bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of recombinant porcine colipase secreted by Pichia pastoris and its application to improve dietary fat digestion and growth of postweaning piglets
Authors: Liu Fang‐Chueh , Chen Hsiao‐Ling , Chong Kowit‐Yu , Hsu A‐Li , Chen Chuan‐Mu 
Published: December 2008

AbstractRecombinant porcine pancreatic colipase (pCoL) was produced in the methylotrophic yeast Pichia pastoris. A synthetic yeast secretion cassette was constructed with the constitutive promoter of the glyceraldehyde‐3‐phosphate‐dehydrogenase (GAPDH) gene and the yeast α‐mating factor signal peptide. The pCoL cDNA corresponding in the coding sequence, excluding a 16‐amino acid segment of the native signal sequence, was cloned into the pGAPZαB vector and integrated into the genome of P. pastoris. Yeast transformants were cultured and bioactive pCoL protein was detected in the supernatant at a high‐level of 126.8 mg/L after 3 days of culture. The transformed yeast containing the highest recombinant colipase level (pCoL yeast) and native yeast GS 115 not containing pCoL (non‐pCoL yeast, as a control group) were separately cultured and the supernatants were adsorbed by dried skim milk. In an animal trial, two concentrations of colipase activity (0 vs. 5,000 U/kg in the diet) were blended with the pig corn‐soybean basal diet and fed to weaned piglets for 4 weeks. The pCoL‐administrated test group gained significantly more weight than piglets in the control group when measured at Day 15 (11.84 ± 0.70 vs. 10.59 ± 0.39 kg, P < 0.05), Day 22 (15.84 ± 0.95 vs. 14.32 ± 0.59 kg, P < 0.01), and Day 28 (20.19 ± 1.47 vs. 18.54 ± 0.92 kg, P < 0.01) after weaning. The blood triglyceride (TG) concentrations were significantly increased in the experimental test group that received recombinant colipase on the 28th day of postweaning when compared with that of the control group (32.50 vs. 16.37 mg/dL; P < 0.0001). These experimental data suggest that the use of recombinant porcine colipase as a dietary supplement provides an alternative approach for improving fat digestion and enhancing growth in postweaning piglets.
The establishment of a controllable expression system in baculovirus: Stimulated overexpression of polyhedrin promoter by LEF‐2
Authors: Wu Yueh‐Lung , Chao Yu‐Chan 
Published: December 2008

AbstractPreviously, controllable gene expression in baculovirus was not possible using an insect system. We found that this was due to a high background activation of minimal promoter by the viral polyhedrin upstream (pu) sequence. Here, by truncation of the pu sequence, regulatory gene expression was established through the tetracycline regulatory expression system. This novel system was used to test the stimulatory function of the polyhedrin promoter by the controlled expression of the late expression factor‐2 (lef‐2). To efficiently trace lef‐2 expression and analyze suppression of this gene, the coding sequences of lef‐2 and enhanced green fluorescent protein (egfp) were ligated together to generate a fusion protein, and an ∼100‐fold suppression of egfp‐lef‐2 expression was achieved by doxycycline treatment. A very low level expression of lef‐2 was found to be sufficient for proper expression of polyhedrin promoter; however, progressively higher levels of lef‐2 expression could stimulate much higher‐than‐original polyhedrin promoter expression in the viral genome. This system was found to exhibit significantly better suppression than the double‐stranded RNA (dsRNA) strategy, and would be useful for expression of foreign or viral genes whose functions require the interaction of multiple and/or unknown baculovirus gene products.
Ultrathin silver nanowires produced by amyloid biotemplating
Authors: Malisauskas Mantas , Meskys Rolandas , Morozova‐Roche Ludmilla A. 
Published: December 2008

AbstractBy using a self‐assembled amyloid from lysozyme as biotemplate we produced an ultrathin silver wire of 1 nm diameter and up to 2 μm in length, which is at the limit attainable in nanobiotechnological manufacturing. We showed that 2,2,2‐trifluoroethanol produces a dual effect: it reduces ionic silver to colloidal nanoparticles with a regular size, depending on the length of incubation, and induces fibrillar assembly into the amyloid scaffold, forming the hollow channel filled with silver.
Host cell protein clearance during protein a chromatography: Development of an improved column wash step
Authors: Shukla Abhinav A. , Hinckley Peter 
Published: December 2008

AbstractHost cell protein (HCP) contaminant clearance is a significant concern during downstream process development for biopharmaceuticals. Protein A chromatography as a capture step for monoclonal antibodies and Fc fusion proteins can clear a large proportion of these impurities from cell culture harvest. Nevertheless, remaining levels of this process‐related impurity class do present significant constraints on the rapid development of effective and robust polishing steps. Conventionally, an intermediate pH wash is employed between column loading and elution to minimize HCP levels after Protein A chromatography. A significant mechanistic finding presented in this work is that HCP contaminants that persist following Protein A capture predominantly comprise species that associate with the product in preference to direct interaction with the chromatographic resin. This suggests that the development of improved column wash techniques to maximize HCP clearance ought to focus on disrupting protein–HCP interactions rather than Protein A–HCP interactions. A higher wash pH to preserve product – Protein A binding along with the use of additive combinations to disrupt interactions between HCPs and the product are investigated. This strategy was successfully applied to develop a broadly applicable wash condition that has the potential for eliminating the need for product specific optimization of wash conditions. A combination of 1 M urea and 10% isopropanol in the wash buffer were successfully applied as a platform wash condition for Protein A chromatography. Use of this (and other similar) wash conditions are anticipated to aid the rapid development of effective downstream processes for monoclonal antibodies and Fc fusion proteins resulting in their rapid introduction into clinical trials.
Enhancing cell affinity of nonadhesive hydrogel substrate: The role of silica hybridization
Authors: Wang Chunming , Bai Jing , Gong Yihong , Zhang Feng , Shen Jiangbo , Wang Dong‐An 
Published: December 2008

AbstractThe aim of this study is to elucidate the functional mechanism of nanosilica‐induced cell affinity for the normally attachment‐fouling hydrogel substrates. Investigations were conducted in the following three major aspects: (1) environmental protein adsorption before cell contacts, which determines the presence of adhesive ligands; (2) integrin expression profile, which reflects the cell responses via surface receptor turning‐over; and (3) vinculin activation and F‐actin assembly, which sketches the induced cytoskeletal organization posing for focal adhesion. The results indicate that the hybridized nanosilica additives are capable of arresting adhesive proteins from the environment. As binding ligands, such immobilized proteins activate α5β1‐specific pathway for cell focal adhesion, but fail in invoking β3‐participated signaling cascade. This partial activation enables modest amount of high‐quality cell adhesion on the modified substrate, by which a conservative cell affinity is established on hydrogel matrices.
Separation of protein charge variants with induced pH gradients using anion exchange chromatographic columns
Authors: Pabst Timothy M. , Carta Giorgio , Ramasubramanyan Natrajan , Hunter Alan K. , Mensah Paul , Gustafson Mark E. 
Published: December 2008

AbstractThis work concerns the chromatographic separation of protein charge variants using pH gradients generated by step changes in buffer composition with weak base anion exchange columns. A local equilibrium model is first developed to describe pH transitions occurring in the column using buffers comprising neutral, zwitterionic or positively charged species. Model predictions, based solely on the resins' titration curves and obtained with the method of characteristics, show, in excellent agreement with experiments, that induced pH gradients of varying durations and shapes can be obtained with a broad range of buffer systems including Tris, Bis‐Tris propane, histidine, and their mixtures and ethanolamine. The separation of protein charge variants is then demonstrated for bovine apo‐transferrin and for a monoclonal antibody. The resolution of the charge variants present in these proteins, demonstrated via isoelectric focusing analyses, is obtained for conditions amenable to scale‐up for preparative purposes; that is larger particle sizes (90 μm), higher flow rates (100–600 cm/h), and higher protein loads (2–5 mg/mL). Because the approach requires only step changes in buffer composition and commonly available, unretained buffers species, practical implementation is straightforward. The focusing effect of the induced pH gradient results in relatively sharp peaks and substantial resolution even for these conditions.
High Resolution Continuous Flow Electrophoresis
Authors: Gobie William A. , Beckwith James B. , Ivory Cornelius F. 
Published: December 2008

AbstractContinuous free‐flow electrophoresis (CFE) provides the biochemist with a powerful technique for preparative‐scale purification of labile biomolecules. However, solute resolution in the classic CFE is limited by the deleterious effects of diffusion, electroosmotic dispersion, and connective dispersion. These problems can be overcome in the CFE by continuously recycling effluent through the chamber in a manner which allows complete recovery of undiluted product at arbitrarily high purities.Mathematical models of the continuous flow device with recycle (RCFE) are derived for the two limiting cases: rapid diffusion and slow diffusion. The analytical solutions of these models are used to predict separation factors under various operating conditions.
Protein Extraction Using Reverse Micelles
Authors: Cöklen Kent E. , Hatton T. Alan 
Published: December 2008

AbstractThe controlled solubilization of proteins in a reverse micelle‐containing organic phase is considered as a method for separation of proteins by liquid‐liquid extraction. Cytochrome‐C was transferred between a bulk aqueous phase and a micellar phase, using the Aerosol OT‐isooctane system of surfactant and solvent. It was observed that the extent of protein solubilization could be controlled by varying the ionic strength of the aqueous phase equilibrated with the micellar phase.The protein transferred into the micellar phase rapidly, under conditions of low ionic strength, and transferred out of the micelles relatively slowly, under conditions of high ionic strength. It is proposed that the effect of the ionic strength was to alter the electrostatic interaction between the surface charge of the protein and the charged interior of the reverse micelle by Debye screening.
Continuous Immobilized Recombinant Protein Production from E. coli Capable of Selective Protein Excretion: A Feasibility Study
Authors: Georgiou G. , Chalmers J. J. , Shuler M. L. , Wilson D. B. 
Published: December 2008

AbstractCurrently genetically engineered bacteria are economically attractive only for the production of high‐value, low‐volume‐proteins. For this technology to be useful for the manufacture of high volume proteins new bioprocessing concepts are necessary. A potential approach is to construct a plasmid containing an inducible promoter and a hybrid gene containing a signal sequence fused to the gene for the protein to be produced. The plasmid is transformed into Escherichia coli. The plasmid‐host interaction is such that the host becomes “leaky” upon introduction. Since the signal sequence allows the recombinant protein to pass the cytoplasmic membrane, a “leaky cell” can allow selective excretion of the desired protein. If the “leaky” cell is immobilized and maintained in a resting state, the sustained continuous production of the target protein is possible. To test this concept we have used plasmid pKK in E. coli RB791 with β‐lactamase as the target protein. Production levels of at least 390 units β‐lactamase/L‐h can be sustained for at least 120 days. Peak productivities of 10,000 unitslL‐h have been obtained. The β‐lactamase constitutes approximately 40% of the protein in solution. Such systems could reduce recombinant protein production costs due both to the continuous nature of the bioreactor and the improved ease of protein recovery.
Continuous Affinity Chromatography Using a Magnetically Stabilized Fluidized Bed
Authors: Burns Mark A. , Graves David J. 
Published: December 2008

AbstractA new processing tool, the magnetically stabilized fluidized bed, offers opportunities for efficient fluid‐solids contact.The bubbling and mixing normally seen in a fluidized bed are eliminated, and continuous countercurrent contact is possible. This technique is especially suitable for biochemical separations, where cellular debris would clog an ordinary fixed bed. Preliminary modeling and experimental work on such a system is discussed, along with details of a relatively simple experimental system.
Aerosol Formation During the Microwave Freeze Dehydration of Beef
Authors: Arsem H. B. , Ma Y. H. 
Published: December 2008

AbstractData are obtained from the combined microwave and radiant freeze drying of beef steaks. Causes for the high drying rates are explained by the entrainment of solid ice crystals in the vapor during the low pressure drying. The effects of solid entrainment on dehydration rate and product quality are discussed in terms of the loss of intercellular salts and proteins and tearing of muscle fibers caused by high vapor velocity.
Flow Cytometry Analysis of Plasmid Amplification in Escherichia coli
Authors: Seo Jin‐Ho , Srienc Friedrich , Bailey James E. 
Published: December 2008

AbstractLarge quantities of cloned DNAs for use in characterization, or in subsequent recombinant plasmid construction, may be obtained by plasmid amplification, which is the increase of cellular plasmid DNA content relative to the amount of host cell chromosomal DNA. Plasmid amplification has been investigated with flow cytometry in chloramphenicol (Cm) treated cell populations of Escherichia coli HB101 carrying plasmids with pMBI replication origins at different copy number levels ranging from 12 to 122. The flow cytometry method is based on the determination of the total cellular DNA content after staining with mithramycin, a DNA‐specific fluororescent dye. This method permits direct measurement of the rate of plasmid amplification and classification of cell populations according to chromosomal replication forks at the time of Cm treatment. Both initial rates of plasmid amplification and final contents of amplified plasmids were found to be proportional to the average initial plasmid copy numbers. The maximum degree of amplification, defined as the ratio of plasmid content after eighteen hours incubation to the preamplification plasmid content at steady‐state growth, was approximately equal for the strains studied.Larger cells, with four active chromosomal replication forks at Cm addition, exhibit faster plasmid replication rates than cells initially at earlier cell cycle states. A kinetic model was proposed that agrees well with the present and previous experimental observations. This work clearly demonstrates the advantage of flow cytometry for experimental analysis of plasmid propagation in recombinant organisms.
Simplified Models for Estimating Isothermal Operating Characteristics of Food Extruders
Authors: Levine Leon , Rockwood Jeffrey 
Published: December 2008

AbstractA model of isothermal food extruder performance is described. Inferences about alternative extruder screw designs and their performance are drawn from the model. The model suggests that thread depth or diameter compression screws are superior in performance to a pitch compression screw. The advantage gained from using diameter compression screws is paid for with significantly higher rates of energy dissipation. The use of the model to characterize screws having both a compression zone and metering zone is described.
Microbial Desulfurization of Coals in a Slurry Pipeline Reactor Using. Thiobacillus ferrooxidans
Authors: Rai Charanjit 
Published: December 2008

AbstractMicrobial desulfurization of Illinois #6 and Indiana #3 bituminous coal having a total sulfur content of 2 to 8% has been investigated using acidophilic microorganism, Thiobacillus ferrooxidans, in laboratory shake flash experiments and in a two‐inch pipeline loop. The results indicate that about 80 to 85% pyritic sulfur removal was achieved with 10 to 25% coal/water slurry recirculated at 6–7 ft/sec at room temperature in 7 to 14 days. The experimental conditions have been optimized for maximum desulfurization. Results from this study show that the rates of bacterial desulfurization from coal samples are higher in the pipeline loop under turbulent flow conditions as compared to the shake‐flask experiments for particle sizes ranging from 43 to 200 μm. It is visualized that the proposed coal slurry pipelines could be used as biological plug flow reactors under aerobic conditions. The laboratory corrosion studies under dynamic test conditions show that use of a corrosion inhibitor will limit the pipeline corrosion rate to acceptable levels.
The Relationship Between Cell Dry Weight Concentration and Culture Turbidity For A Recombinant E. Coli K12 Strain Producing High Levels of Human Alpha Interferon Analogue
Authors: Fieschko John , Ritch Tom , Bengston David , Fenton Dennis , Mann Michael 
Published: December 2008

AbstractA recombinant E. Coli K12 strain carrying a runaway‐type plasmid with the gene for human alpha interferon analogue and capable of producing high levels of recombinant product [1], was grown in rich medium and compared to the host strain without plasmid, and to the strain with the plasmid, but lacking the interferon gene insert. At a non‐inducing temperature, gross cell morphology and the correlation between dry weight and culture turbidity was comparable for the three cultures. Following induction by temperature elevation, both plasmid‐bearing strains displayed significant cell size change, but only the interferon producing strain underwent a decrease in the ratio of cell concentration measured by dry weight to culture turbidity.
Modeling the Stability of Cephamycin C—Producing N. Lactamdurans During Continuous Culture
Authors: Kirpekar A. C. , Kirwan D. J. , Stieber R. W. 
Published: December 2008

AbstractThe reversion of a cephamycin C‐producing strain of Nocardia lactamdurans to a non‐producing variant under continuous culture conditions was examined at dilution rates between 0.025 and 0.045 hr−1. A model incorporating the influence of revertants, when present in the culture in significant numbers, was necessary to adequately describe the dynamics of the reversion process.
Evaluation of Rotational Flow Stabilized Continuous Electrophoresis for Protein Fractionation
Authors: Noble Paul T. 
Published: December 2008

AbstractA research group at Harwell Laboratory have invented a relatively large‐scale electrophoretic separater, which uses angular momentum to stabilize the fluid flow against thermally induced mixing. An earlier review describes the device and shows potential industrial application. Additional experiments and modeling are reported here to help clarify its potential for industrial protein fractionation. The scale‐up potential of the flow stabilization is not considered.
A Novel Bioreactor‐Cell Precipitator Combination for High‐Cell Density, High‐Flow Fermentations
Authors: Stephanopoulos Gregory , San K.‐Y. , Davison B. H. 
Published: December 2008

AbstractA novel bioreactor design has been investigated for carrying out in a single unit a fermentation and cell recycle process simultaneously. The reactor consisted of a typical fermentor and a side‐arm attached at a certain fixed angle to the fermentor. Due to the enhanced sedimentation in the inclined side‐arm, the cells precipitate quickly and flow back into the fermentor. This allowed an essentially cell‐free fermentation broth to be withdrawn at high rates through the sidearm while maintaining a high cell density culture in the fermentor. Continuous flow fermentation runs of S. cerevisiae demonstrated these features and showed that many fold increases in the steady‐state cell density could be achieved using high flowrates, which in an ordinary chemostat would lead to washout. Ethanol productivities were high and can further be increased several‐fold through a straightforward scale‐up of the precipitator sidearm to sustain even larger flow rates. Discussed also are various other reactor features such as the enhanced resistance to contamination, possible reduction of the plasmid instability problems of recombinant microorganisms, potential applications with flocculent strains, steady‐state coexistence of competing populations in mixed culture, and the possibility of carrying out fermentations with stationary cultures under continuous flow conditions.
Stable Competitive Coexistence in a Continuous Fermentor with Size‐Selective Properties
Authors: Davison B. H. , San K.‐Y. , Stephanopoulos Gregory 
Published: December 2008

AbstractA reactor with size‐selective properties was found to allow a mixed culture of Escherichia coli and Saccharomyces cerevisiae to coexist under continuous culture conditions. The reactor consisted of a fermentor with an attached inclined side‐arm to recycle the cells by enhanced sedimentation. The larger size yeast population was retained preferentially and recycled at high efficiencies while the smaller yet faster growing bacteria were removed through the side‐arm. Stability analysis indicated that the coexistence of this system could be stable only if the net yeast removal rate through the side‐arm as a function of the reactor yeast concentration was concave up. This would occur if growth continued in the side‐arm or if settling efficiency decreased at higher cell densities. Another experimental system was devised to measure this net removal rate function. A negative removal rate (i.e., a net addition of yeast to the fermentor) was observed at low biomass indicating growth in the settler and explaining the stability of the coexistence steady state.
An Application of the Method of Lines in Biological Mass Transfer
Authors: Mayes Peter J. D. 
Published: December 2008

AbstractThe method of lines is a numerical technique particularly suited to the solution of parabolic partial differential equations, which has been the subject of much research in recent years, and which has been incorporated into a number of readily available computer library packages. The method has been applied to a number of problems in areas such as chemical engineering, atmospheric physics and nuclear engineering. It does not however, seem to have been widely used in biological mass transfer problems of the type arising in the study of artificial lungs and kidneys. By describing some of the problems tackled by the author and his colleagues, it is hoped to interest a wider audience in the potential benefits of this method.