Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli

Abstract

A gene encoding the protein kinase domain of the epidermal growth factor receptor has been chemically synthesised, cloned and expressed in Escherichia coli. The 942-base-pair gene was constructed by enzymatic ligation of 56 oligonucleotides and cloned into an expression vector downstream of the E. coli trp promoter.Production of active gene product was confirmed by means of a protein kinase assay, demonstrating that the enzymatic activity of the protein kinase domain of the epidermal growth factor receptor is retained after expression in E. coli.A number of the cellular DNAs with sequences similar to transforming genes have been shown to encode growth factors or growth factor receptors, which when either altered or constitutively expressed may cause loss of normal growth control. The receptor for epidermal growth factor (EGF) has extensive similarity with the v-erbB protein, and the neu(cerbB2,HER2) oncogene product is also structurally similar to the EGF receptor and v-erbB protein and is likely to be another transmembrane receptor for an as yet unidentified ligand. Transforming growth factor CI is highly related to EGF, binds to the EGF receptor with similar affinity and induces proliferation of cells bearing the EGF receptor (for a review see [l]).The human EGF receptor is a 170-kDa transmembrane glycoprotein which mediates the mitogenic response of target cells to EGF. Based on the complete amino acid sequence deduced from the nucleotide sequences of cDNA clones [2], it was shown that the receptor consists of an extracellular EGF-binding domain, a hydrophobic transmembrane segment, a cytoplasmic domain which has a catalytic portion possessing tyrosine kinase activity [3] and several autophosphorylation sites. The kinase can mediate autophosphorylation, phosphorylate a variety of other proteins and is activated by EGF-binding. The v-erbB protein is a truncated form of the EGF receptor, lacking most of the EGF-binding domain and the C-terminal portion containing the autophosphorylation sites [l]. Mutagenesis of the tyrosine kinase domain has shown that kinase-negative mutants are defective in the transduction of the mitogenic effect of EGF.