Novel Technologies for Recombinant Protein Overexpression in Escherichia coli
The use of recombinant protein is a cornerstone in many structural and functional studies. The enteric bacterium Escherichia Coli is the most commonly used organism for producing recombinant proteins. E. coli has several advantages over other expression hosts, but also one major disadvantage - the protein of interest does not always adopt its native conformation. Instead the protein might form large insoluble aggregates, inclusion bodies, within the cell. In particular, the heterologous overexpression of eukaryotic and membrane proteins are troublesome.In this thesis, methods are described that can be used to increase the likelihood of overexpressing eukaryotic proteins as well as membrane proteins. In particular, a novel method is described that can distinguish between bacterial colonies expressing soluble proteins from those expressing inclusion bodies. The method utilizes the fact that inclusion bodies are of a considerable size and can be removed by filtration. Using this screening method in combination with methods that alter the physical properties of proteins, we have shown that the likelihood of overexpression in E. coli can be dramatically increased.
Source Type:Doctoral Dissertation
Keywords:NATURAL SCIENCES; Chemistry; Biochemistry
Date of Publication:01/01/2006