Rapid, efficient, and cost-effective protein expression and purification strategies are required for high throughput structural genomics and the production of therapeutic proteins. Fusion protein technology represents one strategy to achieve these goals. Fusion protein technology can facilitate purification, enhance protein expression and solubility, chaperone proper folding, reduce protein degradation, and in some cases, generate protein with a native N-terminus. No technology or reagent is a panacea, however, and establishing tools and optimal conditions for each protein remains an empirical exercise. With this in mind, protein fusions are a leading option to produce difficult-to-express proteins, especially in Escherichia coli.
E. coli, a simple and low cost host, has long been the preferred system for recombinant protein expression. Recently however, difficult-to-express proteins (e.g., G-protein coupled receptors [GPCRs], kinases, ion channels, blood plasma proteins, vaccines, and antibodies) have become the norm instead of the exception. This trend is steering researchers away from E. coli to more costly higher organisms such as baculoviruses and mammalian cells.
The application of gene fusion technology to E. coli, however, offers a promising alternative. Commercial success is more likely when the expression host is simple and inexpensive.
Gene Fusion Technology
Typical problems with expressing difficult-to-express proteins include: low or null expression yields; insoluble protein; purification difficulties; degradation of the expressed protein; and incorrect folding. Some advances in improving recombinant protein expression in E. coli include the development of strong promoters,2 co-expression with chaperones,3 and most influential of all, fusion tags. Examples of popular fusion tags include: glutathione-s-transferase (GST),4 maltose binding protein (MBP),5,6 NusA,7 thioredoxin (TRX),8 polyhistidine (HIS),9-11 small ubiquitin-like modifier (SUMO),12-15 split SUMO, and ubiquitin (Ub).16