Biomedical subjects

A P Middelberg

Publications and source records attributed to A P Middelberg.

At least 19 recordsLinked to original sources

PubMed2001

Direct chemical extraction of a recombinant viral coat protein from Escherichia coli at high cell density.

The release of protein and DNA from nonrecombinant E. coli JM101 and recombinant E. coli HMS174(DE3) expressing L1 (the major viral coat protein of human papillomavirus type 16) as an inclusion body was demonstrated at high cell density (OD(600) = 160). For the nonrecombinant strain, extraction efficiency decreased significantly as cell mass increased, with a high viscosity increase in the postextraction broth. A different dependence on cell concentration was observed for the recombinant strain, with total protein extraction efficiency exceeding 85% for both uninduced and induced cells. Almost complete release of the recombinant L1 protein was achieved at high cell concentration (OD(600) = 80 approximately 160) without the use of reducing agent. This greatly extends the concentration range for chemical extraction.

Biomass↗

PubMed2001

Metabolic and kinetic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli.

A quantitatively repeatable protocol was developed for poly(3-hydroxybutyrate) (PHB) production by Escherichia coli XL1-Blue (pSYL107). Two constant-glucose fed-batch fermentations of duration 25 h were carried out in a 5-L bioreactor, with the measured oxygen volumetric mass-transfer coefficient (k(L)a) held constant at 1.1 min(-1). All major consumption and production rates were quantified. The intracellular concentration profiles of acetyl-CoA (300 to 600 microg x g RCM(-1)) and 3-hydroxybutyryl-CoA (20 to 40 microg x g RCM(-1)) were measured, which is the first time this has been performed for E. coli during PHB production. The kinetics of PHB production were examined and likely ranges were established for polyhydroxyalkanoate (PHA) enzyme activity and the concentration of pathway metabolites. These measured and estimated values are quite similar to the available literature estimates for the native PHB producer Ralstonia eutropha. Metabolic control analysis performed on the PHB metabolic pathway showed that the PHB flux was highly sensitive to acetyl-CoA/CoA ratio (response coefficient 0.8), total acetyl-CoA + CoA concentration (response coefficient 0.7), and pH (response coefficient -1.25). It was less sensitive (response coefficient 0.25) to NADPH/NADP ratio. NADP(H) concentration (NADPH + NADP) had a negligible effect. No single enzyme had a dominant flux control coefficient under the experimental conditions examined (0.6, 0.25, and 0.15 for 3-ketoacyl-CoA reductase, PHA synthase, and 3-ketothiolase, respectively). In conjunction with metabolic flux analysis, kinetic analysis was used to provide a metabolic explanation for the observed fermentation profile. In particular, the rapid onset of PHB production was shown to be caused by oxygen limitation, which initiated a cascade of secondary metabolic events, including cessation of TCA cycle flux and an increase in acetyl-CoA/CoA ratio.

Acetyl Coenzyme A↗

PubMed2000

The mechanical properties of Saccharomyces cerevisiae.

Cell-wall mechanical properties play an integral part in the growth and form of Saccharomyces cerevisiae. In contrast to the tremendous knowledge on the genetics of S. cerevisiae, almost nothing is known about its mechanical properties. We have developed a micromanipulation technique to measure the force required to burst single cells and have recently established a mathematical model to extract the mechanical properties of the cell wall from such data. Here we determine the average surface modulus of the S. cerevisiae cell wall to be 11.1 +/- 0.6 N/m and 12.9 +/- 0.7 N/m in exponential and stationary phases, respectively, giving corresponding Young's moduli of 112 +/- 6 MPa and 107 +/- 6 MPa. This result demonstrates that yeast cell populations strengthen as they enter stationary phase by increasing wall thickness and hence the surface modulus, without altering the average elastic properties of the cell-wall material. We also determined the average breaking strain of the cell wall to be 82% +/- 3% in exponential phase and 80% +/- 3% in stationary phase. This finding provides a failure criterion that can be used to predict when applied stresses (e.g., because of fluid flow) will lead to wall rupture. This work analyzes yeast compression experiments in different growth phases by using engineering methodology.

Cell Wall↗

PubMed2000

Peptide interfacial adsorption is kinetically limited by the thermodynamic stability of self association.

We present a study of the adsorption of two peptides at the octane-water interface. The first peptide, Lac21, exists in mixed monomer-tetramer equilibrium in bulk solution with an appreciable monomer concentration. The second peptide, Lac28, exists as a tetramer in solution, with minimal exposed hydrophobic surface. A kinetic limitation to interfacial adsorption exists for Lac28 at moderate to high surface coverage that is not observed for Lac21. We estimate the potential energy barrier for Lac28 adsorption to be 42 kJ/mol and show that this is comparable to the expected free energy barrier for tetramer dissociation. This finding suggests that, at moderate to high surface coverage, adsorption is kinetically limited by the availability of interfacially active monomeric "domains" in the subinterfacial region. We also show how the commonly used empirical equation for protein adsorption dynamics can be used to estimate the potential energy barrier for adsorption. Such an approach is shown to be consistent with a formal description of diffusion-adsorption, provided a large potential energy barrier exists. This work demonstrates that the dynamics of interfacial adsorption depend on protein thermodynamic stability, and hence structure, in a quantifiable way.

Adsorption↗

PubMed2000

Characterisation of the shrinkage of calcium alginate gel membrane with immobilised Lactobacillus rhamnosus.

To quantify the shrinkage of calcium alginate gel membrane as a support matrix for immobilising cells during the fermentation of Lactobacillus rhamnosus, factors including time, pH, membrane thickness, and the concentrations of immobilised cells, lactic acid, glucose, and calcium chloride were examined by statistical experimental design. A Plackett-Burman design was used for the first screening experiment to identify the important factors which caused the divergent effects. Uniform Design, a powerful modelling design technique, was thus chosen to design the modelling experiments. Through a non-linear step-wise regression analysis, the predictive mathematical model of the shrinkage in membrane thickness was established and the significant main effects and two-factor interactions were identified. However, no significant model equations could be obtained for the shrinkage in area and volume of gel membranes. The methodology developed can be extrapolated to the quantitative characterisation of shrinkage in other immobilised gel matrices, which will be very useful in mathematical modelling, design, operation and scale-up of gel immobilised cell systems.

Alginates↗

PubMed1999

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells.

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized beta-mercaptoethanol, to the permeabilization buffer (6 M urea + 3 mM ethylenediaminetetraacetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. In the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells.

Bioreactors↗

PubMed1999

Control of fed-batch fermentations.

Fed-batch fermentation is used to prevent or reduce substrate-associated growth inhibition by controlling nutrient supply. Here we review the advances in control of fed-batch fermentations. Simple exponential feeding and inferential methods are examined, as are newer methods based on fuzzy control and neural networks. Considerable interest has developed in these more advanced methods that hold promise for optimizing fed-batch techniques for complex fermentation systems.

Journal Article↗

PubMed1998

Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells.

A method is presented for the direct extraction of the recombinant protein Long-R3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (<9). Cell concentration also had a minor effect on Long-R3-IGF-I release and caused an observable increase in viscosity. Advantages of the direct extraction method include its speed, simplicity, and efficiency at releasing product.

Biochemistry↗

PubMed1997

Pilot-scale extraction of PHB from recombinant E. coli by homogenization and centrifugation.

A new method of poly-beta-hydroxybutyrate (PHB) extraction from recombinant E. coli is proposed, using homogenization and centrifugation coupled with sodium hypochlorite treatment. The size of PHB granules and cell debris in homogenates was characterised as a function of the number of homogenization passes. Simulation was used to develop the PHB and cell debris fractionation system, enabling numerical examination of the effects of repeated homogenization and centrifuge-feedrate variation. The simulation provided a good prediction of experimental performance. Sodium hypochlorite treatment was necessary to optimise PHB fractionation. A PHB recovery of 80% at a purity of 96.5% was obtained with the final optimised process. Protein and DNA contained in the resultant product were negligible. The developed process holds promise for significantly reducing the recovery cost associated with PHB manufacture.

Biotechnology↗

PubMed1996

The effect of thermal deactivation on the properties and processing characteristics of E. coli.

Recombinant microorganisms are often employed to produce proteins of commercial significance. It is important to render such organisms non-viable before culture fluids are released from the fermenter. Thermal deactivation is a superficially attractive option because of its simplicity. The effects of such a thermal deactivation step at the end of a recombinant fermentation are reported in this study. In particular, the consequences of this treatment for down-stream process operations, namely homogenisation and centrifugation, are analysed and discussed. Homogenisation efficiency was adversely affected by a simple treatment whereby cells are raised to 65 degrees C from stationary phase. Cell debris size was also significantly increased. These changes could be partially explained by an increase in fractional peptidoglycan crosslinkage and a decrease in mean cell length. A simple process modification removed these detrimental effects. Specifically, the addition of 15 g of glucose 15 mins prior to thermal deactivation enhances downstream processing. Disruption efficiency is increased above that for stationary-phase cells and the resultant cell debris size is significantly reduced, theoretically aiding inclusion body purification. This novel process modification demonstrates that thermal deactivation may be employed to prevent the release of viable recombinant organisms while providing a broth with desirable processing characteristics. It also emphasises the need to optimise any bioprocess as an interconnected sequence of units.

Bioreactors↗

PubMed1996

Centrifugal recovery and dissolution of recombinant Gly-IGF-II inclusion-bodies: the impact of feedrate and re-centrifugation on protein yield.

The impact of centrifuge feedrate and multiple centrifuge passes on protein yield following recombinant Gly-Insulin-like Growth Factor II (Gly-IGF-II) inclusion-body dissolution has been investigated. Altering centrifuge feedrate did not significantly improve the overall protein yield following dissolution. Improved centrifuge recovery at a low feedrate was offset by poorer inclusion body paste purity. This reduced purity resulted in a significant loss of protein during inclusion-body dissolution due to proteolysis. Multiple centrifuge passes improved the inclusion-body paste purity. This resulted in a net improvement in the overall protein yield following dissolution. This work demonstrates that a strong interaction exists between centrifuge performance and inclusion-body dissolution for protease-sensitive products such as Gly-IGF-II.

Bioreactors↗

PubMed1996

Centrifugal processing of cell debris and inclusion bodies from recombinant Escherichia coli.

The settling characteristics of cell debris and inclusion bodies prior to, and following, fractionation in a disc-stack centrifuge were measured using Cumulative Sedimentation Analysis (CSA) and Centrifugal Disc photoSedimentation (CDS). The impact of centrifuge feedrate and repeated homogenisation on both cell debris and inclusion body collection efficiency was investigated. Increasing the normalised centrifuge feedrate (Q/sigma) from 1.32 x 10(-9) m s-1 to 3.97 x 10(-9) m s-1 leads to a 36% increase in inclusion body paste purity. Purity may also be improved by repeated homogenisation. Increasing the number of homogeniser passes results in smaller cell debris size whilst leaves inclusion body size unaltered. At a normalised centrifuge feedrate of 2.65 x 10(-9) m s-1, increasing the number of homogeniser passes from two (2) to ten (10) improved overall inclusion body paste purity by 58%. Grade-efficiency curves for both the cell debris and inclusion bodies have also been generated in this study. The data are described using an equation developed by Mannweiler (1989) with parameters of k = 0.15-0.16 and n = 2.5-2.6 for inclusion bodies, and k = 0.12-0.14 and n = 2.0-2.2 for cell debris. This is the first accurate experimentally-determined grade efficiency curve for cell debris. Previous studies have simply estimated debris grade efficiency curves using an approximate debris size distribution and grade efficiency curves determined with 'ideal particles' (e.g. spherical PVA particles). The findings of this study may be used to simulate and optimise the centrifugal fractionation of inclusion bodies from cell debris.

Cell Fractionation↗

PubMed1995

Process-scale disruption of microorganisms.

Common hosts for the large-scale manufacture of biological products, such as Escherichia coli and Saccharomyces cerevisiae, do not excrete products to the medium. Effective techniques for cell disruption are therefore required. These include physical, chemical, enzymatic and mechanical methods. Mechanical methods such as bead milling, high-pressure homogenization, and microfluidization are preferred. However, gentler, specific methods are receiving increasing attention particularly when used in combination to synergistically exploit their different specificities. Benefits can also be derived by integrating product release and recovery. In all cases it is essential to consider the interaction of the disruption operation with downstream units and to clearly demonstrate the cost benefits of alternative strategies.

Journal Article↗

PubMed1991

Monitoring the centrifugal recovery of recombinant protein inclusion bodies.

The industrial processing of proteins expressed as insoluble inclusion bodies employs a reasonably standard sequence of unit operations. One of these is centrifugation, which serves to concentrate the inclusion bodies after disruption of the host microorganism, and also separates the inclusion bodies from other cellular debris. Monitoring the performance of the centrifuge is essential if excessive product and hence financial loss is to be avoided and a reasonable separation obtained. The analytical disc centrifuge may be used to monitor the centrifugation. This instrument returns the sample size distribution with high resolution and without fouling. By obtaining size distributions of the centrifuge feed, supernatant and concentrate, the fractional collection efficiency of the centrifuge may be determined as a function of the Stokes diameter, and a mass balance constructed.

Cell Fractionation↗

PubMedDate not supplied

Sizing biological samples by photosedimentation techniques.

The performance of the Joyce-Loebl disk centrifuge in the sizing of Escherichia coli cells, protein inclusion bodies, and cell debris is evaluated. The need for a density gradient that extends throughout the entire spin fluid is highlighted, and a set of standard conditions that fulfill this requirement is defined. E. coli cells experience a reduction in their Stokes diameter when exposed to ethanol, indicating that a spin-buffer fluid combination such as glycerol-water is to be preferred for the sizing of bacteria. The instrument baseline is influenced by the presence of particles, and a method of estimating the baseline is described. The sizing of small particles is further complicated by baseline drift due to temperature sensitivity of the optical yoke. An analysis of diffusion in the spin fluid is conducted, and an expression for the sedimentation:diffusive flux ratio is derived. For the current samples, it is shown that diffusion within the spin fluid does not lead to significant errors for 0.15-microns particles, whereas the phenomenon may be significant at the manufacturer's size limit of 0.01 micron.

Centrifugation, Density Gradient↗

PubMedDate not supplied

High-resolution particle size analysis in biotechnology process control.

Many industrially important proteins can now be expressed intracellularly as insoluble protein inclusion bodies. In production, large-scale centrifugation is commonly used to separate and recover the inclusion bodies. Recovery efficiency depends critically on the centrifuge feed rate, which must be optimized to minimize production costs. We have used a disc centrifuge photosedimentometer to make high-resolution measurements of the particle size distribution (PSD) of the supernatant during the production of porcine somatotropin (pST) inclusion bodies. These measurements readily monitor the breakthrough of inclusion bodies into the supernatant and allow the centrifugation operation to be optimized.

Biotechnology↗

PubMedDate not supplied

A correlation for the effective strength of Escherichia coli during homogenization.

A new model for high-pressure homogenization has been previously developed. A key model parameter, the mean effective cell strength, can be correlated with average cell length and peptidoglycan cross-linkage. In this article, we develop a correlation for mean effective strength based on a statistical thermodynamic approach to fracture. The final correlation provides an unbiased estimate. While it offers no numerical advantage over a previous empirical correlation, it is based on a modeling approach and an understanding of wall structure. The variable groups are therefore justifiable.

Bacteriological Techniques↗

PubMedDate not supplied

A simplified model for the disruption of Escherichia coli: the effect of cell septation.

A new model for the disruption of Escherichia coli by high-pressure homogenization has been previously presented. Initial model development assumed a bimodal distribution of effective cell strengths to allow for a possible difference in strength between septated and nonseptated cells. A considerably simpler model is obtained when any difference in strength is neglected and a normal distribution is employed. In this article, the disruption of a culture with an abnormally high septated fraction is examined. Disruption versus pressure curves are predicted using both the bimodal and normal approximations to the strength distribution. An examination of disrupted cultures by optical and electron microscopy suggests that septated cells are indeed weaker, thus implying that a bimodal approximation is strictly correct. However, comparison of the model predictions with the experimental results suggests that the simple normal distribution provides sufficient predictive accuracy even for cultures with a high septated fraction.

Escherichia coli↗