Search PubMed⌕ Search

Biomedical subjects

D I Wang

Publications and source records attributed to D I Wang.

At least 37 records · Page 2Linked to original sources
PubMed1995

Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chains.

The failure of newly synthesized polypeptide chains to reach the native conformation due to their accumulation as inclusion bodies is a serious problem in biotechnology. The critical intermediate at the junction between the productive folding and the inclusion body pathway has been previously identified for the P22 tailspike endorhamnosidase. We have been able to trap subsequent intermediates in the in vitro pathway to the aggregated inclusion body state. Nondenaturing gel electrophoresis identified a sequential series of multimeric intermediates in the aggregation pathway. These represent discrete species formed from noncovalent association of partially folded intermediates rather than aggregation of native-like trimeric species. Monomer, dimer, trimer, tetramer, pentamer, and hexamer states of the partially folded species were populated in the initial stages of the aggregation reaction. This methodology of isolating early multimers along the aggregation pathway was applicable to other proteins, such as the P22 coat protein and carbonic anhydrase II.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1994

Engineering cell shape and function.

An elastomeric stamp, containing defined features on the micrometer scale, was used to imprint gold surfaces with specific patterns of self-assembled monolayers of alkanethiols and, thereby, to create islands of defined shape and size that support extracellular matrix protein adsorption and cell attachment. Through this technique, it was possible to place cells in predetermined locations and arrays, separated by defined distances, and to dictate their shape. Limiting the degree of cell extension provided control over cell growth and protein secretion. This method is experimentally simple and highly adaptable. It should be useful for applications in biotechnology that require analysis of individual cells cultured at high density or repeated access to cells placed in specified locations.

Albumins↗
PubMed1994

Process monitoring of the production of gamma-interferon in recombinant Chinese hamster ovary cells.

The production of recombinant gamma-interferon was monitored using high-performance liquid chromatographic methods. These methods were able to distinguish between glycosylated and non-glycosylated forms of gamma-interferon by complexing the carbohydrate with borate. Sufficient quantities of standard glycosylated gamma-interferon were not available for peak identification so immunological techniques were used to identify gamma-interferon variants. These techniques were validated with the non-glycosylated form. The non-glycosylated form was then shown to be retained only on a cation-exchange column, while the glycosylated form, complexed with borate, was retained only on an anion-exchange column. Samples were drawn at 2-h intervals over a 60-h production cycle and analyzed by both anion- and cation-exchange chromatography. Results indicated that the production of each form was coincidental and that the glycosylated form of gamma-interferon is produced in greater abundance than non-glycosylated.

Animals↗
PubMed1994

Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor.

In our previous work (Xie and Wang, 1994a), a simplified stoichiometric model on energy metabolism for animal cell cultivation was developed. Fed-batch experiments were performed in T-flasks using this model in supplemental medium design (Xie and Wang, 1994b). In this work, the major pathways of glucose and glutamine metabolism were incorporated into the stoichiometric model. Fed-batch culture was conducted in a 2-liter bioreactor with appropriate process control strategies. Nutrient concentrations, especially glucose and glutamine, were maintained at constant but low levels through the automated feeding of a supplemental medium formulated using the improved stoichiometric model. The formation of toxic byproducts, such as ammonia and lactate (Hassell et al., 1991), was greatly reduced. The specific lactate production rate was decreased by 62-fold compared with batch culture in bioreactor and by 8-fold compared to fed-batch culture in T-flask using the previous stoichiometric model. Ammonia formation was also decreased compared with both the batch and fed-batch cultures. Most importantly, the monoclonal antibody concentration reached 900 mg l-1, an increase of 17- and 1.6-fold compared with the batch and fed-batch cultures respectively.

Amino Acids↗
PubMed1993

Automated process monitoring of monoclonal antibody production.

Antifibronectin, monoclonal antibody was monitored through 52 h of production. Samples were automatically drawn from a bioreactor into the injection valve of an HPLC system without prior sample preparation. The hybridoma cell line was nonadherent, so whole cells were injected directly onto the perfusable protein A affinity column. There was only a modest column back pressure (ca. 1700 psi at a linear flow rate of 1.5 cm/s) after over 75 injections over the 52-h experiment. These experiments demonstrate the utility of high-speed chromatography for rapid process monitoring.

Animals↗
PubMed1992

Polyethylene glycol enhanced refolding of bovine carbonic anhydrase B. Reaction stoichiometry and refolding model.

Polyethylene glycol (PEG) inhibited aggregation during refolding of bovine carbonic anhydrase B (CAB) through the formation of a nonassociating PEG-intermediate complex. Stoichiometric concentrations of PEG were required for complete recovery of active protein during refolding at aggregating conditions. For example, a PEG (Mr = 3350) to CAB molar ratio ([PEG]/[CAB]) of 2 was sufficient to inhibit aggregation during refolding at 1.0 mg/ml (33.3 microM) protein and 0.5 M guanidine hydrochloride. In addition, the PEG concentration required for enhancement was dependent upon the molecular weight and only molecular weights between 1000 and 8000 were effective in inhibiting aggregation. In the presence of PEG, the rate of refolding was the same as that observed for refolding without the formation of associated species. Refolding in the presence of PEG resulted in the rapid formation of a PEG complex with the molten globule first intermediate, and this PEG-intermediate complex did not aggregate. The CAB refolding kinetics in the presence of PEG were determined and used to develop a model of the PEG enhanced refolding pathway. The mathematical model was validated by independent activity measurements of CAB refolding. This model predicted that PEG enhanced refolding of CAB occurred by a specific interaction of PEG with the molten globule first intermediate to form a nonassociating complex which continued to fold at the same rate as the first intermediate. The predicted pathway and binding properties of PEG indicate that PEG enhanced refolding may be analogous to chaperonin mediated protein folding.

Animals↗
PubMed1992

Polyethylene glycol enhanced protein refolding.

Previous studies on the refolding of recombinant bovine carbonic anhydrase B (CAB) indicated that polyethylene glycol (PEG) significantly enhanced the recovery of active protein by reducing aggregation. To further test the ability of PEG to enhance refolding, three recombinant human proteins, deoxyribonuclease (rhDNAse), tissue plasminogen activator (rhtPA), and interferon-gamma (rhIFN-gamma) were refolded in the presence of PEG (3350 MW). rhDNAse produced from CHO cells was denatured in 7.2 M urea and refolded by rapid dilution to 4.0 M urea and 0.20 mg/ml protein. When a final PEG to rhDNAse molar ratio of 5 to 1 (0.1 milligram PEG, 3350 MW) was used in the dilution buffer, refolding was improved by 30% to yield complete recovery of active protein. Impure E. coli derived inclusion body preparations of rhDNAse were solubilized in 8 M urea and refolded by dilution to 4 M urea and 0.10 mg/ml protein. Refolding with a dilution buffer which yielded a final PEG to rhDNAse molar ratio of 10 to 1 (0.1 milligram PEG, 3350 MW) resulted in a three-fold increase in the recovery of active protein. When PEG was used in the dilution buffer, aggregation of rhDNAse did not occur during refolding in either case. rhtPA produced from CHO cells was denatured in 5 M guanidine hydrochloride (GuHCl) and refolded by rapid dilution to 0.10 M GuHCl and 0.20 mg/ml protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Refolding and aggregation of bovine carbonic anhydrase B: quasi-elastic light scattering analysis.

Bovine carbonic anhydrase B (CAB) is chosen as the model protein to study the phenomenon of protein aggregation, which often occurs during the refolding process. Refolding of CAB from 5 M GuHCl has been observed by quasi-elastic light scattering (QLS), which confirms the formation of a molten globular protein structure as reported previously [Semisotnov, G. V., Rodionova, N. A., Kutyshenko, V. P., Ebert, B., Blanck, J., & Ptitsyn, O. B. (1987) FEBS Lett. 224, 9-13]. QLS analysis reveals the formation of multimeric species prior to precipitation. Activity and cross-linking studies have confirmed the presence of inactive multimeric protein species. The dimer formation has been determined to be the initiating step in the aggregation of CAB during refolding. Activity studies have indicated that the first intermediate observed in the refolding pathway of CAB aggregates to form the inactive dimer. The rate of formation of the dimer has a stoichiometric dependence on the final protein concentration. The dimer formation rate is a function of the final guanidine hydrochloride (GuHCl) concentration to the inverse 6.7 power, which correlates well with the binding of GuHCl to the native protein in 0.60-0.80 M GuHCl. These rate dependencies require the refolding of CAB to be performed at high GuHCl concentrations (1 M GuHCl) and low protein concentrations (less than 1 mg/mL) to avoid the formation of aggregates. Alternatively, refolding can be performed by allowing the first intermediate to form the second intermediate prior to further dilution or dialysis. The aggregation of a hydrophobic first intermediate species is likely to be common to the refolding of other molten globular proteins.

Animals↗
PubMed1990

Cosolvent assisted protein refolding.

The use of cosolvents in aqueous systems has been shown to enhance protein refolding and decrease aggregation. In this study, we have used polyethylene glycol (PEG) in the molecular weight range of 1000 to 8000 Daltons to effectively increase the rate of refolding and prevent aggregation of the model protein, bovine carbonic anhydrase B (CAB). At concentrations of 3 and 30 g/l, PEG increased the rate of recovery of active protein in the absence of aggregation. Using 3 g/l PEG (3350 MW), the refolding rate was three fold greater than the observed normal refolding rate. The observed rate enhancement was caused by PEG acting on the first intermediate in the CAB refolding pathway to increase the rate of formation of the second intermediate. The interaction of PEG with the first intermediate also prevented its self-association during refolding and at equilibrium. The stabilization of this first intermediate resulted in complete recovery of active protein under normal aggregating conditions.

Animals↗
PubMed1984

Penicillin fermentation in a 200-liter tower fermentor using cells confined to microbeads.

The scale-up of the penicillin fermentation through cell confinement in a 200-L tower fermentor is described. P. chrysogenum spores were adsorbed into Celite microbeads having diameters greater than 180 microns. Fed-batch fermentations were performed using both free and confined cells. Cell growth and penicillin concentrations were measured during the fermentation. In addition, the oxygen transfer rate, the aeration rate, and the level of dissolved oxygen were also measured. Significant improvement in the mass transfer coefficient was found when the cells were anchored onto the microbeads. This improved oxygen transfer rate was accompanied by higher production of penicillin at a lower aeration rate. Besides the improved oxygen transfer rate into the mycelial broth, a reduction of the energy input for the oxygen transfer was observed. The confinement of the cells to this microcarrier furthermore allowed the intermittent harvesting of fermentation broth without reducing the cell mass in the fermentor.

Fermentation↗
PubMed1984

Effective diffusivity of oxygen in microbial pellets.

In a typical submerged aerobic fermentation with microbial pellets, the effective diffusivity of oxygen in the pellets is probably the most important, yet most difficult transport property to characterize experimentally. Its values directly indicate the efficiency or deficiency of oxygen to individual cells, and thus the biological activity of the microorganisms. In the past, it was not possible to assess reliably the effective diffusivity of oxygen in pellets due to several reasons. Firstly, most oxygen electrodes available were coarse, and hence not suitable for in situ measurements. Secondly, there was a lack of methods rigorous enough to characterize the structure of the microbial pellets. A state-of-the-art review of the literature relating to the feature subject is presented. Emphasis is laid upon development and evolution of the means for quantitative characterization of the effective diffusivity of oxygen in microbial pellets.

Journal Article↗
PubMed1984

Elucidation of Growth Inhibition and Acetic Acid Production by Clostridium thermoaceticum.

The production of acetic acid by Clostridium thermoaceticum was studied by using batch fermentations. In a pH-controlled fermentation with sodium hydroxide (pH 6.9), this organism was able to produce 56 g of acetic acid per liter. On the other hand, when the pH was not controlled and was decreased during fermentation to 5.4, the maximum attainable acetic acid concentration was only 15.3 g/liter. To obtain a better understanding of the end product inhibition, various salts were tested to determine their effect on the growth rate of C. thermoaceticum. An inverse linear relationship between the growth rate and the final cell concentration to the sodium acetate concentration was found. By using different concentrations of externally added sodium salts, the relative growth inhibition caused by the anion was found to be in the order of acetate > chloride > sulfate. Various externally added cations of acetate were also examined with respect to their inhibitory effects on growth. The relative magnitude of inhibition on the growth rate was found to be ammonium > potassium > sodium. The combined results have shown that the undissociated acetic acid was much more inhibitory than the ionized acetate ion. Complete growth inhibition resulted when the undissociated acetic acid concentration was between 0.04 and 0.05 M and when the ionized acetate concentration was 0.8 M. Therefore, at low pH (below 6.0), undissociated acetic acid is responsible for growth inhibition, and at high pH (above 6.0), ionized acetate ion is responsible for growth inhibition.

Journal Article↗