Strategies for achieving high-level expression of genes in Escherichia coli.
Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli.
Makrides
S C
SC
Department of Molecular Biology, T Cell Sciences, Inc., Needham, Massachusetts 02194, USA.
D001709:Biotechnology / Q000379:methods*; D002451:Cell Compartmentation; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins