Optimisation of Expression of Cloned Genes

Abstract

In order to maximise production of proteins from cloned genes it is essential that both transcription and translation are optimised for an appropriate microbial host. This not only improves the yield of such proteins from fermentations, but also assists protein recovery and other aspects of downstream processing. A variety of high-expression vectors has been constructed, principally for E. coli, but, increasingly, for those organisms traditionally used in industry. Such expression vectors function by placing the cloned sequence under the control of a strong promoter and a ribosome-binding site that are efficiently recognised by the host machinery for transcription and translation respectively. Gene expression can usually be enhanced by using cloning vectors that alter the dosage of the cloned sequence and ensure that the sequence is maintained stably throughout growth of cultures. This chapter describes improvements in yield of proteins that can be achieved by adopting such strategies. Genetic manipulations necessary to circumvent post-translational barriers to high-level expression of genes in heterologous backgrounds and to assist in the purification of desired proteins from microbial cells are also considered.