Title: The pET expression system revisited - how to boost soluble recombinant protein expression in E. coli
Other Titles: Verbesserung der Löslichkeit von rekombinantem Protein produziert durch das pET Expressionssystem
Language: English
Authors: Veiter, Lukas Jakob August 
Qualification level: Diploma
Advisor: Spadiut, Oliver 
Assisting Advisor: Wurm, David Johannes 
Issue Date: 2016
Number of Pages: 62
Qualification level: Diploma
This Thesis deals with the production of an antibody fragment in Escherichia coli in preferably soluble form. Full length antibodies feature glycosylation and thus are typically produced in mammalian cells which usually involves low space time yields, risk of contamination and considerable cost for equipment and media. Since desired antigen-binding properties are located in the fragment antigen binding (Fab) region, recombinant expression of only a Fab or single chain fragment variable (scFv) instead of a whole antibody is promising and can be accomplished in E. coli. The gut bacteria E. coli is one of the most widely used expression hosts for the production of recombinant proteins. It is superior to mammalian cells in several regards such as easier genetic manipulation and higher productivity in high cell density fermentations. The pET expression system features an inducible lac promotor which enables optimal cell growth in the Upstream Process (USP) until induction of gene expression, usually with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) as inducer. Induction by IPTG is used in most industrial fermentation processes as it cannot be metabolized by the cells and thus can be added in a simple one pulse addition. It however imposes a strong metabolic load onto the cells and is toxic at higher concentrations. Furthermore over-expression of recombinant protein in the cytoplasm results in inclusion body (IB) formation, containing the target protein in wrongly folded, non-functional form. Alternatively the lac operon's natural inducer lactose can induce expression of recombinant proteins equally effective. Regarding IB formation it was found that solubility of recombinant protein can be increased by modification of fermentation parameters like decreasing the growth rate (µ). Also studies indicate that lactose induction of the lac operon results in enhanced solubility (SP) of recombinant protein. The aim of this Thesis was to develop a mixed substrate feeding strategy for the production of a novel single chain fragment variable (scFv) using glucose and lactose in high density fermentation cultures and thereby increase the amount of soluble product as opposed to IPTG. Advantages of this strategy include a higher fraction of soluble protein providing significant improvement in Downstream Process (DSP) complexity. Furthermore knowledge on the link between substrate uptake and product formation as IB or SP leads to tunability of protein production rate and form. Such a fermentation setup is challenging because lactose needs to be fed continuously since it serves both as substrate and inducer. Consequently concomitant substrate uptake must be taken into account to avoid over-feeding, as in our experience glucose presence is necessary for lactose uptake. We found that lactose not only favors the recombinant production of soluble scFv when compared to IPTG, but furthermore that formation of soluble product can be tuned by the specific uptake rate of glucose during induction. On this basis a mechanistic correlation between specific uptake rates of lactose and glucose was determined in order to develop a corresponding model to be potentially valid for other E. coli strains as well.
Keywords: E. coli; lactose; mixed feed; expression tuning; substrate uptake rate; recombinant protein expression; concomitant substrate uptake; protein solubility; inclusion bodies; antibody fragment
URI: https://resolver.obvsg.at/urn:nbn:at:at-ubtuw:1-3967
Library ID: AC13247405
Organisation: E166 - Inst. f. Verfahrenstechnik, Umwelttechnik und Techn. Biowissenschaften 
Publication Type: Thesis
Appears in Collections:Thesis

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