Epitope-Tagged Metallothionein: Its Biochemical and Immunocytochemical Applications
We developed epitope-tagged metallothionein IIA cDNA by the addition of an 8-amino acid sequence, DYKDDDDK, at the N-terminus metallothionein using polymerase chain reaction. The fusion proteins were expressed by transfection in human embryonic kidney 293 (HEK-293) cells and were specifically recogn...
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| Published in | Journal of Health Science Vol. 49; no. 2; pp. 156 - 159 |
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| Main Authors | , |
| Format | Journal Article |
| Language | English |
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The Pharmaceutical Society of Japan
2003
Pharmaceutical Society of Japan |
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| Abstract | We developed epitope-tagged metallothionein IIA cDNA by the addition of an 8-amino acid sequence, DYKDDDDK, at the N-terminus metallothionein using polymerase chain reaction. The fusion proteins were expressed by transfection in human embryonic kidney 293 (HEK-293) cells and were specifically recognized by commercially available antibody, an anti-Flag M2 monoclonal antibody. Furthermore, the fusion protein was detectable on a Western blot, whose migration was expectedly at 7000-8000 in an SDS-denatured form. Thus, availability of the epitope-tagged metallothionein will facilitate the investigation of cellular mechanisms of the protein such as its nuclear localization mechanism. |
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| AbstractList | We developed epitope-tagged metallothionein IIA cDNA by the addition of an 8-amino acid sequence, DYKDDDDK, at the N-terminus metallothionein using polymerase chain reaction. The fusion proteins were expressed by transfection in human embryonic kidney 293 (HEK-293) cells and were specifically recognized by commercially available antibody, an anti-Flag M2 monoclonal antibody. Furthermore, the fusion protein was detectable on a Western blot, whose migration was expectedly at 7000-8000 in an SDS-denatured form. Thus, availability of the epitope-tagged metallothionein will facilitate the investigation of cellular mechanisms of the protein such as its nuclear localization mechanism. We developed epitope-tagged metallothionein IIA cDNA by the addition of an 8-amino acid sequence, DYKDDDDK, at the N-terminus metallothionein using polymerase chain reaction. The fusion proteins were expressed by transfection in human embryonic kidney 293 (HEK-293) cells and were specifically recognized by commercially available antibody, an anti-Flag M2 monoclonal antibody. Furthermore, the fusion protein was detectable on a Western blot, whose migration was expectedly at 7000-8000 in an SDS-denatured form. Thus, availability of the epitope-tagged metallothionein will facilitate the investigation of cellular mechanisms of the protein such as its nuclear lo-alization mechanism. INTRODUCTION Metallothioneins, a metalloprotein of low molecular weight (6000-7000), are cysteine rich (30%) intracellular proteins with high affinity for certain metals 1-3). High levels of an endogenous metallothionein bound to zinc and copper have been detected in mammalian liver during gestation and early postnatal period in various species, suggesting a biological role for metallothionein in metal homeostasis 1-3). Metallothionein is shown to sequester large amounts of toxic metals such as cadmium intracellularly and therefore it may play a significant role in metal detoxification 1-3). |
| Author | Ishido, Masami Sayato-Suzuki, Junko |
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| Cites_doi | 10.1146/annurev.biochem.55.1.913 10.1073/pnas.95.15.8428 10.1248/jhs.48.381 10.1016/S0378-4274(00)00178-8 10.1046/j.1471-4159.1998.71062585.x 10.1016/S0024-3205(98)00621-3 10.1248/jhs.47.583 10.1016/0304-4157(91)90013-M 10.1002/1439-7633(20000818)1:2<86::AID-CBIC86>3.0.CO;2-A 10.1074/jbc.C000593200 10.1248/jhs.47.9 10.1016/0012-1606(83)90067-2 10.1016/0167-4838(85)90098-6 10.1016/0014-5793(91)80597-V 10.1093/carcin/22.7.1043 10.1016/0024-3205(95)00100-X 10.1016/0092-8674(85)90051-0 10.1038/227680a0 10.1289/ehp.0211037 10.1016/0300-483X(82)90048-8 10.1016/S0024-3205(98)00382-8 |
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| References_xml | – ident: 2 doi: 10.1146/annurev.biochem.55.1.913 – ident: 3 doi: 10.1073/pnas.95.15.8428 – ident: 11 doi: 10.1248/jhs.48.381 – ident: 22 doi: 10.1016/S0378-4274(00)00178-8 – volume: 71 start-page: 2585 issn: 0022-3042 issue: 6 year: 1998 ident: 13 publication-title: J. Neurochem. doi: 10.1046/j.1471-4159.1998.71062585.x contributor: fullname: KUNIMOTO M – volume: 1 start-page: 13 year: 2000 ident: 17 publication-title: Res. Adv. Neurochem. contributor: fullname: Ishido, M. and Kunimoto, M. – ident: 21 doi: 10.1016/S0024-3205(98)00621-3 – ident: 14 doi: 10.1248/jhs.47.583 – volume: 1071 start-page: 83 issn: 0005-2728 issue: 1 year: 1991 ident: 9 publication-title: Biochim. Biophys. Acta doi: 10.1016/0304-4157(91)90013-M – volume: 18 start-page: 86 issn: 1439-4227 year: 2000 ident: 10 publication-title: Chembiochem doi: 10.1002/1439-7633(20000818)1:2<86::AID-CBIC86>3.0.CO;2-A contributor: fullname: Blobel, G. – ident: 5 doi: 10.1074/jbc.C000593200 – ident: 23 doi: 10.1248/jhs.47.9 – volume: 97 start-page: 95 issn: 0012-1606 issue: 1 year: 1983 ident: 7 publication-title: Dev. Biol. doi: 10.1016/0012-1606(83)90067-2 contributor: fullname: Panemangalore, M., Banerjee, D., On – volume: 55 start-page: 101 issn: 0098-4108 year: 1998 ident: 20 publication-title: J. Toxicol. Environ. Health contributor: fullname: Ishido, M., Homma, S., Tohyama, C. – volume: 827 start-page: 36 issn: 0005-2728 issue: 1 year: 1985 ident: 4 publication-title: Biochim. Biophys. Acta doi: 10.1016/0167-4838(85)90098-6 contributor: fullname: Thornalley, P. J. and Vasak, M. – ident: 8 doi: 10.1016/0014-5793(91)80597-V – volume: 22 start-page: 1043 issn: 0143-3334 issue: 7 year: 2001 ident: 12 publication-title: Carcinogenesis doi: 10.1093/carcin/22.7.1043 contributor: fullname: ISHIDO M – ident: 19 doi: 10.1016/0024-3205(95)00100-X – volume: 41 start-page: 9 issn: 0092-8674 issue: 1 year: 1985 ident: 1 publication-title: Cell doi: 10.1016/0092-8674(85)90051-0 contributor: fullname: Karin, M. – volume: 290 start-page: 923 issn: 0022-3565 issue: 2 year: 1999 ident: 15 publication-title: J. Pharmacol. Exp. Ther. contributor: fullname: ISHIDO M – volume: 227 start-page: 680 issn: 0028-0836 year: 1970 ident: 18 publication-title: Nature (London) doi: 10.1038/227680a0 contributor: fullname: Laemmli, U. K. – volume: 110 start-page: 37 issue: 1 year: 2002 ident: 16 publication-title: Environ. Health Perspect. doi: 10.1289/ehp.0211037 contributor: fullname: ISHIDO M – ident: 6 doi: 10.1016/0300-483X(82)90048-8 – ident: 24 doi: 10.1016/S0024-3205(98)00382-8 |
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| Title | Epitope-Tagged Metallothionein: Its Biochemical and Immunocytochemical Applications |
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