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A study of the recovery and purification of a recombinant protein

Pierce, James Jonathan; (1997) A study of the recovery and purification of a recombinant protein. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Recombinant DNA technology has allowed the cloning and expression of heterologous proteins in a host cell. Further manipulation of these foreign proteins by genetic engineering allows them to be expressed into the periplasmic space of Escherichia coli. This compartmentalisation of the cell contents exploits a number of key bioprocess advantages. There is a reduction in the process volume, the level of protein and protease contamination is lower, the oxidative environment allows the correct folding of the protein and it is protected from denaturing conditions, such as air-liquid interfaces, during production. However, release from the periplasm at large scale is not straight forward and the balance of advantages and disadvantages can only be assessed by examination of complete processes. This thesis examines some of the process options available for the recovery of selectively released recombinant proteins using a model system comprising E. coli expressing a thermostable α-amylase derived from Streptomyces thermoviolaceus CUB 74. Selective release using lysozyme has been examined with results indicating that the amounts of lysozyme used can be reduced five-fold, from those used in previous studies, to 2.8 mg lysozyme g-1 cells at cell concentrations between 30 to 70 g 1-1. Adjustment of the composition of the periplasmic lysis mixture shows that release was still achieved where no lysozyme use was possible. The possibility of recycling lysozyme for repeated use in periplasmic release was also addressed. The project examines the issues in processing periplasmically secreted recombinant proteins particularly from process liquors that incorporate periplasmic lysis mixtures. Comparison of selective release with homogenisation and secretion, from Streptomyces lividans, was carried out as process alternatives for the recovery of recombinant protein.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: A study of the recovery and purification of a recombinant protein
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Applied sciences; Recombinant protein
URI: https://discovery.ucl.ac.uk/id/eprint/10098795
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