In vivo resolution of oligomers with fluorescence photobleaching recovery histograms.

Simple independent enzyme-catalyzed reactions distributed homogeneously throughout an aqueous environment cannot adequately explain the regulation of metabolic and other cellular processes in vivo. Such an unstructured system results in unacceptably slow substrate turnover rates and consumes inordinate amounts of cellular energy. Current approaches to resolving compartmentalization in living cells requires the partitioning of the molecular species in question such that its localization can be resolved with fluorescence microscopy. Standard imaging approaches will not resolve localization of protein activity for proteins that are ubiquitously distributed, but whose function requires a change in state of the protein. The small heat shock protein sHSP27 exists as both dimers and large multimers and is distributed homogeneously throughout the cytoplasm. A fusion of the green fluorescent protein variant S65T and sHSP27 is used to assess the ability of diffusion rate histograms to resolve compartmentalization of the 2 dominant oligomeric species of sHSP27. Diffusion rates were measured by multiphoton fluorescence photobleaching recovery. Under physiologic conditions, diffusion rate histograms resolved at least 2 diffusive transport rates within a living cell potentially corresponding to the large and small oligomers of sHSP27. Given that oligomerization is often a means of regulation, compartmentalization of different oligomer species could provide a means for efficient regulation and localization of sHsp27 activity.

Concentration-dependent internalization of a cytokine/cytokine receptor complex in human hematopoietic cells.

Soluble steel factor (SF) is a potent stimulator of hematopoietic progenitor cell proliferation in vitro, and cytokine combinations that include SF can support extensive expansions of hematopoietic cells. Recently, we showed that very primitive progenitor cells from normal human bone marrow require exposure to very high concentrations of cytokines to maintain their primitive status while proliferating. These cells also display higher cell-specific cytokine uptake rates than more differentiated types of hematopoietic cells. As a first step toward identifying the mechanisms involved in mediating such cytokine dose-dependent effects, we have now investigated the kinetics of SF receptor (c-kit) internalization by human Mo7e cells exposed to different extracellular concentrations of soluble SF. Transfer of Mo7e cells to a higher concentration of SF caused an initially rapid downregulation of cell surface c-kit which was accompanied by a rapid depletion of extracellular SF. Confocal microscopy showed a concomitant increase in the number and intensity of intracellular c-kit aggregates. After the first 30 min, the cells continued to deplete SF from the medium but at a much slower rate. During this period, there was a gradual recovery of expression of c-kit on the cell surface. A mathematical analysis of bulk medium to cell-surface SF-mass transport indicated that the cytokine-depletion rates measured were not likely to have significantly depleted the SF concentration in the microenvironment of the cells. Taken together, these results underscore the importance of monitoring and appropriately regulating cytokine concentrations in hematopoietic cell expansion cultures. They may also help to explain the different biological responses exhibited by primitive hematopoietic cells exposed to different types and concentrations of cytokines for periods of days.

Cellulose as an inert matrix for presenting cytokines to target cells: production and properties of a stem cell factor-cellulose-binding domain fusion protein.

A chimaera of stem cell factor (SCF) and a cellulose-binding domain from the xylanase Cex (CBDCex) effectively immobilizes SCF on a cellulose surface. The fusion protein retains both the cytokine properties of SCF and the cellulose-binding characteristics of CBDCex. When adsorbed on cellulose, SCF-CBDCex is up to 7-fold more potent than soluble SCF-CBDCex and than native SCF at stimulating the proliferation of factor-dependent cell lines. When cells are incubated with cellulose-bound SCF-CBDCex, activated receptors and SCF-CBDCex co-localize on the cellulose matrix. The strong binding of SCF-CBDCex to the cellulose surface permits the effective and localized stimulation of target cells; this is potentially significant for long-term perfusion culturing of factor-dependent cells. It also permits the direct analysis of the effects of surface-bound cytokines on target cells.

Heat resistance of Salmonella weltevreden in low-moisture environments.

The heat resistance of Salmonella weltevreden inoculated into flour and heated in hot air was determined for (a) an initial water activity (aw) range of 0.20 to 0.60 prior to heating, (b) a range of storage relative humidities of 6.0 to 35.5% prior to heating, and (c) temperatures of 57 to 77 degrees C. The death curves obtained were biphasic, demonstrating an initial rapid decline in the numbers of survivors (1.0- to 1.5-log reductions) during the first 5 to 10 min of heating for all the temperature-water activity combinations tested. Following this initial rapid decline in the number of cells, a linear survivor curve was obtained where inactivation occurred at a slower rate. The initial decline in survivors coincided with a rapid decrease in the water activity of all the samples tested. Irrespective of the initial water activity level in the samples prior to heating, the aw decreased to < 0.2 during the first 5 to 10 min of heating. The D values obtained for these experimental parameters ranged from a D60-62 of 875 min at an initial aw of 0.4 to a D63-65 of 29 min at an initial aw of 0.5. The results demonstrated that, for any temperature, as the initial water activity of the sample prior to heating decreased, the heat resistance of the cells increased. The z values obtained from these data ranged from 15.2 to 53.9 degrees C. The relative humidity during storage prior to heating did not appear to have a significant effect on the heat resistance of S. weltevreden in flour. These results demonstrate that the amount of available water in foods that are considered to be "dry" (i.e., with a water activity less than 0.60) will significantly influence the effectiveness of the heat processing of foods and, in addition to the temperature, the aw prior to heating is a critical controlling factor during these processes.

Surface diffusion of cellulases and their isolated binding domains on cellulose.

The surface diffusion rate of bacterial cellulases from Cellulomonas fimi on cellulose was quantified using fluorescence recovery after photobleaching analysis. Studies were performed on an exo-beta-1-4-glycanase (Cex), an endo-beta-1-4-glucanase (CenA), and their respective isolated cellulose-binding domains (CBDs). Although these cellulose-binding domains bind irreversibly to microcrystalline cellulose, greater than 70% of bound molecules are mobile on the cellulose surface. Surface diffusion rates are dependent on surface coverage and range from a low of 2 x 10(-11) to a maximum of 1.2 x 10(-10) cm2/s. The fraction of mobile molecules increases only slightly with increasing fractional surface coverage density. Results demonstrate that the packing of C. fimi cellulases and their isolated binding domains onto the cellulose surface is a dynamic process. This suggests that the exclusion of potential CBD binding sites on the cellulose due to steric effects of neighboring bound CBDs may not fully explain the apparent negative cooperativity exhibited in CBD adsorption isotherms. Comparison with the kinetics of cellulase hydrolysis of crystalline substrate suggests that surface diffusion rates do not limit cellulase activity.

Very high-level production and export in Escherichia coli of a cellulose binding domain for use in a generic secretion-affinity fusion system.

A novel expression vector pTugA, previously constructed in our laboratory, was modified to provide kanamycin resistance (pTugK) and used to direct the synthesis of polypeptides as fusions with the C- or N-terminus of a cellulose binding domain which serves as the affinity tag in a novel secretion-affinity fusion system. Fed-batch fermentation strategies were applied to production in recombinant E. coli TOPP5 of the cellulose binding domain (CBD) from the Cellulomonas fimi cellulase Cex. The pTugK expression vector, which codes for the Cex leader sequence that directs the recombinant protein to the periplasm of E. coli, was shown to remain stable at very high-cell densities. Recombinant cell densities in excess of 90 g (dry cell weight)/L were achieved using media and feed solutions optimized using a 2(n) factorial design. Optimization of inducer (isophenyl-thio-beta-D-galactopyranoside) concentration and the time of induction led to soluble, fully active CBD(Cex) production levels in excess of 8 g/L.

On-line monitoring and control of methanol concentration in shake-flask cultures of Pichia pastoris.

The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol.

Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven.

Isothermal titration microcalorimetry is combined with solution-depletion isotherm data to analyze the thermodynamics of binding of the cellulose-binding domain (CBD) from the beta-1,4-(exo)glucanase Cex of Cellulomonas fimi to insoluble bacterial microcrystalline cellulose. Analysis of isothermal titration microcalorimetry data against two putative binding models indicates that the bacterial microcrystalline cellulose surface presents two independent classes of binding sites, with the predominant high-affinity site being characterized by a Langmuir-type Ka of 6.3 (+/-1.4) x 10(7) M-1 and the low-affinity site by a Ka of 1.1 (+/-0.6) x 10(6) M-1. CBDCex binding to either site is exothermic, but is mainly driven by a large positive change in entropy. This differs from protein binding to soluble carbohydrates, which is usually driven by a relatively large exothermic standard enthalpy change for binding. Differential heat capacity changes are large and negative, indicating that sorbent and protein dehydration effects make a dominant contribution to the driving force for binding.

Strategies for maximizing metallothionein promoter regulated recombinant protein production in mammalian cell cultures.

A stably transformed BHK cell line, engineered to produce a human transferrin half-molecule under the control of a mouse metallothionein (MT) promoter, was used as a model system to develop strategies to increase inducible recombinant protein production. Gene expression regulated by the MT promoter is induced by heavy metals (e.g. Zn(+2) or Cd(+2)) in a dose dependent fashion. However, at high concentrations these metals are toxic to cells. Culture protocols which balance these counteractive effects are needed to maximize transferrin production. Fully induced cells produced up to 0.7 pg transferrin/cell·h, a 3-fold increase in production over uninduced levels. Cell growth was inhibited at Cd(+2) dosages above 1 fmol/cell; prolinged exposure at this dosage was cytotoxic. Cell specific transferrin productivities decreased within 48 h following induction with Cd(+2) although cell-associated Cd(+2) levels remain high. Further addition of Cd(+2) to cultures restored cell specific transferrin production rates. This suggests that cell associated Cd(+2) is sequestered into a form which does not stimulate the MT promoter. Cd(+2) dosing regimes which maintained cell associated Cd(+2) concentrations between 0.2 and 0.35 fmol/cell ensured cell growth and high cell specific productivities which maximized final product titers. For routine batch culture, initial Cd(+2) loadings of 0.8 fmol/cell gave near-maximum transferrin production levels. For extended culture, repeated small doses of 0.5 fmol/cell every 24 to 48 h maximized transferrin synthesis with this cell line.

The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose.

CenA is a bacterial cellulase (beta-1,4-glucanase) comprised of a globular catalytic domain joined to an extended cellulose-binding domain (CBD) by a short linker peptide. The adsorption of CenA and its two isolated domains to crystalline cellulose was analyzed. CenA and CBD.PTCenA' (the CBD plus linker) adsorbed rapidly to cellulose at 30 degrees C, and no net desorption of protein was observed during the following 16.7 h. There was no detectable adsorption of the catalytic domain. Scatchard plots of adsorption data for CenA and for CBD.PTCenA were nonlinear (concave upward). The adsorption of CenA and CBD.PTCenA exceeded 7 and 8 mumol/g cellulose, respectively, but saturation was not attained at the highest total protein concentrations employed. A new model for adsorption was developed to describe the interaction of a large ligand (protein) with a lattice of overlapping potential binding sites (cellobiose residues). A relative equilibrium association constant (Kr) of 40.5 and 45.3 liter.g cellulose-1 was estimated for CenA and CBD.PTCenA, respectively, according to this model. A similar Kr value (33.3 liter.g-1) was also obtained for Cex, a Cellulomonas fimi enzyme which contains a related CBD but which hydrolyzes both beta 1,4-xylosidic and beta-1,4-glucosidic bonds. It was estimated that the CBD occupies approximately 39 cellobiose residues on the cellulose surface.

Rapid immunoassay technique for process monitoring of animal cell fermentations.

A calibration and quality control technique suited to process monitoring with immunoassay is demonstrated. The particle concentration fluorescence immunoassay (PC-FIA) is shown to provide a sensitive and rapid method for the quantification of specific biomolecules in cell cultures. Smoothing of linear calibration parameters is performed by forming weighted averages of standard points as the run progresses. These estimates are then used to determine slope and intercept values for improved calibration. The nonuniformity of the fluorescent signal variance is also considered, and a weight model is developed to describe the relationship between signal fluorescence and signal variance for weighted linear curve fitting. Pooling calibration results over the process run improves overall assay performance as determined by using standard control chart analysis. This method is suitable for semicontinuous monitoring of animal cell fermentations and has been used here to measure cell-associated and culture supernatant concentrations of monoclonal antibody (Ab) from hybridoma cells. The cell-associated Ab concentration correlates with cell-specific production rate. Assay times on the order of 10 min for supernatant and 25-30 min for cell-associated Ab concentrations can be achieved, making this procedure suitable for process monitoring and control. Under these conditions the assay has a detection limit of approximately 10 ng/mL, providing a sensitive and specific method for the quantification of cell culture constituents.