Influence of specific signal peptide mutations on the expression and secretion of the alpha-amylase inhibitor tendamistat in Streptomyces lividans.
The Streptomyces alpha-amylase inhibitor tendamistat is secreted by a signal peptide with an amino-terminal charge of +3. To elucidate the influence of the charged residues on protein secretion in Streptomyces, the amino-terminal charge was varied from +6 to neutral net charge. The effects of charge variation were analyzed in combination with three Streptomyces promoters and two transcriptional terminators. Introduction of additional positive charges significantly decreased the amount of secreted tendamistat. On the contrary, a charge reduction to +2 resulted in the doubling of inhibitor production. After exclusion of transcriptional effects, the observed alterations of inhibitor secretion by the mutants with a charge of +6 to +2 were attributed to a modulation of precursor synthesis. Furthermore, a tight coupling of synthesis and export was stated. Charge reduction to +1 or neutral charge generally reduced the yield of secreted tendamistat, yet remarkable differences were found for mutants with identical net charge. Elimination of the positive charge at a defined position resulted in the release of tendamistat precursor protein, which suggested a specific uncoupling of synthesis and translocation.
Fass
S H
SH
Institut für Organische Chemie, Johann Wolfgang Goethe-Universität, D-60439 Frankfurt am Main, Federal Republic of Germany.
Engels
J W
JW
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors; D004926:Escherichia coli; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D004791:Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000516:alpha-Amylases; C030526:tendamistate