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

Arasteh, Shaghayegh (2012). Periplasmic expression of D1.3 Fab antibody in E. coli. University of Birmingham. M.Phil.

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Abstract

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:

Thesis (Masters by Research > M.Phil.)

Award Type:

Masters by Research > M.Phil.

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