Applying Fusion Protein Technology to E. coli - Protein fusions are a leading option to produce difficult-to-express proteins, especially in Escherichia coli. - BioPharm International


Applying Fusion Protein Technology to E. coli
Protein fusions are a leading option to produce difficult-to-express proteins, especially in Escherichia coli.

BioPharm International

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

Recombinant proteins often are considered unwanted by cells and are subjected to proteolytic degradation.45 Several strategies have been developed to protect recombinant proteins from degradation, including the use of protease inhibitors,46 secretion into the periplasm or culture medium,47,48 and generating protective fusions.12,28,49–53 The compartmentalization hypothesis describes the mechanism by which gene fusions protect against proteolytic degradation.54 Fusions can promote the translocation of their partner proteins to different cellular compartments, thereby decreasing the concentration of the recombinant protein in the protease-rich cytosol. For example, SUMO can translocate from the cytosol to the nucleus and maltose binding protein (MBP) can translocate to the membrane compartment of the cell.55,56

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