Difference between revisions of "Part:BBa K1166005"
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Codes for the T7 expression cassette for Apoptin composed of a N-terminal TAT translocation peptide, followed by a linker (GGGGS), a histidine tag (6x) and by the same linker fused to Apoptin. Apoptin, also called VP3, is a protein from the Chicken Anemia Virus (CAV) known to cause p53-independent apoptosis in more than 70 human cancer cell lines while leaving normal cells unharmed (Backendorf, et al., 2008). | Codes for the T7 expression cassette for Apoptin composed of a N-terminal TAT translocation peptide, followed by a linker (GGGGS), a histidine tag (6x) and by the same linker fused to Apoptin. Apoptin, also called VP3, is a protein from the Chicken Anemia Virus (CAV) known to cause p53-independent apoptosis in more than 70 human cancer cell lines while leaving normal cells unharmed (Backendorf, et al., 2008). | ||
| − | Apoptin contains a bipartite nuclear localization signal (NLS1: aa 82-88 and NLS2: aa 111-121) and a nuclear export signal (NES) (aa 97-105) (Heckl, et al., 2008). In cancer cells, Apoptin localizes to the nucleus while in normal cells it's found in the cytoplasm; it is thought that the bipartite NLS is activated by phosphorylation at Threonine 108 which only happens in cancer cells. | + | Apoptin contains a bipartite nuclear localization signal (NLS1: aa 82-88 and NLS2: aa 111-121) and a nuclear export signal (NES) (aa 97-105) (Heckl, et al., 2008). In cancer cells, Apoptin localizes to the nucleus while in normal cells it's found in the cytoplasm; it is thought that the bipartite NLS is activated by phosphorylation at Threonine 108 which only happens in cancer cells (Rohn, et al., 2002). |
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| + | ==Results: Therapeutic proteins== | ||
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| + | Expression of therapeutic proteins | ||
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| + | The presence of the therapeutic proteins TRAIL and TAT-APOPTIN in cell lysates was confirmed by Western Blot analysis. Protein samples were obtained from 2 different batches of Escherichia coli BL21 (DE3) cultures, from both the soluble and insoluble products of the sonicated culture. | ||
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| + | [[File:apoptin-1.png]] | ||
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| + | Figure 1: Western Blot, probed with anti-His6x antibody HRP conjugated. Protein samples were recovered from soluble fractions of E.coli BL21 (DE3) lysates, unless otherwise stated. Lane2: Negative control (non-transformed E.coli BL21); Lane3: Positive control (previously confirmed HIS-GFP); Lane4: HIS-TAT-APOPTIN (from Batch 2, transformant 2); Lane5: HIS-TAT-APOPTIN (from Batch 2, transformant 1); Lane6: HIS-TRAIL (from Batch 2, insoluble fraction); Lane7: HIS-TRAIL (from Batch2); Lane8: Amersham High-Range Molecular Weight Marker; Lane9: HIS-TAT-APOPTIN (from Batch 1); Lane10: HIS-TRAIL (from Batch 1, insoluble fraction) | ||
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| + | In Figure 1, we show the expression of TRAIL and TAT-APOPTIN from different cultures of E.coli BL21. | ||
==References== | ==References== | ||
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Heckl S, Regenbogen M, Sturzu A, Gharabaghi A, Feil G, Beck A, Echner H, Nagele T. (2008). Value of apoptin's 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells.Apoptosis. 13(4):495-508 | Heckl S, Regenbogen M, Sturzu A, Gharabaghi A, Feil G, Beck A, Echner H, Nagele T. (2008). Value of apoptin's 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells.Apoptosis. 13(4):495-508 | ||
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| + | Rohn JL, Zhang YH, Aalbers RI, Otto N, Den Hertog J, Henriquez NV, Van De Velde CJ, Kuppen PJ, Mumberg D, Donner P, Noteborn MH. (2002). A tumor-specific kinase activity regulates the viral death protein Apoptin. J Biol Chem. 277(52):50820-7 | ||
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Latest revision as of 03:51, 28 September 2013
TAT-Apoptin
Codes for the T7 expression cassette for Apoptin composed of a N-terminal TAT translocation peptide, followed by a linker (GGGGS), a histidine tag (6x) and by the same linker fused to Apoptin. Apoptin, also called VP3, is a protein from the Chicken Anemia Virus (CAV) known to cause p53-independent apoptosis in more than 70 human cancer cell lines while leaving normal cells unharmed (Backendorf, et al., 2008).
Apoptin contains a bipartite nuclear localization signal (NLS1: aa 82-88 and NLS2: aa 111-121) and a nuclear export signal (NES) (aa 97-105) (Heckl, et al., 2008). In cancer cells, Apoptin localizes to the nucleus while in normal cells it's found in the cytoplasm; it is thought that the bipartite NLS is activated by phosphorylation at Threonine 108 which only happens in cancer cells (Rohn, et al., 2002).
Results: Therapeutic proteins
Expression of therapeutic proteins
The presence of the therapeutic proteins TRAIL and TAT-APOPTIN in cell lysates was confirmed by Western Blot analysis. Protein samples were obtained from 2 different batches of Escherichia coli BL21 (DE3) cultures, from both the soluble and insoluble products of the sonicated culture.
Figure 1: Western Blot, probed with anti-His6x antibody HRP conjugated. Protein samples were recovered from soluble fractions of E.coli BL21 (DE3) lysates, unless otherwise stated. Lane2: Negative control (non-transformed E.coli BL21); Lane3: Positive control (previously confirmed HIS-GFP); Lane4: HIS-TAT-APOPTIN (from Batch 2, transformant 2); Lane5: HIS-TAT-APOPTIN (from Batch 2, transformant 1); Lane6: HIS-TRAIL (from Batch 2, insoluble fraction); Lane7: HIS-TRAIL (from Batch2); Lane8: Amersham High-Range Molecular Weight Marker; Lane9: HIS-TAT-APOPTIN (from Batch 1); Lane10: HIS-TRAIL (from Batch 1, insoluble fraction)
In Figure 1, we show the expression of TRAIL and TAT-APOPTIN from different cultures of E.coli BL21.
References
Backendorf C, Visser AE, de Boer AG, Zimmerman R, Visser M, Voskamp P, Zhang YH, Noteborn M. (2008). Apoptin: therapeutic potential of an early sensor of carcinogenic transformation. Annu Rev Pharmacol Toxicol. 48:143-69.
Heckl S, Regenbogen M, Sturzu A, Gharabaghi A, Feil G, Beck A, Echner H, Nagele T. (2008). Value of apoptin's 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells.Apoptosis. 13(4):495-508
Rohn JL, Zhang YH, Aalbers RI, Otto N, Den Hertog J, Henriquez NV, Van De Velde CJ, Kuppen PJ, Mumberg D, Donner P, Noteborn MH. (2002). A tumor-specific kinase activity regulates the viral death protein Apoptin. J Biol Chem. 277(52):50820-7
Sequence and Features
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- 1000COMPATIBLE WITH RFC[1000]