Expression of a wheat alpha-amylase gene in Escherichia coli: recognition of the translational initiation site and the signal peptide.
Transcription of a full-length cDNA clone of wheat alpha-amylase using a lac promoter in Escherichia coli results in synthesis of a precursor alpha-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 beta-lactamase gene when it is fused to alpha-amylase. The alpha-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 alpha-amylase signal peptide with a beta-lactamase signal peptide also enables the bacterial cell to secrete the enzyme. The presence of the beta-lactamase and the alpha-amylase signal peptides in tandem results in secretion of the enzyme and removal of both signal peptides.
D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013045:Species Specificity; D014908:Triticum / Q000201:enzymology / Q000235:genetics; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics
D010940:Plant Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA; D000516:alpha-Amylases; D001618:beta-Lactamases