Heterologous Expression of a Human Cap-Binding Protein eIF4E in Escherichia coli

Abstract

A crucial step in the initiation of cap dependent translation of eukaryotic mRNA is the interaction of the mRNA cap-binding protein, eIF4E, with eIF4G, two integral components of the mRNA cap-binding complex. The present study was aimed to express human eIF4E at high level in E. coli. For high-level expression, the E. coli cells were first transformed with an expression vector that contains the cloned gene, human eIF4E. Following transformation, the recombinant plasmid was purified and stepwise digestion of the recombinant plasmid revealed that the plasmid was harboring an insert, equal to the length of the eIF4E gene. As the vector contained a heat-inducible promoter, high-level expression of human eIF4E was carried out by heat induction at 42°C. Expression of recombinant protein was observed by comparing the cell extracts collected before and after heat induction and their subsequent analysis on a denatured polyacrylamide gel. The appearance of a 24-kDa protein following induction of transformed cells confirmed the expression of the cloned gene, human eIF4E.