3: Protein Expression and Production

Knowledge of the full sequence of many genomes has led to the identification of thousands of genes encoding proteins with unknown or poorly known activity, which can only be elucidated by expression of the genes and analysis of the expressed protein by various methodologies. Producing recombinant proteins in forms that are either suitable for elucidating function for investigative purposes or in amounts useful for therapeutic applications is a key challenge. Approaches and hazards relating to the production of the protein in good yield and in the right form are evaluated, including consideration of host-related issues and the use of cell-free systems. Expression vectors, particularly pBAD and pET and their derivatives, are described, including their use in one-step cloning and expression systems. Fusion proteins formed from the protein of interest are appraised in relation to tags that enhance solubility and/or purification and the ease with which they may be subsequently removed. Consideration of eukaryotic and cell-free expression systems is also included. Finally, proteomic requirements through high-throughput methodologies are described.