Gene expression using Gram-negative bacteria

Abstract

The technology for expressing heterologous gene products has developed to the point where it may be successfully applied to almost any Gram-negative bacterial system. Yet, when one thinks of gene expression in Gramnegative bacteria, Escherichia coli is the organism that typically first comes to mind. E. coli has been, and continues to be, the workhorse of the gene expression field. No other system has been developed that can not only express a large number of known gene products at levels sufficient for detailed biochemical analysis or product development, but also express undefined coding sequences (open reading frames) in amounts sufficient to determine the identity of the gene product and to characterize its function. Indeed, literally hundreds of papers are published each year in which it has been reported that genes of prokaryotic or eukaryotic origin have been cloned and expressed in E. coil The ability to express this myriad of genes has been aided by the development of numerous expression vector systems which use highly efficient transcriptional and translational regulatory signals to optimize production to levels that may reach 30% of total cellular protein (for a review of expression vector technology see [ 1] ). However, over the last year or two, the focus of improving expression in E, coli has shifted from the optimization of these regulatory signals to the manipulation of the coding sequence itself and the quality of the protein being produced. This review highlights advances made over the last two years in our ability to optimize the expression of heterologous gene products in E. coli with respect to both the quantity and quality (solubility, folding, activity, and homogeneity) of the proteins being produced. In addition, it will spotlight our emerging abilities to express heterologous gene products in other Gram-negative organisms where the aim is not so much to overproduce these proteins but rather to impart a useful new phenotype or function to these organisms.