New approaches to the production and separation of recombinant proteins

Hsu, Chia-Chang (2014). New approaches to the production and separation of recombinant proteins. University of Birmingham. Ph.D.

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Abstract

Fab fragments are invaluable for therapeutic and diagnosis purposes. The robust and low cost Escherichia coli expression system for Fab production seems promising. However, Fab production in E. coli currently suffers from low yield and poor solubility.
In this study, E. coli strain CLD048 was initially employed for the production of Fab D1.3 target yield of ≥ 100 mg/L. Unfortunately, the fermentation was unsuccessful. Thus, several strategies for improving Fab production were explored. Consequently, the majority of Fab was accumulated in the periplasm, the overall Fab yield was increased and the solubility of Fab D1.3 was improved. Subsequently, optimising release conditions for periplasmic proteins was investigated.
A successful fermentation was demonstrated by expressing A33 Fab' in E. coli strain W3110 (yield ≥ 300 mg/L in the periplasm). Various selective protein release methods were investigated. Crude E. coli periplasmic extract containing A33 Fab' was directly employed in the AMTPS, which successfully demonstrated the feasibility of continuous protein separation with a highly purified final product (~98%).
In other work, thermo-responsive polymer brush surface modified M-PVA were benchmarked against Chelating Sepharose Fast Flow. Two coloured proteins were employed to exploit the feasibility of thermally mediated target protein elution under binding conditions.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):