Leader peptide efficiency correlates with signal recognition particle dependence in Saccharomyces cerevisiae

• Published in: Biotechnology and bioengineering Vol. 59; no. 3; pp. 286 - 293
• Main Authors: Arnold, Caron E., Parekh, Rajesh N., Yang, Wenli, Wittrup, K. Dane
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 05-08-1998; Wiley
• Subjects: Amino Acid Sequence; Animals; Aprotinin - biosynthesis; Aprotinin - chemistry; Aprotinin - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Cattle; Cloning, Molecular - methods; ER membrane translocation; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; HSP70 Heat-Shock Proteins - metabolism; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; Plasmids; Protein Sorting Signals - chemistry; Protein Sorting Signals - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Saccharomyces cerevisiae - genetics; signal peptide; signal recognition particle; Signal Recognition Particle - metabolism; ubiquitin translocation assay; Ubiquitins - metabolism; Leader peptide; Yeast; Serine endopeptidases; Bovine; Signal recognition particle; Enzyme; Secretion; Enzyme inhibitor; Gene expression; Membrane translocation; Fungi; Peptidases; Vertebrata; Trypsin; Mammalia; Hydrolases; Ascomycetes; Artiodactyla; Recombinant protein; Pancreas; Fusion protein; Saccharomyces cerevisiae; Ungulata; Thallophyta
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(19980805)59:3<286::AID-BIT4>3.0.CO;2-7

Secretion of bovine pancreatic trypsin inhibitor (BPTI) in Saccharomyces cerevisiae was examined with four different leader peptides: the invertase signal peptide, the mfα1 signal peptide, a synthetic signal peptide, and a synthetic pre pro leader. BPTI secretion from a low‐copy CEN plasmid varies from 1.8 to 10.4 μg/mL among these constructs. Secretion titers correlate with dependence on signal recognition particle (SRP), with greatest secretion from the most SRP‐dependent construct. Examination of co‐ vs post‐translational translocation pathways and overall translocation efficiency by ubiquitin translocation assay (UTA) does not provide insight into the variation in BPTI secretion efficiency, perhaps due to alteration in translocation kinetics from the additional polypeptide fusion required by the assay. BPTI translocation efficiency (as measured by UTA) is found to drop markedly upon depletion of Srp54p, prior to any observable growth defect. Subsequent to stress response induction and the onset of slow growth (15‐h doubling time), BPTI translocation efficiency recovers to the level observed prior to SRP depletion. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:286–293, 1998.