A systematic approach for testing expression of human full-length proteins in cell-free expression systems

In a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%.We have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield.With the sequencing of the human genome completed and with mRNA/cDNA identification rapidly progressing, many potential novel genes have been discovered and attention has turned to the function and structure of the predicted proteins [1-4]. In order to study these novel gene products, sufficient amounts of protein generally obtained through recombinant protein expression are required. The (high-throughput) expression and characterisation of unknown and poorly characterised human proteins is a main objective of recombinant proteomic studies today.Escherichia coli is the most commonly used prokaryotic expression system for the high-level production of recombinant proteins in vivo [5] and has already been used successfully in high-throughput protein expression and purification studies [4,6]. The use of E. coli has many advantages, including the ease of growth and manipulation of the organism and the availability of many different vectors and host strains that have been developed over the years. Howeve