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Development of Preparative Microfluidic Techniques for Lysis of Microbial Cells and Affinity Purification of Proteins

El-Sabbahy, H; (2013) Development of Preparative Microfluidic Techniques for Lysis of Microbial Cells and Affinity Purification of Proteins. Doctoral thesis (PhD), UCL (University College London). Green open access

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Abstract

In order to fully realise the benefits of microscale mammalian cell culture and microbial fermentation systems, a device capable of online sample preparation to enable further investigation of product quality is a key requirement. The aim of this work is to move toward such a device by designing and characterising a microfluidic lysis device and microaffinity chromatography device that are compatible with each other. The resulting microfluidic lysis device is useful for preparatory lysis of microbial cells. It works by mixing a lysis reagent (BugBuster MastermixTM), with microbial culture, using a T-Piece connection. Lysis takes place in a 700µm internal diameter fused silica capillary. The device was able to successfully lyse microbial cells with similar active Glutathione S Transferase release to sonication. The operating flowrate range of the device was 3.207µL min-1 to 6.414 µL min-1 and the device volume was 30µL - 60µL. The microaffinity chromatography column performed well in studies with pure Glutathione S Transferase. It showed good loading and elution behaviour. The breakthrough and elution curves, and quantity of protein eluted per unit bed volume, were similar to lab scale. The difference being as a result of experimental error. The column also performed well with a 100% clarified Escherichia coli lysate containing recombinant Glutathione S Transferase from Schistosoma japonicum. The eluate had a purity of 55% and concentration of 2.24 mg/ml. The column was fabricated from inexpensive fused silica capillary. It had an internal diameter of 700µm, a length of 5cm (the same length as a typical lab scale Glutathione Affinity column), and a bed volume of approximately 19µL. The operating flowrate range for the column was the same as the microlysis device.

Type: Thesis (Doctoral)
Qualification: PhD
Title: Development of Preparative Microfluidic Techniques for Lysis of Microbial Cells and Affinity Purification of Proteins
Open access status: An open access version is available from UCL Discovery
Language: English
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/1419100
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