Electric chips for rapid detection and quantification of nucleic acids.

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli.

A simple pulse-based method for the determination of the maximum uptake capacities for glucose and oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where specific growth rate was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when specific growth rate declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.

Physiological responses to mixing in large scale bioreactors.

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Monitoring of genes that respond to process-related stress in large-scale bioprocesses.

In large-scale aerobic fed-batch processes, cells are exposed to local zones of high glucose concentrations that can also cause local oxygen limitations at high cell densities. The mRNA levels of four stress genes (clpB, dnaK, uspA, and proU) and three genes responding to oxygen limitation or glucose excess (pfl, frd, and ackA) were investigated in an industrial 20-m(3) Escherichia coli process and in a scale-down reactor with defined high-glucose and low-oxygen zones. The mRNA levels of ackA and proU were high during the batch growth phase, but declined drastically when glucose became limited, whereas the mRNA levels of the other stress genes were relatively constant throughout the process. In the industrial-scale reactor, the stress gene mRNA levels were, in most cases, highest in the middle part and at the top of the reactor, where the substrate was fed. Cells passing through the high glucose zone of the scale-down reactor had elevated mRNA levels for the oxygen limitation genes and had also elevated heat-shock gene mRNA levels. Both responses to stress occurred within seconds. The approach presented in this study offers a tool for monitoring process-related changes in the transcriptional regulation of genes.

Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli.

Industrial 20-m3-scale and laboratory-scale aerobic fed-batch processes with Escherichia coli were compared. In the large-scale process the observed overall biomass yield was reduced by 12% at a cell density of 33 g/l and formate accumulated to 50 mg/l during the later constant-feeding stage of the process. Though the dissolved oxygen signal did not show any oxygen limitation, it is proposed that the lowered yield and the formate accumulation are caused by mixed-acid fermentation in local zones where a high glucose concentration induced oxygen limitation. The hypothesis was further investigated in a scale-down reactor with a controlled oxygen-limitation compartment. In this scaledown reactor similar results were obtained: i.e. an observed yield lowered by 12% and formate accumulation to 238 mg/l. The dynamics of glucose uptake and mixed-acid product formation (acetate, formate, D-lactate, succinate and ethanol) were investigated within the 54 s of passage time through the oxygen-limited compartment. Of these, all except succinate and ethanol were formed; however, the products were re-assimilated in the oxygen-sufficient reactor compartment. Formate was less readily assimilated, which accounts for its accumulation. The total volume of the induced-oxygen-limited zones was estimated to be 10% of the whole liquid volume in the large bioreactor. It is also suggested that repeated excretion and re-assimilation of mixed-acid products contribute to the reduced yield during scale-up and that formate analysis is useful for detecting local oxygen deficiency in large-scale E. coli processes.

Modeling of overflow metabolism in batch and fed-batch cultures of Escherichia coli.

A dynamic model of glucose overflow metabolism in batch and fed-batch cultivations of Escherichia coli W3110 under fully aerobic conditions is presented. Simulation based on the model describes cell growth, respiration, and acetate formation as well as acetate reconsumption during batch cultures, the transition of batch to fed-batch culture, and fed-batch cultures. E. coli excreted acetate only when specific glucose uptake exceeded a critical rate corresponding to a maximum respiration rate. In batch cultures where the glucose uptake was unlimited, the overflow acetate made up to 9. 0 +/- 1.0% carbon/carbon of the glucose consumed. The applicability of the model to dynamic situations was tested by challenging the model with glucose and acetate pulses added during the fed-batch part of the cultures. In the presence of a glucose feed, E. coli utilized acetate 3 times faster than in the absence of glucose. The cells showed no significant difference in maximum specific uptake rate of endogenous acetate produced by glucose overflow and exogenous acetate added to the culture, the value being 0.12-0.18 g g-1 h-1 during the entire fed-batch culture period. Acetate inhibited the specific growth rate according to a noncompetitive model, with the inhibition constant (ki) being 9 g of acetate/L. This was due to the reduced rate of glucose uptake rather than the reduced yield of biomass.

Stabilization of a proteolytically sensitive cytoplasmic recombinant protein during transition to downstream processing.

The influence of aeration and glucose feeding on the stability of recombinant protein A in Escherichia coli during the transition period from a fed-batch cultivation to downstream processing was studied. Neither interruption of the feeding under aerobic conditions nor anaerobic conditions in presence of glucose could stabilize protein A completely and the intracellular ATP pool did not decrease to less than 0.75-1 mM by this treatment. On the other hand, the absence of both oxygen and glucose resulted in a decrease of the ATP pool to less than 0.5 mM and almost complete stabilization of protein A. The decrease of ATP was more severe when sulfite was used instead of nitrogen gas to create anaerobic conditions in presence of glucose. This also resulted in nearly complete stabilization of protein A, which might be explained by an inhibiting effect of sodium sulfite on fermentation. Therefore, protein stabilization and decrease of the ATP pool were correlated in experiments in vivo. The concentrations of ADP and AMP increased during starvation and may also play a role in stabilization of the protein in vivo. ATP may be a limiting factor of proteolysis also during further steps of downstream processing. Its concentration decreases by 80-90% during harvesting and centrifugation of biomass and even further during disruption of cells. However, neither addition nor regeneration of ATP in cell disintegrate was enough to restore degradation of protein A, indicating that an additional factor limits proteolysis in vitro.

Growth and energy metabolism in aerobic fed-batch cultures of Saccharomyces cerevisiae: simulation and model verification.

A kinetic model of overflow metabolism in Saccharomyces cerevisiae was used for simulation of aerobic fed-batch cultivations. An inhibitory effect of ethanol on the maximum respiration of the yeast was observed in the experiments and included in the model. The model predicts respiration, biomass, and ethanol formation and the subsequent ethanol consumption, and was experimentally validated in fed-batch cultivations. Oscillating sugar feed with resulting oscillating carbon dioxide production did not influence the maximum respiration rate, which indicates that the pyruvate dehydrogenase complex is not involved as a bottleneck causing aerobic ethanol formation.

In vitro complex-formation between the molecular chaperone DnaK and staphylococcal protein A derivatives produced in Escherichia coli and its use in the purification of DnaK.

Complex-formation between a truncated staphylococcal Protein A produced in Escherichia coli and a native E coli molecular chaperone, DnaK, can be used for the purification of DnaK by IgG-affinity chromatography. The half-time constant for in vitro formation of the Protein A-DnaK complex is about 14 min. Complex-formation in the presence of ATP is faster, but pre-incubation of DnaK with ATP decreases the final amount of the complex. A second complex with a slower migration on native PAGE is formed when the ratio of DnaK to Protein A is increased. A derivative of Protein A, ZZ, which essentially contains only two modified domains of Protein A, did not bind DnaK. After insertion of a tryptophan-rich peptide close to the C-terminus, the resulting protein, ZZT3, became able to bind DnaK. The binding of these three proteins to DnaK correlates with proteolysis in E coli, indicating a possible role for the binding of DnaK in the control of proteolysis.

Impact of plasmid presence and induction on cellular responses in fed batch cultures of Escherichia coli.

Fed batch cultivations of plasmid-free and recombinant Escherichia coli were employed in order to determine cellular responses and effects of plasmid presence and induction on the host cell physiology. While plasmid presence was shown to have minor influence on overall biomass yield, induction with 0.1 mM IPTG led to a marked reduction. The number of dividing cells, measured as colony forming ability, was influenced by plasmid presence and to a larger extent by induction. The latter caused a decline in the number of dividing cells to less than 10% of the population within 10 h. However, this cell segregation did not affect the specific rate of product formation, which was approximately constant throughout the cultivations. Analysis of the in vivo degradation rate of the product indicated that it was proteolytically stable. The cellular content of the stringent response signal substance, ppGpp, peaked immediately after transition from batch to fed batch mode to stabilise at a higher value than in the batch phase. When the specific growth rate declined below 0.06 h-1 an additional rise in ppGpp concentration was observed.

Cell segregation and lysis have profound effects on the growth of Escherichia coli in high cell density fed batch cultures.

Cell segregation into nondividing states and lysis was found to dominate the growth behavior of high cell density fed batch cultures of Escherichia coli. When the specific growth rate declined below a critical value, the biomass production, oxygen consumption, and carbon dioxide formation rates declined sharply. Concomitantly, an extensive loss of colony-forming ability (cfu) and accumulation of extracellular proteins was observed. A segregated model that considered different physiological states, including dividing, nondividing, and lysed cells, was developed and applied to experimental data from high cell density cultures of E. coli.

Response of guanosine tetraphosphate to glucose fluctuations in fed-batch cultivations of Escherichia coli.

A rapid transient increase of guanosine 3'-diphosphate 5'-diphosphate (ppGpp) in Escherichia coli was found in response to short-term glucose fluctuations that may occur in large-scale fed-batch cultivations. The concentration of ppGpp was measured in laboratory-scale glucose limited fed-batch cultivations. Starvation zones were imitated by using an intermittent feeding scheme or a two-compartment reactor system. The cellular concentration of ppGpp per biomass increased from 80 nmol to 300-600 nmol per g cell dry weight within only 1 min after consumption of the residual glucose in dependence on the test system, which is much faster than earlier described in literature. Readdition of glucose caused immediate reduction of the ppGpp to the basic level which did not differ in cultivations with simulated starvation zones from control cultivations. Possible physiological consequences by an enhanced stringent response in cultivations with limited mass transfer have to be considered.

The influence of energy sources on the proteolysis of a recombinant staphylococcal protein A in Escherichia coli.

The kinetics of proteolysis of a recombinant staphylococcal protein A in Escherichia coli were studied by a Western-blotting-based method. The proteolysis constants obtained from this method are very close to those obtained from the traditional radioactive pulse-chase technique. Protein A was selectively degraded to a great extent, while host proteins were quite stable after heat induction of protein A expression. The proteolysis of protein A was much faster in the presence of energy sources compared to when cells were starved of energy. The degradation rate constants are 2.8 h-1 in the presence of 10 g/l glucose and about 0.4 h-1 in the absence of any external carbon source. The supplementation of glucose to the medium at 0-100 mg/l caused a gradual increase of proteolysis of protein A, but the proteolysis was saturated when the concentration of glucose exceeded 200 mg/l at a cell concentration of about 0.36 g/l. The respiration inhibitor sodium azide completely inhibited the degradation of protein A in glucose-free salt medium but had almost no effect in the presence of glucose. Therefore, the proteolysis process is energy dependent but the energy supply rate obtained by fermentation of glucose is enough to meet this requirement. The proteolysis rate increased with the temperature in the interval 5-45 degrees C but was then reduced due to damage of the proteolysis system by high temperature. At 60 degrees C, the proteolysis ceased completely within 30 min.

Proteolysis of fusion proteins: stabilization and destabilization of staphylococcal protein A and Escherichia coli beta-galactosidase.

The product yield of staphylococcal Protein A reached only 1.8% of the cell dry weight, while the corresponding value was 14% for a fusion protein composed of Protein A and Escherichia coli beta-galactosidase [1], when produced in the same E. coli host strain, with the same promoter and under identical process conditions. Measurement of the stability of Protein A in vivo showed that it was quickly degraded in the cell with a half-life of 30 min when the protein was expressed alone, but after fusion to beta-galactosidase, the Protein A part became considerably stabilized. In spite of the fast intracellular proteolysis of Protein A, few degradation products could be identified on Coomassie Brilliant Blue-stained SDS/PAGE gels after IgG purification, indicating an even faster degradation of the Protein A fragments. Such degradation products, however, accumulated during incubation of the disintegrated cells. Intracellular degradation intermediates could be demonstrated with the more sensitive Western-blot technique. This technique also revealed that a slow degradation took place not only in the Protein A moiety of the fusion protein, but also in the beta-galactosidase moiety. A control with native beta-galactosidase also showed a weak in vivo proteolysis of this molecule, but it was more stable in free form than in the fused form. This means that the proteolytically very sensitive Protein A was stabilized by fusion with beta-galactosidase, but the originally rather stable beta-galactosidase became slightly more susceptible to proteolysis after the fusion.

Influence of substrate oscillations on acetate formation and growth yield in Escherichia coli glucose limited fed-batch cultivations.

A Large bioreactor is an inhomogenous system with concentration gradients which depend on the fluid dynamics and the mass transfer of the reactor, the feeding strategy, the saturation constant, and the cell density. The responses of Escherichia coli cells to short-term oscillations of the carbon/energy substrate in glucose limited fed-batch cultivations were studied in a two-compartment reactor system consisting of a stirred tank reactor (STR) and an aerated plug flow reactor (PFR) as a recycle loop. Short-term glucose excess or starvation in the PFR was simulated by feeding of glucose to the PFR or to the STR alternatively. The cellular response to repeated short-term glucose excess was a transient increase of glucose consumption and acetate formation. But, there was no accumulation of acetate in the culture, because it was consumed in the STR part where the glucose concentration was growth limiting. However, acetate accumulated during the cultivation if the oxygen supply in the PFR was insufficient, causing higher acetate formation. The biomass yield was then negatively influenced, which was also the case if the PFR was used to simulate a glucose starvation zone. The results suggest that short-term heterogeneities influence the cellular physiology and growth, and can be of major importance for the process performance. (c) 1995 John Wiley & Sons, Inc.

Effects of amino acid insertions on the proteolysis of a staphylococcal protein A derivative in Escherichia coli.

In vivo proteolysis of protein ZZT0, derived from the B domain of staphylococcal protein A, was investigated in Escherichia coli before and after insertion of 1-3 multiples of the tetrapeptide Ala-Trp-Trp-Pro close to the C-terminus of ZZT0. Before insertion, ZZT0 was proteolytically stable as judged from the purity of IgG binding proteins up to 1 h after inhibition of protein synthesis with chloramphenicol. Insertion of 1-3 units of Ala-Trp-Trp-Pro into ZZT0 increased progressively the sensitivity to proteolysis and induced DnaK and GroEL binding to the protein. The time for 50% in vivo hydrolysis of the full length protein derivative that was most susceptible to proteolysis, i.e. with three tetrapeptide units, was about 40 min when cultivated in a bioreactor and about 4 min in a shaken flask culture. Molecular masses and N-terminal sequences of the main degradation products indicated that protein ZZT0 is cleaved at identical sites irrespective of the number of inserted tetrapeptide units and that the cleavage sites are located far from the insertion point. Insertion of another hydrophobic amino acid, isoleucine, as the tetrapeptide Ala-Ile-Ile-Pro, only induced a slight proteolysis of the ZZT0 molecule under similar conditions. This indicates that the insertion of tryptophan residues, rather than of a general hydrophobic segment, plays an essential role in the induced proteolysis of the ZZT0 protein.

Modeling of high cell density fed batch cultivation.

Escherichia coli was grown in carbon- and energy source-limited fed batch cultures to study the effect of osmotic stress and different feed rates on the growth kinetics. An unstructured model based on the linear equation for substrate consumption provided an adequate description of the bacterial growth during the first phase of biomass production (20 h), except for cultures exposed to osmotic stress by the addition of 0.5 M NaCl. The addition of salt to the culture media had a large effect on the energetics, that could not simply be described in terms of an increased maintenance requirement. In the later phase of growth, an extensive decline in viability for all cultures was observed. Coincidentally, the specific sugar uptake rate approached a lower limit. It is concluded that the total obtainable biomass in a fed batch culture is strongly affected by the magnitudes of the substrate feed rate and the ionic strength of the culture medium.

The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli.

The production of the fusion protein staphylococcal protein A/E. coli beta-galactosidase in Escherichia coli was studied in batch and fed batch cultivations. Batch cultivation of a recombinant E. coli strain yielded a final cell dry weight of 16.4 g l-1 with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the cell dry weight. Fed batch cultivation made it possible to increase the final cell dry weight to 77.0 g l-1. The intracellular product concentration (25%) was lower as compared to batch cultivation resulting in a total concentration of recombinant protein of 19.2 g l-1.

Control of in vivo proteolysis in the production of recombinant proteins.

Proteolytic degradation of protein products causes many problems in the bioprocess industry. The increasing production of proteins in heterologous hosts through the use of recombinant DNA technology, has recently brought the problem into focus, since it seems that heterologous proteins are more prone to proteolysis. The result of proteolytic attack may vary from complete hydrolysis of the product to minor truncation of the protein without impairment of its biological function. The degree to which such partial proteolysis is a problem depends on the requirements of the ultimate use of the protein product.

Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli using a dual affinity fusion strategy.

A dual affinity fusion approach has been used to study the expression and secretion of labile recombinant proteins in Escherichia coli. Here we show that three small eukaryotic proteins (human proinsulin, a thioredoxin homologous domain of rat protein disulfide isomerase, and the extracellular domain of the alpha 1.2-chain of a human T-cell receptor) are stabilized in vivo using a dual affinity fusion strategy, where the gene encoding the desired product is fused between two genes encoding two different affinity domains. Relatively high yields of full-length product were obtained for all three proteins as compared to when fused to a single fusion partner. Despite the use of a signal peptide, significant amounts of the disulfide protein isomerase and T-cell receptor gene products were maintained in the cytoplasm, while the proinsulin fusion was efficiently secreted to the periplasm. Interestingly, the E. coli heat shock proteins DnaK and GroEL were associated with the fusion proteins isolated from the cytoplasm.