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K D Wittrup

Publications and source records attributed to K D Wittrup.

At least 37 records · Page 2Linked to original sources
PubMed1998

Secretion efficiency in Saccharomyces cerevisiae of bovine pancreatic trypsin inhibitor mutants lacking disulfide bonds is correlated with thermodynamic stability.

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.

Amino Acid Sequence↗
PubMed1998

Thermodynamic characterization of affinity maturation: the D1.3 antibody and a higher-affinity mutant.

Understanding the structural and dynamic determinants of binding free energy in the antigen-antibody bond is of great interest. Much work has focused on selective mutations in order to locate key interaction residues, but this generally results in reduced affinity. The present work instead examines a higher-affinity mutant to characterize the thermodynamic pathway of the affinity maturation process. We have compared the antigen binding energetics of scFv D1.3, an anti-hen egg lysozyme single chain antibody, with a higher-affinity mutant (Hawkins, R. E., Russell, S. J., Baier, M. and Winter, G. (1993). J. Mol. Biol. 234, 958-964). The mutant has five-fold higher affinity for lysozyme but nearly the same enthalpy and heat capacity change upon binding, as measured by isothermal titration calorimetry. Thus, much of the binding free energy difference can be attributed to entropic effects. Fluorescence quenching with acrylamide indicates that this more favorable entropy change may result from a more flexible mutant-lysozyme complex and thus be a configurational entropy effect.

Animals↗
PubMed1998

Purification and functional characterization of a chaperone from Methanococcus jannaschii.

A chaperone from Methanococcus jannaschii has been purified to homogeneity with a single chromatographic step. The chaperone was identified and characterized using activity assays for characteristic chaperone abilities. The M. jannaschii chaperone binds unfolded proteins, protects proteins against heat-induced aggregation, and has a strongly temperature dependent ATPase activity. The chaperone has also been shown to inhibit the spontaneous refolding of a mesophilic protein at low temperatures. The purified chaperone complex has a M(r) of about 1,000,000 and consists of a single type of subunit with an approximate M(r) of 60,000. Analysis of partial sequence data reveals that this chaperone is the predicted protein product of the previously identified chaperonin gene in M. jannaschii (BULT et al., 1996). To our knowledge, this is the first functional characterization of a chaperone from a methanogen.

Adenosine Triphosphatases↗
PubMed1998

Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments.

We have produced single-chain antibody fragments (scFv) in Saccharomyces cerevisiae at levels up to 20 mg/L in shake flask culture by a combination of expression level tuning and overexpression of folding assistants. Overexpression of the chaperone BiP or protein disulfide isomerase (PDI) increases secretion titers 2-8 fold for five scFvs. The increases occur for scFv expression levels ranging from low copy to ER-saturating overexpression. The disulfide isomerase activity of PDI, rather than its chaperone activity, is responsible for the secretion increases. A synergistic increase in scFv production occurs upon cooverexpression of BiP and PDI.

Amino Acid Substitution↗
PubMed1997

Yeast surface display for screening combinatorial polypeptide libraries.

Display on the yeast cell wall is well suited for engineering mammalian cell-surface and secreted proteins (e.g., antibodies, receptors, cytokines) that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity. C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library. A eukaryotic host should alleviate expression biases present in bacterially propagated combinatorial libraries. Quantitative flow cytometric analysis enables fine discrimination of kinetic parameters for protein binding to soluble ligands.

Animals↗
PubMed1997

Isolation of anti-T cell receptor scFv mutants by yeast surface display.

Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.

Antibodies, Monoclonal↗
PubMed1996

Reduction of BiP levels decreases heterologous protein secretion in Saccharomyces cerevisiae.

Increased levels of the endoplasmic reticulum-resident protein folding chaperone BiP would be expected to either increase protein secretory capacity by improved solubilization of folding precursors or decrease secretory capacity by binding and retaining misfolded proteins. To address this question, the relationship between BiP levels and heterologous secretion in yeast was determined. A yeast strain was constructed in which BiP expression is tunable from 5 to 250% of wild-type levels, and this strain was used to explore the effect of varying BiP level on overall secretion of three heterologous proteins: human granulocyte colony-stimulating factor, Schizosaccharomyces pombe acid phosphatase, and bovine pancreatic trypsin inhibitor. For all three proteins examined, reduction in BiP expression below wild-type level diminished overall secretion, whereas 5-fold BiP overexpression from a constitutive glycolytic promoter did not substantially increase or decrease secretion titers. These results are consistent with a positive role for BiP in promoting membrane translocation and solubilization of folding precursors but are inconsistent with a negative role in proofreading and improper retention of heterologous secreted proteins.

Animals↗
PubMed1995

Disulfide bond formation and eukaryotic secretory productivity.

The relationship between disulfide bond formation and the exit of proteins from the endoplasmic reticulum may prove critical to maximizing the productivity of eukaryotic expression systems. During the past year, manipulation of redox active foldase enzymes, global inhibition of disulfide formation with dithiothreitol, and removal of specific disulfides via site-directed mutagenesis have all been shown to result in surprising effects on the rate and efficiency of protein secretion in eukaryotic hosts.

Animals↗
PubMed1994

The stress response to loss of signal recognition particle function in Saccharomyces cerevisiae.

It has been shown previously that growth and endoplasmic reticulum (ER) translocation defects occur in response to depletion of the 54-kDa subunit of signal recognition particle (SRP54) in Saccharomyces cerevisiae (Hann, B. B., and Walter, P. (1991) Cell 67, 131-144). We report here that cells depleted of SRP54p undergo a general stress response, the onset of which is observed almost two-cell doublings after SRP54 protein levels fall below the limits of detection. The stress response to SRP54p depletion occurs in two distinct phases, unlike the response to other stressors such as heat shock. In the initial phase, the cytoplasmic Hsp70 levels are drastically increased coincident with an abrupt slowing of growth and accumulation of untranslocated species of the ER-resident chaperone BiP. During this first response, levels of the yeast DnaJ homolog Ydj1p are also increased. In the second phase, which is detected 5 h later, levels of the cytoplasmic heat shock proteins Hsp82 and Hsp104 are increased. BiP is also induced during this second phase, while the ER levels of the resident foldase protein disulfide isomerase are significantly reduced. Since only those cytoplasmic stress proteins which have been shown to participate in membrane translocation are induced in the first phase, these findings indicate the presence of a stress response specific to accumulation of secretory protein precursors in the cytoplasm.

Blotting, Western↗
PubMed1994

Fluorescence array detector for large-field quantitative fluorescence cytometry.

A prototypic fluorescence array detector (FAD) has been designed and constructed which is capable of quantifying single-cell fluorescence emissions from a statistically significant population of cell-sized objects (over 10(3)) on a solid substrate. The system is comprised of a cryogenically cooled CCD, 50 mW air-cooled argon ion laser, and optics that image a large (1 x 1 cm) field at 1:1 (no magnification). The CCD is effectively treated as a two-dimensional array of 2.7 x 10(5) independent 20 x 20 microns photodetectors, with each cell-sized object imaged across only a few CCD pixels. Algorithms have been developed for focusing, image segmentation, shading correction, and noise rejection; performance data for the FAD with fluorescent calibration beads are presented. The FAD is a simple alternative to microscope-based imaging cytometry, allowing large-field imaging without a scanning stage.

Flow Cytometry↗
PubMed1994

Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae.

Overexpression of protein disulfide isomerase (PDI) from a single chromosomally integrated copy in Saccharomyces cerevisiae results in ten-fold higher levels of secretion of human platelet derived growth factor B homodimer, and a four-fold increase in secretion of Schizosaccharomyces pombe acid phosphatase. This result provides evidence that inefficient protein folding limits the secretion of some heterologous proteins, and that manipulation of the endoplasmic reticulum lumenal environment can help overcome this limitation.

Cloning, Molecular↗
PubMed1989

Application of flow cytometric measurement of surface IgG in kinetic analysis of monoclonal antibody synthesis and secretion by murine hybridoma cells.

The kinetics of antibody synthesis and secretion from a murine hybridoma cell line were studied using measurements of total cell-associated IgG, surface IgG, and IgG secreted into the medium. Kinetic analysis of IgG secretion demonstrates approximately constant secretion rate per viable cell over the entire batch cultivation. A correlation was observed (r2 = 0.74) between mean surface immunofluorescence and the total cell-associated IgG determined by ELISA of detergent-extracted cell lysates. No correlation was found between specific secretion rate and mean surface IgG level estimated by immunofluorescence flow cytometry measurements. Material balances on cellular IgG demonstrated that about 7% of the antibody which was synthesized during exponential batch growth was not released to the growth medium. Distributions of single-cell surface antibody content showed two subpopulations, one with very low surface IgG. The fraction of the population with low surface IgG increased throughout a batch cultivation.

Animals↗
PubMed1989

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

DNA Polymerase I↗
PubMed1988

A mathematical model of recombinational amplification of the 2 mu plasmid in the yeast Saccharomyces cerevisiae.

A mathematical model of 2 mu plasmid recombinational amplification in Saccharomyces cerevisiae has been developed, based on mechanisms of 2 mu recombination and replication presented in the literature. A probabilistic description reveals the limits inherent in the recombinational mode of plasmid amplification. These limits correspond well with values calculated from reported results. In the model, copy number control is effected by the constitutive expression of a repressor of recombinase expression. Estimation of the model parameters is accomplished via a set of heuristic rules which restrict the feasible parameter space considerably. It is demonstrated that many parameter sets arbitrarily chosen from the feasible parameter space reproduce the observed characteristics of 2 mu plasmid amplification: rapid correction of downward copy number deviations, with a lack of strict control of steady-state copy number.

DNA Replication↗
PubMed1988

A single-cell assay of beta-galactosidase activity in Saccharomyces cerevisiae.

A novel assay of single-cell exogenous beta-galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin-beta-D-galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X-100, and the assay is performed at 0 degrees C. These conditions were chosen to minimize intercellular fluorescence communication. Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells. Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate. A quantitative correlation between fluorescence and beta-galactosidase activity is obtained under optimized assay conditions.

Cell Separation↗
PubMedDate not supplied

Stochastic stimulation of 2 mu multicopy plasmid amplification in yeast.

The 2 mu plasmid of Saccharomyces cerevisiae has been used extensively as a vector for foreign protein expression because of its natural amplification to 60-100 copies per cell. The amplification of the 2 mu plasmid occurs through a recombinational mechanism which we have simulated using two free parameters: the FLP recombinase DNA-binding probability and FLP specific enzymatic activity. Model results indicate that a single plasmid amplifies to 5.5-7 copies on average at maximal FLP concentrations and that amplification is approximately proportional to the probability of FLP attachment but independent of its specific enzymatic activity. Thus, FLP overexpression should lead to maximal plasmid amplification. The model predicts the existence of topologically novel replication intermediates.

Computer Simulation↗
PubMedDate not supplied

Constitutive overexpression of secreted heterologous proteins decreases extractable BiP and protein disulfide isomerase levels in Saccharomyces cerevisiae.

High-level gene expression does not always lead to corresponding high-level secretion of heterologous proteins in yeast. The rate-limiting step in many cases has been shown to exit from the endoplasmic reticulum (ER). Within the ER, the correct folding of secreted proteins is required for export competence; hence, the cellular proteins involved in these events are likely to be important for efficient secretion. We have found that the extractable levels of two ER-resident proteins involved in folding--heavy chain binding protein (BiP) and protein disulfide isomerase (PDI)--are significantly reduced by prolonged constitutive overexpression of human granulocyte colony stimulating factor (GCSF), human erythropoietin, or Schizosaccharomyces pombe acid phosphatase. However, the rate of BiP synthesis measured in pulse--chase radiolabeling experiments is not reduced by GCSF overexpression, and galactose-directed transcription of the BiP gene does not restore normal BiP protein levels once they have been depleted. The observed loss of lumenal resident proteins, either by proteolysis or irreversible aggregation, is expected to contribute significantly to the inefficiency of foreign protein secretion in yeast.

Acid Phosphatase↗