Protein fusion tags are often used to purify proteins from crude extracts. GST and MBP are two examples. Other protein fusion
systems, such as NusA, SUMO, Split SUMO, thioredoxin, and ubiquitin require an affinity tag, such as polyhistidine (HIS).
Numerous examples of affinity purification exist for fusion proteins, including nickel-nitriloacetic acid to isolate hexahistidine-fused
proteins,22 amylose to isolate MBP-fused proteins,23 and GSH-sepharose to isolate glutathione (Table 3).24 Successful purification schemes achieve high quality and quantity with inexpensive, high capacity resins and mild elution
conditions. For effective purification, high affinity between the fusion tag and resin is essential, but affinity must not
be too high, because harsh elution conditions can disrupt tertiary structure.
Table 3. Affinity Tags Influence Protein Expression Yield and Activity.
Enhancing Recombinant Protein Expression
Protein expression depends on transcriptional regulation, mRNA stability, and translational efficiency, whereas enhanced recombinant
protein expression is governed by a high mRNA copy number, efficient translational initiation and elongation, stability of
the mRNA, and the translational enhancers (reviewed by Makrides).25 Codon bias is another factor that affects expression,26 yet it has been overcome by engineering new strains or cell lines that contain rare tRNAs or by altering the problematic
codons to more common prokaryotic codons.27
Promoters also play a fundamental role in the transcription of heterologous genes and recombinant protein expression. Strong
and highly regulated promoters are now commonplace for E. coli, yeast, and insect cells.28-30 On the other hand, there is still much to be learned about gene fusion technology, which has been shown to dramatically
enhance expression.15,28,31 The exact mechanism by which fusion proteins enhance expression remains unknown. Some speculate that it is the result of
the highly conserved structure of the fusion tag.32
Protein Folding and Enhanced Solubility
Cost and simplicity are the primary driving forces when choosing a recombinant expression organism. As a result, E. coli is usually the first choice. However, E. coli has various shortcomings as a recombinant expression organism. Many eukaryotic proteins, especially proteins with disulfide
bridges or sugar moieties, cannot be expressed as soluble, active, and properly folded proteins in E.
coli.33 Over-expression in E. coli often yields macromolecular crowding (200–300 mg/mL in the cytoplasm), which presents an unfavorable environment for protein
folding and results in a high concentration of incorrectly folded proteins that form undesirable inclusion bodies that require
re-folding. Inclusion bodies afford protection from proteolytic degradation, which may be their only advantage.
To circumvent these problems, several strategies have been implemented to enhance solubility, promote properly folded protein,
and reduce the percentage of inclusion bodies. These strategies include co-expression of molecular chaperones and foldases,
expression of secreted proteins, and expression of protein fusions.34-37
Protein fusion tags have been shown to act as solubility enhancers and chaperones.38 Neither mechanism is well understood, but the hypotheses include:
- Fusion of a stable or conserved structure to an insoluble recombinant protein may serve to stabilize and promote proper folding
of the recombinant protein.
- Fusion tags may act as a nucleus of folding ("molten globule hypothesis").39,40
It should be noted that even though fusion partners promote solubility, this is not a universal indicator of correct folding,
and researchers recommend taking additional measurements (including monodispersity by light scattering,41 NMR,42,43 CD spectropolarimetry,
bis-ANS binding,44 ligand binding or enzymatic activity) to provide supporting evidence for correct folding.
Protection From Degradation