Thioredoxin and Related Proteins as Multifunctional Fusion Tags for Soluble Expression in E. coli

Escherichia coli has traditionally been a popular host for the production of heterologous proteins because of its ease of genetic manipulation and growth. Recombinant proteins produced in E. coli have been useful for a variety of purposes, including the study of protein tertiary structure, structure-function experiments, enzymology, and as bio-pharmaceuticals. Despite an impressive body of literature describing the production of numerous non-native proteins in E. coli , successful results are in no way assured. The use of E. coli as a robust expression system has been hampered by several integral pitfalls. Low or undetectable expression levels can often be caused by inefficient translation initiation of eukaryotic mRNAs on bacterial ribosomes (1 ). Recombinant proteins produced in E. coli sometimes retain the N-terminal initiator methionine residue, as they may be a poor substrate for the host methionine aminopeptidase (2 ). In addition, individual purification schemes must be devised for each native recombinant protein produced in E. coli. But perhaps most importantly, it is very common for recombinant proteins expressed in E. coli to form insoluble, misfolded cytoplasmic complexes known as “inclusion bodies” (3 ). The likelihood of inclusion body formation is unpredictable but appears to increase in proportion to the size and complexity of the protein.