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 recombinant therapeutic protein business had sales of more than $34 billion in 2004.1 This total excludes therapeutic antibodies, vaccines, DNA–RNA synthetics, small molecules, and gene and cell therapies.
Projections to 2010 show sales amounting to $52 billion. Table 1 lists top selling recombinant therapeutic proteins.
Table 1. Top eight recombinant therapeutic proteins and their global sales between 2002 and 2004.
In the future, the industry is likely to be hampered by an imposing and growing bottleneck resulting from an inability to
efficiently express large quantities of biologically active protein at low costs. The so-called "low hanging fruits" have
been picked and a new era involving difficult-to-express proteins is upon us. This situation may drive the industry to use
more costly higher organisms for protein production.
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
Gene fusion 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. Nevertheless, protein expression remains
an arduous task that involves a complex decision tree. Whether or not to use gene fusion technology is just one choice. Other
factors include the expression system, host strain, mRNA stability, codon bias, inclusion body formation and prevention, site-specific
proteolysis, secretion, post-translational modification, and co-overexpression. The complexity is compounded by the diversity
of proteins. To date, no technology or reagent is a panacea. Thus, establishing tools and optimal conditions for each protein
remains an empirical exercise.