In vitro activation and inhibition of recombinant EGFR tyrosine kinase expressed in Escherichia coli

Abstract

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-(32)P]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.

Figure 1

GST-TKJM and GST-TKJMΔ expression in E. coli. E. coli were transformed with the pGEX-TKJM (TK), pGEX-TKJMΔ (Δ), and the pGEX-6-P-1 (empty) plasmids, and the recombinant proteins are expressed as indicated in Section 2. The proteins were run in a 10% SDS-PAGE and stained with Coomassie blue (a) or subjected to western blot using an anti-GST antibody (b). Several E. coli clones expressing TKJM (TK) or TKJMΔ (Δ) are shown. The arrow points to the TKJM and TKJMΔ recombinant proteins.

Figure 2

Dimerization and tyrosine kinase assays. Total protein extracts of E. coli transformed by pGEX-TKJMΔ (Δ) were treated with BS3 or glutaraldehyde (GLU) as indicated and analyzed by western blot with the anti-GST antibody (a). A control (C) without cross-linkage agent is also shown. The positions of the protein size marker (Bio-Rad) are indicated from 30 to 97 kD. The recombinant proteins were assayed with [γ-³²P]ATP on immobilized glutathione-Sepharose beads as described in Section 2 (b). The empty pGEX-6P plasmid was used as control (P). Genistein (Genist) was added where indicated.

Figure 3

Recombinant protein purification. The recombinant GST-TKJMΔ and GST proteins were purified as described in Section 2. Lane 1: flow through; lane 2: ATP-wash; and lane 3: eluted proteins. The samples were run in 10% SDS/PAGE and stained with Coomassie blue. The arrow points to the TKJMΔ recombinant protein.