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

by Råvik, Mattias

Abstract (Summary)
AbstractThe 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.
Bibliographical Information:

Advisor:

School:Kungliga Tekniska högskolan

School Location:Sweden

Source Type:Master's Thesis

Keywords:ODONTOLOGY; Biochemistry; Downstream processing; cell surface properties; surface plasmon resonance; contact angle measurements; microelectrophoresis; aqueous two-phase partitioning; filtration; EBA adsorbents; high-pressure homogenisation

ISBN:91-7178-321-0

Date of Publication:01/01/2006

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