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1.
J Biol Chem ; 273(31): 19453-8, 1998 Jul 31.
Article in English | MEDLINE | ID: mdl-9677365

ABSTRACT

A fraction of each secreted protein is retained and degraded by the endoplasmic reticulum (ER) quality control apparatus that restricts export to correctly folded proteins. The intrinsic biophysical attributes that determine efficiency of escape from this proofreading process have been examined by expressing mutants of bovine pancreatic trypsin inhibitor (BPTI) in yeast. Secretion efficiency is strongly correlated with thermodynamic stability for a series of six point mutations of BPTI. No correlation of secretion efficiency with either oxidative folding or refolding rates in vitro is found; both the rapidly folded Y35L BPTI mutant and the slowly unfolded G36D BPTI mutant exhibit low secretion efficiency. Elimination of cysteines 14 and 38 by mutagenesis does not increase secretion efficiency, indicating that intramolecular thiol/disulfide rearrangements are not primarily responsible for retention and degradation of destabilized BPTI variants. Mutant yeast strains with diminished ER-associated degradation do not secrete BPTI more efficiently, indicating that retention and degradation are separable processes. These data support a model for ER quality control, wherein protein folding is functionally reversible and the relative rates of folding, unfolding, vesicular export, and retention determine secretion efficiency.


Subject(s)
Aprotinin/genetics , Endoplasmic Reticulum/physiology , Protein Folding , Aprotinin/metabolism , Cysteine/genetics , Disulfides/chemistry , Kinetics , Mutagenesis, Site-Directed/genetics , Protein Disulfide-Isomerases/genetics , Saccharomyces cerevisiae/physiology
2.
Biochemistry ; 37(5): 1264-73, 1998 Feb 03.
Article in English | MEDLINE | ID: mdl-9477952

ABSTRACT

Bovine pancreatic trypsin inhibitor (BPTI) has been widely used as a model protein to investigate protein structure and folding pathways. To study the role of its three disulfide bonds in folding, proofreading, and secretion of BPTI in an intact eucaryotic cell, BPTI was expressed and secreted from a synthetic gene in the yeast Saccharomyces cerevisiae. Site-directed mutagenesis was used to create all possible single and pairwise cysteine to alanine BPTI mutants, and the effect of these mutations on secretion efficiency was determined. The 5-55 disulfide bond is found to be essential for secretion-loss of either Cys5, Cys55, or both prevents secretion. Removal of the 14-38 disulfide bond results in a small reduction of secretion, but individual Cys14 or Cys38 replacements reduce secretion efficiency by 30%. Cys30 and Cys30-51 mutants are secreted at half the level of wild-type BPTI, while secretion of the Cys51 mutant is reduced by 90%. BPTI containing only a single disulfide bond (5-55) is not secreted. No relationship is observed between secretion efficiency and in vitro folding or unfolding rates, but mutant BPTI secretion is directly correlated with the in vitro unfolding temperature Tm and the free energy of stabilization provided by each of the three disulfides. These results indicate that structural fluctuations rather than the time-averaged structure observed by NMR or X-ray crystallography may determine recognition of a protein as misfolded and subsequent retention and degradation.


Subject(s)
Aprotinin/genetics , Aprotinin/metabolism , Disulfides , Saccharomyces cerevisiae/genetics , Thermodynamics , Amino Acid Sequence , Animals , Aprotinin/chemistry , Cattle , Gene Expression Regulation , Intracellular Fluid/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Disulfide-Isomerases/biosynthesis , Protein Disulfide-Isomerases/genetics , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolism
3.
Biotechnol Bioeng ; 59(3): 286-93, 1998 Aug 05.
Article in English | MEDLINE | ID: mdl-10099339

ABSTRACT

Secretion of bovine pancreatic trypsin inhibitor (BPTI) in Saccharomyces cerevisiae was examined with four different leader peptides: the invertase signal peptide, the mfalpha1 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 microgram/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.


Subject(s)
Aprotinin/biosynthesis , Protein Sorting Signals/metabolism , Signal Recognition Particle/metabolism , Amino Acid Sequence , Animals , Aprotinin/chemistry , Aprotinin/genetics , Base Sequence , Cattle , Cloning, Molecular/methods , HSP70 Heat-Shock Proteins/metabolism , Molecular Sequence Data , Plasmids , Protein Sorting Signals/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Saccharomyces cerevisiae/genetics , Ubiquitins/metabolism
4.
Biotechnol Prog ; 13(2): 117-22, 1997.
Article in English | MEDLINE | ID: mdl-9104035

ABSTRACT

The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture.


Subject(s)
Aprotinin/biosynthesis , Aprotinin/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/physiology , Amino Acid Sequence , Animals , Cattle , Intracellular Fluid/metabolism , Molecular Sequence Data , Protein Folding , RNA, Messenger/metabolism
5.
Biotechnol Prog ; 12(1): 16-21, 1996.
Article in English | MEDLINE | ID: mdl-8845105

ABSTRACT

We have constructed a yeast integration vector targeted to chromosomal Ty delta sequences and used it to create Saccharomyces cerevisiae strains with stable tandem integrations ranging from 1 to 30 vector copies. The vector carries the bacterial NEO gene, allowing copy number to be tuned by varying G418 resistance, which generally increases with copy number as determined by quantitative Southern blot. Tandem integration into a single site is most commonly observed, but single-copy and two-site integration is also observed. Bovine pancreatic trypsin inhibitor was constitutively expressed and secreted using the NEO-based delta vector, and secretion levels were 2-10-fold improved relative to commonly used 2 mu multicopy yeast plasmids. The NEO-based Ty delta vector is a powerful tool for stable heterologous protein expression and secretion in yeast.


Subject(s)
Chromosomes, Artificial, Yeast/genetics , Genetic Vectors , Saccharomyces cerevisiae/genetics , Animals , Base Sequence , Blotting, Southern , Cattle , Culture Media , Gene Amplification , Gene Expression , Kanamycin Resistance/genetics , Minisatellite Repeats , Molecular Sequence Data , Saccharomyces cerevisiae/metabolism , Transformation, Genetic , Trypsin Inhibitor, Kazal Pancreatic/biosynthesis , Trypsin Inhibitor, Kazal Pancreatic/genetics
7.
Prosthet Orthot Int ; 17(2): 101-6, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8233765

ABSTRACT

This study examined the energy cost of ambulation using the reciprocating gait orthosis (RGO) and the modified Isocentric RGO in paraplegic spinal cord injured subjects. In 4 subjects, the rates of O2 consumption per minute, O2 cost per metre, heart rate (HR), respiratory exchange ratio, velocity, and physiologic cost index (PCI) were measured during ambulation with the two orthotic devices. PCI was calculated by dividing the difference between walking and resting HR by velocity. PCI was significantly lower during ambulation trials with the Isocentric RGO compared to the RGO, but was the only measurement that detected a significant difference between the two orthotic devices. These results indicate that energy costs of ambulation at self-selected speeds were lower with the Isocentric RGO compared to the standard RGO. Furthermore, PCI could be used as a sensitive indicator of gait efficiency in spinal cord injury subjects.


Subject(s)
Energy Metabolism , Gait , Orthotic Devices , Paraplegia/metabolism , Paraplegia/rehabilitation , Spinal Cord Injuries/complications , Adult , Efficiency , Evaluation Studies as Topic , Heart Rate , Humans , Male , Monitoring, Physiologic , Orthotic Devices/classification , Orthotic Devices/standards , Oxygen Consumption , Paraplegia/etiology , Paraplegia/physiopathology , Prosthesis Design , Pulmonary Gas Exchange
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