In Vitro Activation and Inhibition of Recombinant EGFR Tyrosine Kinase Expressed in Escherichia coli
The present work concerns the heterologous expression of the intracellular domain harbouring the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Protein expression was improved thanks to the deletion of a 13-amino acid peptide of the juxtamembrane region (JM). The recombinan...
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| Format: | Article |
| Language: | English |
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Wiley
2013-01-01
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| Series: | The Scientific World Journal |
| Online Access: | http://dx.doi.org/10.1155/2013/807284 |
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| _version_ | 1832558847398510592 |
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| author | Jihene Elloumi-Mseddi Karim Jellali Sami Aifa |
| author_facet | Jihene Elloumi-Mseddi Karim Jellali Sami Aifa |
| author_sort | Jihene Elloumi-Mseddi |
| collection | DOAJ |
| description | The present work concerns the heterologous expression of the intracellular domain harbouring the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Protein expression was improved thanks to the deletion of a 13-amino acid peptide of the juxtamembrane region (JM). The recombinant proteins were produced as a glutathione S-transferase (GST) fusion in Escherichia coli, and the solubilisation was performed by sarkosyl addition during extraction. The produced proteins spontaneously dimerize allowing the activation of the tyrosine kinase domain in the presence of [γ-32P]ATP. The activity assay has revealed the autophosphorylation of EGFR proteins which was decreased in the presence of genistein. Our system could facilitate the screening of EGFR inhibitors without the need of adding an exogenous substrate. |
| format | Article |
| id | doaj-art-2c802c45a2954c7eb400a8c4008f5cb1 |
| institution | Kabale University |
| issn | 1537-744X |
| language | English |
| publishDate | 2013-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | The Scientific World Journal |
| spelling | doaj-art-2c802c45a2954c7eb400a8c4008f5cb12025-02-03T01:31:25ZengWileyThe Scientific World Journal1537-744X2013-01-01201310.1155/2013/807284807284In Vitro Activation and Inhibition of Recombinant EGFR Tyrosine Kinase Expressed in Escherichia coliJihene Elloumi-Mseddi0Karim Jellali1Sami Aifa2Centre of Biotechnology of Sfax, P.O. Box 1177, 3018 Sfax, TunisiaCentre of Biotechnology of Sfax, P.O. Box 1177, 3018 Sfax, TunisiaCentre of Biotechnology of Sfax, P.O. Box 1177, 3018 Sfax, TunisiaThe present work concerns the heterologous expression of the intracellular domain harbouring the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Protein expression was improved thanks to the deletion of a 13-amino acid peptide of the juxtamembrane region (JM). The recombinant proteins were produced as a glutathione S-transferase (GST) fusion in Escherichia coli, and the solubilisation was performed by sarkosyl addition during extraction. The produced proteins spontaneously dimerize allowing the activation of the tyrosine kinase domain in the presence of [γ-32P]ATP. The activity assay has revealed the autophosphorylation of EGFR proteins which was decreased in the presence of genistein. Our system could facilitate the screening of EGFR inhibitors without the need of adding an exogenous substrate.http://dx.doi.org/10.1155/2013/807284 |
| spellingShingle | Jihene Elloumi-Mseddi Karim Jellali Sami Aifa In Vitro Activation and Inhibition of Recombinant EGFR Tyrosine Kinase Expressed in Escherichia coli The Scientific World Journal |
| title | In Vitro Activation and Inhibition of Recombinant EGFR Tyrosine Kinase Expressed in Escherichia coli |
| title_full | In Vitro Activation and Inhibition of Recombinant EGFR Tyrosine Kinase Expressed in Escherichia coli |
| title_fullStr | In Vitro Activation and Inhibition of Recombinant EGFR Tyrosine Kinase Expressed in Escherichia coli |
| title_full_unstemmed | In Vitro Activation and Inhibition of Recombinant EGFR Tyrosine Kinase Expressed in Escherichia coli |
| title_short | In Vitro Activation and Inhibition of Recombinant EGFR Tyrosine Kinase Expressed in Escherichia coli |
| title_sort | in vitro activation and inhibition of recombinant egfr tyrosine kinase expressed in escherichia coli |
| url | http://dx.doi.org/10.1155/2013/807284 |
| work_keys_str_mv | AT jiheneelloumimseddi invitroactivationandinhibitionofrecombinantegfrtyrosinekinaseexpressedinescherichiacoli AT karimjellali invitroactivationandinhibitionofrecombinantegfrtyrosinekinaseexpressedinescherichiacoli AT samiaifa invitroactivationandinhibitionofrecombinantegfrtyrosinekinaseexpressedinescherichiacoli |