Expression of a wheat α-amylase gene in Escherichia coli: recognition of the translational initiation site and the signal peptide

Abstract

Transcription of a full-length cDNA clone of wheat α-amylase using a lac promoter in Escherichia coli results in synthesis of a precursor α-amylase polypeptide of the correct size, indicating that translation initiates correctly. Recognition of the plant translational initiation site by E. coli ribosomes is 15–20% as efficient as the ribosome-binding site of the β-lactamase gene when it is fused to α-amylase. The α-amylase signal peptide is recognised in E. coli resulting in secretion of the enzyme into the periplasmic space; deletion of the signal peptide prevents secretion. Replacement of the α-amylase signal peptide with a β-lactamase signal peptide also enables the bacterial cell to secrete the enzyme. The presence of the β-lactamase and the α-amylase signal peptides in tandem results in secretion of the enzyme and removal of both signal peptides.