Nuclear Import Analysis of Two Different Fluorescent Marker Proteins into Hepatocyte Cell Lines (HuH-7 Cell)

https://doi.org/10.22146/ijbiotech.7557

Aris Haryanto(1*), Michael Kann(2)

(1) Department of Biochemistry, Faculty of Veterinary Medicine, Gadjah Mada University.Jl. Olahraga, Karangmalang, Yogyakarta 55281, Indonesia
(2) Institute of Medical Virology, Justus-Liebig-University, Giessen, Germany.Frankfurterstrasse 107, 35392 Giessen, Germany
(*) Corresponding Author

Abstract


The application of fluorescent proteins as expression markers and protein fusion partners has proved immensely valuable for resolving the organization of biological events in living cells. EGFP and DsRed2 are commonly fluorescent marker protein which is used for biotechnology and cell biology research. The present study was designed to identify the expression vector that suitable to ligate with DNA encoding HBV core protein for intracellular localization study in hepatocyte cell, which were expressed as fusion proteins. We also compared and quantified the expressed fluorescent protein which predominantly localized in the cell compartment. The results indicated that DsRed2 shown as less than ideal for intracellular localization study of than EGFP, because of its tetrameric structure of the fluorescent protein and when fused to a protein of interest, the fusion protein often forms aggregates in the living cells. In contrast, EGFP fluorescent protein shown a much higher proportion of cytoplasmic localization, thus being more suitable for analysis of intracellular localization than DsRed2 fluorescent protein. EGFP fluorescent protein is also capable to produce a strong green fluorescence when excited by blue light, without any exogenously added substrate or cofactor, events inside living cell can thus be visualized in a non-invasive way. Based on our present quantitative data and some reasons above shown that EGFP is more suitable than DsRed2 as a fluorescent marker protein for intracellular localization study into HuH-7 cell.

Keywords


EGFP, DsRed2 fluorescent protein , HuH-7 cell, HBV, intracellular localization

Full Text:

PDF


References

Baird, G. S., Zacharias, D.A., and Tsien, R.Y., 2000. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA., 97, 11984– 11989.

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C., 1994. Green fluorescent proteoin as marker for gene expression. Science, 263, 802-803.

Chudakov, D., Lukyanov, S., and Lukyanov K., 2005. Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol., 23 (12), 605-13.

Dingwall, C. and Laskey, R., 1992. The nuclear membrane. Science, 258, 942-947.

Gross, L.A., Baird, G.S., Hoffman, R.C., Baldridge, K.K., and Tsien, R.Y., 2000. The structure of the chromophore within DsRed,a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA., 97, 11990–11995.

Matz, M.V., Fradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov, M.L., and Lukyanov, S.A., 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol., 17, 969-973.

Mayer, G., Launhardt, H., and Munder, T., 1999. Application of the green fluorescent protein as a reporter for AceI-based, two hibrid studies. Bio- Techniques, 27, 84-86.

Mizuno, H., Sawano, A., Eli, P., Hama, H., and Miyawaki,A., 2001. Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. Biochemistry, 27, 2502– 2510.

Niedenthal, R.K., Riles, L., Johnston, M., and Hegemann, J.H., 1996. Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast. Yeast, 12, 773-786.

Phillips, G., 2001. Green fluorescent protein—a bright idea for the study of bacterial protein localization. FEMS Microbiol. Lett., 204 (1), 9-18.

Prendergast, F. and Mann, K., 1978. Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskålea. Biochemistry, 17 (17), 3448-53.

Tsien, R.Y., 1998. The green fluorescent protein. Annu. Rev. Biochem., 67, 509–544.

Yuste, R., 2005. Fluorescence microscopy today. Nat. Methods, 2 (12), 902-4.



DOI: https://doi.org/10.22146/ijbiotech.7557

Article Metrics

Abstract views : 1405 | views : 1470

Refbacks

  • There are currently no refbacks.


Copyright (c) 2015 Indonesian Journal of Biotechnology