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Influence of Escherichia coli feedstock properties on the performance of primary protein purification
KTH, School of Biotechnology (BIO), Bioprocess Technology.
2006 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2006. , 56 p.
Keyword [en]
Downstream processing, cell surface properties, surface plasmon resonance, contact angle measurements, microelectrophoresis, aqueous two-phase partitioning, filtration, EBA adsorbents, high-pressure homogenisation
National Category
Dentistry
Identifiers
URN: urn:nbn:se:kth:diva-3941ISBN: 91-7178-321-0 (print)OAI: oai:DiVA.org:kth-3941DiVA: diva2:10116
Presentation
2006-05-12, Sal FA32, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 14:00
Opponent
Supervisors
Note
QC 20101129Available from: 2006-05-09 Created: 2006-05-09 Last updated: 2012-02-17Bibliographically approved
List of papers
1. A method for microbial cell surface fingerprinting based on surface plasmon resonance
Open this publication in new window or tab >>A method for microbial cell surface fingerprinting based on surface plasmon resonance
2007 (English)In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 70, no 4, 595-604 p.Article in journal (Refereed) Published
Abstract [en]

A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants 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 mutants were observed. At the same time, the physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and compared to 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).

Keyword
microbial cell surface properties; surface plasmon resonance; contact angle measurements; microelectrophoresis; aqueous two-phase partitioning; multivariate data analysis
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-5671 (URN)10.1016/j.jbbm.2007.01.016 (DOI)000246933400009 ()2-s2.0-34247613490 (Scopus ID)
Note
QC 20100824 Uppdaterad från submitted till published (20100824)Available from: 2006-05-09 Created: 2006-05-09 Last updated: 2017-12-14Bibliographically approved
2. Effect of bacterial cell surface properties on membrane filtration and chromatography media interaction
Open this publication in new window or tab >>Effect of bacterial cell surface properties on membrane filtration and chromatography media interaction
(English)Manuscript (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5672 (URN)
Note
QC 20101129Available from: 2006-05-09 Created: 2006-05-09 Last updated: 2010-11-29Bibliographically approved
3. High-pressure homogenisation of Escherichia coli and its effect on viscosity and DNA fragmentation
Open this publication in new window or tab >>High-pressure homogenisation of Escherichia coli and its effect on viscosity and DNA fragmentation
(English)Manuscript (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5673 (URN)
Note
QC 20101129Available from: 2006-05-09 Created: 2006-05-09 Last updated: 2010-11-29Bibliographically approved

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