| 1. |
- Ahlqvist, Josefin, et al.
(författare)
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Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies
- 2006
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 122:2, s. 216-225
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Tidskriftsartikel (refereegranskat)abstract
- A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B.. 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.
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| 2. |
- Basselet, Pascal, et al.
(författare)
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Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)
- 2008
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859 .- 1475-2859. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Background: Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days. Results: A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies. The sample processing comprises first 2.5 min of ultrasonic treatment, DNA extraction (2x), and afterwards additional 5 min ultrasonication. Thus, the total sample preparation time for a confirmative analysis of EHEC is nearly 10 min. Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes. Six strains with distinct pathogenic properties were selected for this study. At last, the EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. This should permit to sense all currently accessible variants of the selected sequences in EHEC types and subtypes. Conclusion: In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems.
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| 3. |
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| 4. |
- Bollok, Monika, et al.
(författare)
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Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris
- 2005
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Ingår i: Applied Biochemistry and Biotechnology. - : Springer Science and Business Media LLC. - 0273-2289 .- 1559-0291. ; 126, s. 61-77
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Tidskriftsartikel (refereegranskat)abstract
- The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15 degrees C instead of 22 degrees C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.
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| 5. |
- Castan, A., et al.
(författare)
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Characteristics of a DO-controlled fed-batch culture of Escherichia coli
- 2000
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Ingår i: Bioprocess engineering (Berlin. Print). - : Springer Science and Business Media LLC. - 0178-515X .- 1432-0797 .- 1615-7591. ; 22:6, s. 509-515
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Tidskriftsartikel (refereegranskat)abstract
- The DO-controlled glucose limited fed-batch technique was investigated in an E. coli process for production of a recombinant protein. The k(L)ac* value (oxygen transfer rate at zero oxygen concentration) was calculated from on-line gas analysis data during the process. In the investigated processes with induced production of recombinant protein, the k(L)ac* value decreased drastically several hours after induction. The reason for the decrease was found in increasing concentrations of DNA in the medium and increased viscosity due to cell lysis. The consequences of such a dramatic decrease in the volumetric oxygen transfer coefficient on the glucose feed and specific rates are described in computer simulations and experimental data.
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| 6. |
- Castan, A., et al.
(författare)
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Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli
- 2002
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 77:3, s. 324-328
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Tidskriftsartikel (refereegranskat)abstract
- Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.
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| 7. |
- Castan, A., et al.
(författare)
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Oxygen enriched air supply in Escherichia coli processes : production of biomass and recombinant human growth hormone
- 2002
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Ingår i: Enzyme and microbial technology. - 0141-0229 .- 1879-0909. ; 30:7, s. 847-854
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Tidskriftsartikel (refereegranskat)abstract
- In order to investigate the impact of high oxygen and carbon dioxide concentrations, Escherichia coli was grown in batch cultivations where the air supply was enriched with either oxygen or carbon dioxide. The effect of elevated concentrations of oxygen and carbon dioxide on stochiometric and kinetic constants was studied this way. The maximum growth rate was significantly reduced, the production of acetic acid and the biomass yield coefficient on glucose increased in cultures with carbon dioxide enriched air, compared to reference cultivations and cultivations with oxygen enriched air. The application of oxygen enriched air was studied in high cell density cultivations of Escherichia coli. Two production processes were chosen to investigate the impact of oxygen enrichment. Biomass concentration, specific growth rate, yield coefficient, respiration, mixed acid fermentation products and the product yield and quality for the recombinant product were investigated. First, a process for the production of biomass was investigated. Exponential growth could proceed for a longer time and higher growth rates could be maintained with oxygen enriched air supply. However, a higher specific oxygen consumption rate per glucose was measured after the start of the oxygen enrichment, indicating higher maintenance and consequently the growth rate and yield coefficient decreased drastically in the end of the process. Second, a process for the production of recombinant human growth hormone (rhGH) was investigated. Although the glucose feed rate and all medium components were doubled, the amount of produced biomass could only be increased by 77% when oxygen enriched air (40% oxygen) supply was applied. This was due to a decreased yield coefficient of biomass per glucose. The total amount of produced product was decreased by almost 50% compared to the control, although less proteolytically degraded variants were produced.
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| 8. |
- Castan, A., et al.
(författare)
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The use of flow cytometry to detect nucleic acids attached to the surface of Escherichia coli in high cell density fed-batch processes
- 2002
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Ingår i: Biotechnology letters. - 0141-5492 .- 1573-6776. ; 24:3, s. 219-224
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Tidskriftsartikel (refereegranskat)abstract
- E. coli was grown in an aerobic fed-batch process for the production of a recombinant protein (rhGH). The cells were examined by flow cytometry and PI (propidium iodide) staining. The fluorescence of the PI-stained cells increased with increasing concentrations of DNA in the medium. Furthermore, DNA and RNA attached to the cell could partly be degraded with DNase/RNase and the fluorescence decreased. Formate excretion during the aerobic processes may be due to DNA and possibly also RNA attached to the cell surface, so creating diffusion resistance.
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| 9. |
- Charoenrat, Theppanya, et al.
(författare)
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Increased total air pressure versus oxygen limitation for enhanced oxygen transfer and product formation in a Pichia pastoris recombinant protein process
- 2006
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Ingår i: Biochemical engineering journal. - : Elsevier BV. - 1369-703X .- 1873-295X. ; 30:2, s. 205-211
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Tidskriftsartikel (refereegranskat)abstract
- Two strategies to increase the productivity of secreted Thai Rosewood beta-glucosidase in Pichia pastoris processes were evaluated. Both methods were based on increasing the oxygen transfer rate (OTR) in the process by simple means. Increasing the driving force for the diffusion from the air bubbles to the medium by elevating the air pressure, from 1.2 to 1.9 bar increased the oxygen uptake rate (OUR) by 59% while increasing the driving force by accepting oxygen limitation increased the OUR by 35%. The OTR increased less than in proportion to the increased solubility in the high-pressure process, which indicates that air bubble compression reduces the volumetric oxygen transfer coefficient (K(L)a). Even though the methanol consumption increased almost in proportion to the OTR in both processes the biomass production did not increase as much. This is explained as a higher maintenance demand for methanol in the oxygen limited (0.027 g g(-1) g(-1)) and high-pressure processes (0.035 g g(-1) g(-1)), compared to 0.022 g g(-1) g(-1) in the methanol limited reference process. However, in spite of the low effect of increasing OTR on the biomass production the total beta-glucosidase yield increased almost in proportion to the increased methanol consumption and reached highest value in the high-pressure process, while the beta-glucosidase purity was highest in the oxygen-limited process due to release of less contaminating proteins.
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| 10. |
- Charoenrat, Theppanya, et al.
(författare)
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Oxygen-limited fed-batch process : an alternative control for Pichia pastoris recombinant protein processes
- 2005
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Ingår i: Bioprocess and biosystems engineering (Print). - : Springer Science and Business Media LLC. - 1615-7591 .- 1615-7605. ; 27:6, s. 399-406
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Tidskriftsartikel (refereegranskat)abstract
- An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood beta-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q(O)/q(O,max)), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90-95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.
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