Illicit secretion of a cytoplasmic protein into the periplasm of Escherichia coli requires a signal peptide plus a portion of the cognate secreted protein. Demarcation of the critical region of the mature protein.
The beta-lactamase signal peptide alone is not sufficient to direct secretion of chicken muscle triosephosphate isomerase, a normally cytoplasmic protein, into the periplasm of Escherichia coli. The signal peptide and at least the first 3 residues of the mature beta-lactamase are required before any secretion of the isomerase can be observed. At this point the level of secretion is very low, but the addition of further residues of the mature beta-lactamase enhances the secretion of the hybrid protein. The maximum level of secretion is achieved when 12 or more residues of the mature beta-lactamase intervene between the signal peptide and the isomerase. It is the proximity of an arginine residue at position 3 of the isomerase that is responsible for the blockade to secretion of these hybrid proteins (see Summers, R.G., Harris, C.R., and Knowles, J.R. (1989) J. Biol. Chem. 264, 20082-20088). With 12 residues of the mature beta-lactamase between the signal peptide and the isomerase, the offending arginine now lies at position 15 of the hybrid. The 14 residues that immediately follow the signal peptide therefore define a region of constrained properties that is critical to the secretability of proteins from E. coli.
Summers
R G
RG
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Knowles
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D002238:Carbohydrate Epimerases / Q000235:genetics*; D002645:Chickens; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010449:Peptide Mapping; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013104:Spheroplasts / Q000201:enzymology; D014305:Triose-Phosphate Isomerase / Q000096:biosynthesis / Q000235:genetics*; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics*
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase