SavedAdd your journal

Journals / International Advanced Researches and Engineering Journal / 2019 / Cilt: 3 - Sayı: 1

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

Rizvan İMAMOĞLU
İsa GÖKÇE
Bahadır ATAMAN
Duygu DÜZGÜN
Hülya KUDUĞ
Pages
20–25
Publication Date
2022-10-19
DOI
—
🏛️Bibliographic metadata and full text access for this article are provided via DergiPark.
DergiPark Record ↗
✨

AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

High throughput expression of 81 mg fluorescent protein from a liter of E. coli culture carried out in bioreactor.

📌 3 Key Takeaways

🎯
Objective & Hypothesis:New and improved genetic engineered variants of fluorescent proteins (FPs) have become useful tools for bioimaging in biomedical researches.
📊
Key Findings & Data: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.
💡
Conclusion & Impact:High throughput expression of 81 mg fluorescent protein from a liter of E. coli culture carried out in bioreactor.
🔬Design:Research Article
📍Scope:Comprehensive Sample
⚙️Variables:Bioreactor · E.coli · Fluorescent Proteins

Abstract

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.

🔖

Cite This Article

İMAMOĞLU, R., GÖKÇE, İ., ATAMAN, B., DÜZGÜN, D., KUDUĞ, H. (2022). 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, Cilt: 3 - Sayı: 1, 20–25. https://ajindex.com/articles/539373

📄 Full Text PDF Viewer

📄

PDF görüntüleyici hazırlanıyor...

References (13)

  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 t