Heterologous Gene Expression in E.coli

Katharina Karste, Maren Bleckmann, Joop van den Heuvel

Nicola A. Burgess-Brown

Recombinant protein expression is not limited to E. coli or other prokaryotic systems. It is inevitable to use eukaryotic systems in order to express challenging mammalian proteins. Eukaryotic systems are able to perform complex posttranslational modifications like protein processing, phosphorylation, glycosylation, which are essential for stability and functionality of many proteins. Different eukaryotic protein expression systems employing yeast, insect, or mammalian cell lines are established with each having its own advantages and disadvantages. Often it is quite difficult to decide which will be the most optimal expression system as this depends highly on the protein itself. Expression in stable cell lines requires substantial screening of expressible constructs prior to developing a stable expression cell line. To achieve fast screening by transient expression in multiple hosts, versatile vectors can be applied. In this chapter, we present an overview of the most common multi-host vectors, which allow for fast expression analysis without tedious (re)cloning of the gene of interest in several different protein production systems. The protocols in this chapter describe the latest methods for fast transient expression in insect and mammalian cell lines.