods, 2005. 2(12): p. 905–909.
Primary Language en
Subjects Engineering, Multidisciplinary
Journal Section Research Articles
Authors

Orcid: 0000-0003-0365-2760
Author: Hülya Kuduğ (Primary Author)
Institution: GAZİOSMANPAŞA ÜNİVERSİTESİ, MÜHENDİSLİK VE DOĞA BİLİMLERİ FAKÜLTESİ
Country: Turkey


Orcid: 0000-0002-8782-2706
Author: Bahadır Ataman
Institution: GAZİOSMANPAŞA ÜNİVERSİTESİ, MÜHENDİSLİK VE DOĞA BİLİMLERİ FAKÜLTESİ
Country: Turkey


Orcid: 0000-0002-6306-4760
Author: Rizvan İmamoğlu
Institution: BARTIN ÜNİVERSİTESİ, FEN FAKÜLTESİ
Country: Turkey


Orcid: 0000-0002-5998-8397
Author: Duygu Düzgün
Institution: GAZİOSMANPAŞA ÜNİVERSİTESİ, MÜHENDİSLİK VE DOĞA BİLİMLERİ FAKÜLTESİ
Country: Turkey


Orcid: 0000-0002-5023-9947
Author: İsa Gökçe
Institution: GAZİOSMANPAŞA ÜNİVERSİTESİ, MÜHENDİSLİK VE DOĞA BİLİMLERİ FAKÜLTESİ
Country: Turkey


Dates

Application Date : June 1, 2018
Acceptance Date : September 24, 2018
Publication Date : April 15, 2019

Bibtex @research article { iarej429547, journal = {International Advanced Researches and Engineering Journal}, issn = {}, eissn = {2618-575X}, address = {}, publisher = {Ceyhun YILMAZ}, year = {2019}, volume = {3}, pages = {20 - 25}, doi = {}, title = {Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization}, key = {cite}, author = {Kuduğ, Hülya and Ataman, Bahadır and İmamoğlu, Rizvan and Düzgün, Duygu and Gökçe, İsa} }
APA 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 (1) , 20-25 . Retrieved from https://dergipark.org.tr/en/pub/iarej/issue/44303/429547
MLA Kuduğ, H , Ataman, B , İmamoğlu, R , Düzgün, D , Gökçe, İ . "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 (2019 ): 20-25 <https://dergipark.org.tr/en/pub/iarej/issue/44303/429547>
Chicago Kuduğ, H , Ataman, B , İmamoğlu, R , Düzgün, D , Gökçe, İ . "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 (2019 ): 20-25
RIS TY - JOUR T1 - Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization AU - Hülya Kuduğ , Bahadır Ataman , Rizvan İmamoğlu , Duygu Düzgün , İsa Gökçe Y1 - 2019 PY - 2019 N1 - DO - T2 - International Advanced Researches and Engineering Journal JF - Journal JO - JOR SP - 20 EP - 25 VL - 3 IS - 1 SN - -2618-575X M3 - UR - Y2 - 2018 ER -
EndNote %0 International Advanced Researches and Engineering Journal Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization %A Hülya Kuduğ , Bahadır Ataman , Rizvan İmamoğlu , Duygu Düzgün , İsa Gökçe %T Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization %D 2019 %J International Advanced Researches and Engineering Journal %P -2618-575X %V 3 %N 1 %R %U
ISNAD Kuduğ, Hülya , Ataman, Bahadır , İmamoğlu, Rizvan , Düzgün, Duygu , Gökçe, İsa . "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 / 1 (April 2019): 20-25 .
AMA Kuduğ H , Ataman B , İmamoğlu R , Düzgün D , Gökçe İ . Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization. IAREJ. 2019; 3(1): 20-25.
Vancouver Kuduğ H , Ataman B , İmamoğlu R , Düzgün D , Gökçe İ . 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. 2019; 3(1): 25-20.