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Periplasmic expression of D1.3 Fab antibody in E. coli

Arasteh, Shaghayegh (2012)
M.Phil. thesis, University of Birmingham.

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Periplasmic expression of anti-lysozyme D1.3 Fab fragment antibody was studied as a model for antibody fragment production in general since the periplasm provides a suitable oxidative environment for disulphide bond formation. However, the secretory expression has its own bottlenecks given that the translocation step puts additional burden on cells and while the synthesis rate is not tuned to the translocation capacity, it ends to the inner-membrane accumulation of premature proteins. The process-based strategy here was to decrease the fermentation temperature at the point of induction in a comparative study with conducting the whole cultivation process at a sub-optimal temperature. Both strategies ended in similar final product concentration of 26 mg.mL\(^-\)\(^1\) with its majority being directed to the extracellular medium. Temperature downshift improved the volumetric productivity by almost two-folds (1.7 against 0.85 mg(Fab).mL\(^-\)\(^1\).h\(^-\)\(^1\)) and the periplasmic retention by 18%. Since the two strategies differed only in the temperature of the growth phase, higher antibody leakage by cultivation at low constant temperature can be explained in terms of the physiological-changes triggered by low growth temperature enhancing protein leakage. In addition, in order to examine the chance of lowering detrimental effects of induction by inducing early through logarithmic phase, preliminary studies were performed in form of shake-flask cultivations. The induction takes place early through logarithmic phase exposed cells to severe energy limitations activating genetic programs that mediate cell segregation and lysis. However, inducing at high enough biomass offers the advantage of separating the growth phase from the production phase and consequently leading higher productivity as observed for fed-batch fermentations performed in stirred-tank reactor

Type of Work:M.Phil. thesis.
Supervisor(s):Thomas, Owen and Overton, Tim
School/Faculty:Colleges (2008 onwards) > College of Engineering & Physical Sciences
Department:Department of Biochemical Engineering
Subjects:QD Chemistry
QH301 Biology
QR180 Immunology
Institution:University of Birmingham
ID Code:3714
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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