Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

https://doi.org/10.22146/ijbiotech.7412

Aris Haryanto(1*)

(1) Department of Biochemistry, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta 55281
(*) Corresponding Author

Abstract


Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs through the nuclear pore complex (NPC) and typically requires a specific nuclear localization signal (NLS). In this study, it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B virus core protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLS by co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion protein were expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. The results indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolated from E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transport study shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc and rHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm into the nucleus.

Keywords


NPC; NLS; importin α; importin β; isoforms importin α as 6x histidin-tagged fusion protein; WT rHBc; SV40 Tag; co-immunoprecipitation; westernblotting

Full Text:

PDF


References

Ben-Efraim, I. and Gerace, L., 2001. Gradient of increasing affinity of importin b for nucleoporins along the pathway of nuclear import. J. Cell. Biol., 152, 411- 418.

Chelsky, D., Ralph, R. and Jonak, G., 1989. Sequence requirements for synthetic peptide-mediated translocation to the nucleus. Mol. Cell. Biol., 9, 2487-2492.

Conti, E., Uy, M., Leighton, L., Blobel, G. and Kriyan, J., 1998. Crytallographic analysis of the recognition a nuclear localization signal by the nuclear import factor karyopherin alpha. Cell, 94, 193-104.

Conti, E. and Kuriyan, J., 2000. Crystallographic and lysis of specific yet versatile recognition of distint nuclear localization signal by karyopherin alpha. Structure, 8, 329-338.

Cuomo, C.A., Kirch, S.A., Gyuris, J., Bread, R. and Oettinger, M.A., 1994. Rch1, a protein that specifially interacts with the RAG-1 recombination activating protein. Proc. Natl. Acad. Sci., 91, 6156- 6160.

Dang, C.V. and Lee, W.H., 1989. Nuclear and nucleolar targeting sequences of c-erb A, c-myb, n-myc, p53, HSP70 and HIV tat proteins. J. Biol. Chem., 264, 18019- 18023.

Fried, H. and Kutay, U., 2003. Nucleocytoplasmic transport: taking an inventory. Cell. Mol. Life. Sci., 60, 1659-1688.

Görlich, D., Prehn, S., Laskey, R.A. and Hartmann, E., 1994. Isolation of a protein that is essensial for the first step of nuclear protein import. Cell 79, 767- 778.

Görlich, D., Vogel, F., Mills, A.D., Hartmann, E. and Laskey, R.A., 1995. Distinct functions for the two importin subunit in nuclear protein import. Nature, 377, 246-248.

Görlich, D., Henklein, P., Laskey, R.A. and Hartmann E., 1996. A 41 amino acid motif in importin alpha confers binding to importin beta and hence transit into the nucleus. EMBO J., 15, 1810-1817.

Görlich, D. and Kutay, U. 1999. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell. Dev. Biol., 15, 607-660.

Haryanto, A., 2004. Generation and expression of recombinant glutathione- S-transferase full-length importin a-3 (GST-importin a-3) fusion protein and study of its role on the nuclear transport of protein. I. J. Biotech., ed. June. 681- 720.

Herold, A., Truant, R., Wiegand, H. and Cullens, B.R., 1998. Determination of the functional domain organisation of the importin alpha nuclear import factor. J. Cell. Biol., 143, 309-318.

Kobe, B., 1999. Autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin alpha. Nat. Struct. Biol., 6, 388- 397.

Köhler, M., Ansieau, S., Prehn, S., Leutz, A., Haller, H. and Hartmann, E., 1997. Cloning of two novel importin a subunit and analysis of the expression pattern of the importin a protein family. FEBS lett., 417, 104-108.

Lee, S.J., Matsuura, Y., Liu, S.M. and Stewart, M. 2005. Structural basis for nuclear import complex dissociation by RanGTP. Nature, 435 (7042), 693-696.

Makkerh, J.P., Dingwall, C. and Laskey, R.A. 1996. Comparative mutagenesis of nuclear localization signals reveals the



DOI: https://doi.org/10.22146/ijbiotech.7412

Article Metrics

Abstract views : 1459 | views : 1510

Refbacks

  • There are currently no refbacks.


Copyright (c) 2015 Indonesian Journal of Biotechnology