Cell‐free production of a therapeutic protein: Expression,purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell‐free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell‐free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell‐free system using instrumented mini‐bioreactors for highly reproducible protein production. We achieved recombinant protein production (～600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell‐free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag‐free self‐cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose‐level production of therapeutic proteins at the point‐of‐care.     

Distribution of the CD4 Alleles in Sus scrofa Demonstrates the Genetic Profiles of Western Breeds and Miniature Pigs

Widely used antipig CD4 monoclonal antibodies (mAbs) fail to recognize CD4 alleles characteristic of miniature pig lines such as the National Institutes of Health (NIH) miniature pigs and microminipigs. We surveyed polymorphisms in the coding sequence of the porcine CD4 gene among Western and Oriental pig breeds and Japanese wild boars and investigated their distribution. Of the 13 alleles that we identified among the 47 animals, 2 in group I and 3 in group II were found exclusively in Western breed pigs. Group IV alleles, which included mAb-nonbinding alleles, were found frequently in Oriental breed pigs, suggesting that the mAb-nonbinding allele arose from the gene pool of Oriental pigs. Group IV alleles were also found in Duroc and Large White pigs, suggesting genetic inflow from Oriental pig breeds into Western breeds. Comparison of the CD4 sequences of species in Cetartiodactyla suggested that the group IV alleles in Sus scrofa occurred before the divergence of this species from the other artiodactyls. The different antibody specificities of the various CD4 alleles may facilitate the discrimination of T-cell populations in transplantation studies using miniature pigs. The significance of the preservation of CD4 polymorphisms to immune function in pigs warrants further investigation.     

Evaluation and applications of optical cell density probes in mammalian cell bioreactors

On-line optical cell density probes were implemented to continuously monitor the cell densities in mammalian cell bioreactor and to achieve advanced bioreactor controls. We tested cell density probes from six manufacturers in high cell density bioreactors. When externally calibrated, Aquasant and Ingold backscattering probes produced the most linear probe responses (PR) versus cell density (CD), followed by the ASR and Cerex laser probes. Monitek and Wedgewood transmission probes had lower resolutions. All probes were tested in two murine hybridoma fermentations. Cell densities varied between 1 x 10(6) cells/mL to 20 x 10(6) cells/mL and the bioreactors were operated for 5 to 7 weeks. For our bioreactors, Aquasant, Ingold, ASR, Wedgewood, and Monitek probes gave satisfactory responses. Little fouling was observed with any probe at the end of 2 weeks. Fouling was a possibility after 3 weeks in one bioreactor but its effect can be easily corrected. Cell density control and specific perfusion control of bioreactors based on the Aquasant probe were achieved. Implementation of cell density probe based perfusion control, instead of "step perfusion adjustments" based on manual hemacytometer control, will result in smoother operation, healthier cultures, increased medium delivery efficiency, and reduced operational excursions. (c) 1995 John Wiley & Sons, Inc.     

Protocol for determining bioavailability and biokinetics of organic pollutants in dispersed, compacted and intact soil systems to enhance in situ bioremediation

The development of effective in situ and on-site bioremediation technologies can facilitate the cleanup of chemically-contaminated soil sites. Knowledge of biodegradation kinetics and the bioavailability of organic pollutants can facilitate decisions on the efficacy of in situ and on-site bioremediation of contaminated soils and determine the attainable treatment end-points. Two kinds of compounds have been studied: (1) phenol and alkyl phenols, which represent hydrophilic compounds, exhibiting high water solubility and moderate to low soil partitioning; and (2) polycyclic aromatic hydrocarbons which are hydrophobic compounds with low water solubility and exhibit significant partitioning in soil organic carbon. Representative data are given for phenol and naphthalene. The results provide support for a systematic multi-level protocol using soil slurry, wafer and porous tube or column reactors to determine the biokinetic parameters for toxic organic pollutants. Insights into bioremediation rates of soil contaminants in compact soil systems can be attained using the protocol. Received 04 December 1995/ Accepted in revised form 31 January 1997     

An internal sedimentation bioreactor for laboratory-scale removal of toxic metals from soil leachates using biogenic sulphide precipitation

An internal-sedimentation bioreactor was employed to provide biomass feedback and process intensification in a laboratory-scale sulphide-bioprecipitation system for toxic metals (Cd, Cr, Cu, Mn, Ni, Zn) present in acid leachates from metal-contaminated soil. Biomass feedback was improved by addition of a cationic polymer flocculant and the activity of the sulphate-reducing bacterial culture was increased by the addition of cornsteep in addition to the ethanol used as carbon/energy substrate. A mass-balance was carried out for carbon and sulphur in the system. Sulphate reduction in the reactor was able to remove acidity at moderate sulphate concentrations up to 50 mM although it was insufficient at the highest levels tested. When presented with a simulated toxic metal-containing leachate, the reactor was able to precipitate metals efficiently under all of the conditions of sulphate concentration and pH tested, producing an effluent with metal concentrations suitable for environmental discharge. Received 29 October 1996/ Accepted in revised form 31 March 1997     

Long-term liver cell cultures in bioreactors and possible application for liver support

Hybrid artificial liver systems are being developed as a temporary extracorporeal liver support therapy. A short overview is given which emphasizes the development of hepatocyte culture models for bioreactors, subsequent in vitro studies, animal studies and the clinical application of hybrid liver support systems.An own bioreactor construction has been designed for the utilization of hepatocytes and sinusoidal endothelial cells. The reactor is based on capillaries for hepatocyte aggregate immobilization, coated with biomatrix. Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network. Cells are perfused via independent capillary membrane compartments. Decentralized oxygen supply and carbon dioxide removal with low gradients is possible. There is a decentralized co-culture compartment for nonparenchymal liver cells. The use of identical parallel units to supply a few hepatocytes facilitates scale-up.     

Simple method for simultaneous analysis of different fibroblast strains on one slide

Summary Microscopic slides were mounted with paraffin on Teflon plates that contain 18 holes of 6 mm diameter each. This arrangement is reversible and yields 18 individual cell culture well on one slide, thus allowing a simultaneous culture of different cell lines under identical conditions. The glass-paraffin junction can be easily detached and the paraffin sticks exclusively to the Teflon. This investigation was supported in part by research grants from the Deutsche Forschungsgemeinschaft.     

The cellular basis of wing size modification in Drosophila: The effects of the miniature gene,crowding, and temperature

To elucidate the mechanisms whereby genes and environment influence wing size, we investigated the effects of various rearing temperatures and larval crowding conditions on the wings of the mutant miniature and wild-type fruit flies. In adults we monitored wing size, cell number, wing thickness, cell density; in larval imaginal discs we looked for cell death. Cell density was inversely proportional to wing size. Of particular interest was the finding that smaller wings tend to be thicker. Electron microscope studies showed that the miniature wing layers are grossly abnormal. We hypothesize that these abnormalities are due to abnormal cell flattening of the wing epithelial cells, and we conclude that gene and environmental effects on cell flattening may be an important component in determining cell density and hence organ size.     

The problems associated with high biomass levels in plant cell suspensions

The development of plant cell cultures as an alternative supply of phytochemicals has been difficult. Although there has been some very suitable targets, the yields of these compounds has remained low despite considerable efforts. One of the main constituents of a process is its productivity which is the sum of the process run time (growth rate), yield, and biomass levels. The effect of changes in all three of these components on productivity has been demonstrated.Of the three components making up productivity, biomass is perhaps the easiest to increase. However, high biomass levels will increase the viscosity, which will affect both mixing and oxygen supply. Therefore, this will require more vigorous mixing which may increase the shear within the bioreactor. All these parameters need further investigation at high biomass concentrations.     

Characterization of swine leukocyte antigen alleles and haplotypes on a novel miniature pig line,Microminipig

Microminipigs are extremely small‐sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo‐ and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA‐defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA‐1, SLA‐2 and SLA‐3) and two class II (SLA‐DRB1 and SLA‐DQB1) genes of 14 parental Microminipigs using a high‐resolution nucleotide sequence‐based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp‐31.0 (SLA‐1*1502–SLA‐3*070102–SLA‐2*1601) and Hp‐0.37 (SLA‐DRB1*0701–SLA‐DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.