Influence of Escherichia coli feedstock properties on the performance of primary protein purification

Abstract The aim of the present study was to increase the understanding of how the cell surface properties affect the performance of unit operations used in primary protein purification. In particular, the purpose was to develop, set up and apply methods for studies of cell surface properties and ce...

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Main Author: Råvik, Mattias
Format: Others
Language:English
Published: KTH, Bioprocessteknik 2006
Subjects:
Online Access:http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3941
http://nbn-resolving.de/urn:isbn:91-7178-321-0
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spelling ndltd-UPSALLA1-oai-DiVA.org-kth-39412013-01-08T13:10:56ZInfluence of Escherichia coli feedstock properties on the performance of primary protein purificationengRåvik, MattiasKTH, BioprocessteknikStockholm2006Downstream processingcell surface propertiessurface plasmon resonancecontact angle measurementsmicroelectrophoresisaqueous two-phase partitioningfiltrationEBA adsorbentshigh-pressure homogenisationBiochemistryBiokemiAbstract The aim of the present study was to increase the understanding of how the cell surface properties affect the performance of unit operations used in primary protein purification. In particular, the purpose was to develop, set up and apply methods for studies of cell surface properties and cell interactions. A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli strains were used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cells and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the strains were observed. The physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and were compared with the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA). Studies of the behaviour of the model cells on stirred cell filtration and in an interaction test with different expanded bed adsorption (EBA) adsorbents were performed. It could be concluded that especially one of the strains behaved differently. Differences in the properties of the model cells were indicated by microelectrophoresis and aqueous two-phase partitioning which to some extent correlated with observed differences in behaviour during filtration and in an interaction test with EBA adsorbents. The impact of high-pressure homogenisation of E. coli cell extract was examined, with a lab scale and a pilot scale technique. The DNA-fragmentation, visualised with agarose gel electrophoresis, and the resulting change in viscosity was analysed. A short homogenisation time resulted in increased viscosity of the process solution that correlated with increased concentration of released non-fragmented DNA. With longer homogenisation time the viscosity decreased with increasing degree of DNA-fragmentation. The results show that strain dependant cell surface properties of E. coli may have an impact on several primary steps in downstream processing. QC 20101129Licentiate thesis, comprehensive summaryinfo:eu-repo/semantics/masterThesistexthttp://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3941urn:isbn:91-7178-321-0application/pdfinfo:eu-repo/semantics/openAccess
collection NDLTD
language English
format Others
sources NDLTD
topic Downstream processing
cell surface properties
surface plasmon resonance
contact angle measurements
microelectrophoresis
aqueous two-phase partitioning
filtration
EBA adsorbents
high-pressure homogenisation
Biochemistry
Biokemi
spellingShingle Downstream processing
cell surface properties
surface plasmon resonance
contact angle measurements
microelectrophoresis
aqueous two-phase partitioning
filtration
EBA adsorbents
high-pressure homogenisation
Biochemistry
Biokemi
Råvik, Mattias
Influence of Escherichia coli feedstock properties on the performance of primary protein purification
description Abstract The aim of the present study was to increase the understanding of how the cell surface properties affect the performance of unit operations used in primary protein purification. In particular, the purpose was to develop, set up and apply methods for studies of cell surface properties and cell interactions. A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli strains were used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cells and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the strains were observed. The physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and were compared with the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA). Studies of the behaviour of the model cells on stirred cell filtration and in an interaction test with different expanded bed adsorption (EBA) adsorbents were performed. It could be concluded that especially one of the strains behaved differently. Differences in the properties of the model cells were indicated by microelectrophoresis and aqueous two-phase partitioning which to some extent correlated with observed differences in behaviour during filtration and in an interaction test with EBA adsorbents. The impact of high-pressure homogenisation of E. coli cell extract was examined, with a lab scale and a pilot scale technique. The DNA-fragmentation, visualised with agarose gel electrophoresis, and the resulting change in viscosity was analysed. A short homogenisation time resulted in increased viscosity of the process solution that correlated with increased concentration of released non-fragmented DNA. With longer homogenisation time the viscosity decreased with increasing degree of DNA-fragmentation. The results show that strain dependant cell surface properties of E. coli may have an impact on several primary steps in downstream processing. === QC 20101129
author Råvik, Mattias
author_facet Råvik, Mattias
author_sort Råvik, Mattias
title Influence of Escherichia coli feedstock properties on the performance of primary protein purification
title_short Influence of Escherichia coli feedstock properties on the performance of primary protein purification
title_full Influence of Escherichia coli feedstock properties on the performance of primary protein purification
title_fullStr Influence of Escherichia coli feedstock properties on the performance of primary protein purification
title_full_unstemmed Influence of Escherichia coli feedstock properties on the performance of primary protein purification
title_sort influence of escherichia coli feedstock properties on the performance of primary protein purification
publisher KTH, Bioprocessteknik
publishDate 2006
url http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3941
http://nbn-resolving.de/urn:isbn:91-7178-321-0
work_keys_str_mv AT ravikmattias influenceofescherichiacolifeedstockpropertiesontheperformanceofprimaryproteinpurification
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