Strategies for optimizing heterologous protein overexpression in bacteria

Abstract

Not every gene can be efficiently expressed in bacteria. The protein yield could be decreased due to the differences in translational efficiency of an mRNA, difference in codon usage of the both organisms, the potential toxicity of the produced protein for the host and degradation of the produced proteins. Part one of this overview describes the role of different hosts and expression expression systems in improving the protein yield. Furthermore improving translational efficiency of an mRNA, mRNA stability, protein folding and reducing the degradation of the produced protein by proteases are described. E. coli is a common used host for heterologous protein overexpression. However other hosts such as Anabaena sp., Bacillus subtillis and A. migulanus are also useful for heterologous protein overexpression. Anabaena sp. is very useful for the production of radioactive proteins. Bacillus subtillis is an FDA approved host, well studied and suitable for pharmaceutical protein overexpression. The use of a A. migulanus strain with modified signal peptide is very suitable for overexpression studies. However this bacterium is not FDA approved. Therefore, it is not useful for pharmaceutical protein production. The inducibility of the T7 RNA polymerase based expression system can be improved by adding a plasmid coding for T7 lysozymes or by adding glucose to the medium. The arabinose inducible expression systems can be optimized by using a constitutive promoter for the arabinose transporters. Increasing the expression of the chaperones and heat-shock proteins DnaK, DnaJ, GrpE, ClpB, GroEL and GroES can improve mRNA stability and protein folding and prevent aggregation of proteins in the cytoplasm. Part two of the thesis contains a text version of a troubleshooting guide in the form of an FAQ (Frequently Asked Questions) for molecular cloning.