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.     

Propagation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae: flow cytometry studies and segregated modeling

Efficient expression of a foreign protein product by the yeastSaccharomyces cerevisiaerequires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromereplasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, evenin a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that amaximum of 4-6 centromere plasmids per cell can be stably partitionedat cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.     

Rolling adhesion kinematics of yeast engineered to express selectins

Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin-expressing yeast on a ligand-coated surface. E-selectin-expressing yeast roll specifically on surfaces bearing sialyl-Lewis-x ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large-budded yeast "walk" across the surface, alternately pivoting about bud and parent. Small-budded yeast "wobble" across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin-expressing cells are rolling rather than slipping across ligand-coated surfaces.     

Rapid method for measuring ScFv thermal stability by yeast surface display

We have characterized a simplified method to determine the relative thermal stability of single-chain antibodies by following the irreversible denaturation of scFv fusions on the surface of yeast by flow cytometry. The method was highly reproducible and correlated well with other methods used to monitor thermal denaturation of the soluble proteins. We found a range of thermal stabilities for wild-type single-chain antibodies with half-maximum denaturation temperatures between 43 and 61 degrees C. The ability to quantitate thermal stability of antibodies or other proteins that are immobilized on the surface of yeast allows rapid comparisons of primary structural information with stability. Thermal denaturation could be a useful parameter to consider in the choice of scFv fragments for various applications.     

Interleukin-2 mutants with enhanced alpha-receptor subunit binding affinity

Stimulation of T-cells by IL-2 has been exploited for treatment of metastatic renal carcinoma and melanoma. However, a narrow therapeutic window delimited by negligible stimulation of T-cells at low picomolar concentrations and undesirable stimulation of NK cells at nanomolar concentrations hampers IL-2-based therapies. We hypothesized that increasing the affinity of IL-2 for IL-2Ralpha may create a class of IL-2 mutants with increased biological potency as compared with wild-type IL-2. Towards this end, we have screened libraries of mutated IL-2 displayed on the surface of yeast and isolated mutants with a 15-30-fold improved affinity for the IL-2Ralpha subunit. These mutants do not exhibit appreciably altered bioactivity at 0.5-5 pM in steady-state bioassays, concentrations well below the IL-2Ralpha equilibrium binding constant for both the mutant and wild-type IL-2. A mutant was serendipitously identified that exhibited somewhat improved potency, perhaps via altered endocytic trafficking mechanisms described previously.     

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

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.     

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.     

Directed evolution of a secretory leader for the improved expression of heterologous proteins and full‐length antibodies in Saccharomyces cerevisiae

Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in Saccharomyces cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three‐ to tenfold improvements over wild‐type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFα1pp) is screened for the enhanced secretion of a single‐chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to 16‐fold over wild type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a 180‐fold improvement over previous reports in the secretion of full‐length, functional, glycosylated human IgG₁. The production of full‐length IgG₁ at milligram per liter titers in a simple, laboratory‐scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs. Biotechnol. Bioeng. 2009;103: 1192–1201. © 2009 Wiley Periodicals, Inc.