Applying Fusion Protein Technology to E. coli

May 2, 2007
John P. Hall

Volume 2007 Supplement, Issue 3

Protein expression remains an arduous task that involves a complex decision tree. Establishing tools and optimal conditions for each protein remains an empirical exercise.

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.

Recently, several comparative studies have examined the effects of various fusion partners on total and soluble expression yield (Table 2). Marblestone et al. evaluated the expression and solubility of three model proteins fused to the C termini of MBP, GST, TRX, NusA, Ub, and SUMO tags.16 The tags were ranked in terms of increased total expression as TRX > SUMO ~ NusA > Ub ~ MBP ~ GST and increased soluble expression as SUMO ~ NusA > Ub ~ GST ~ MBP ~ TRX. Hammarstrom et al. cloned 27 human proteins (MW < 20 kDa) into various expression vectors and ranked the tags' ability to promote soluble expression as TRX ~ MBP ~ Gb1 > ZZ > NusA > GST > His6.17 Braun et al. compared the expression of 32 human proteins (molecular weight of which varied from 17 to 110 kDa) and ranked tags in terms of increased expression and yield after purification as GST ~ MBP > CBP > HIS6.18 Shih et al., in a study of 40 different proteins with eight different tags, observed that MBP gave the best overall results in terms of total and soluble expression.19 In one of the studies in Dyson et al., the solubility of 20 mammalian proteins was compared and the fusion tags were ranked in terms of increased soluble expression as TRX ~ MBP > HIS10 > GST > GFP.20 De Marco et al. demonstrated that NusA was better than GST at enhancing the solubility and stability of recombinant proteins.21

Table 2. Summary of several comparative studies that examine the effects of various fusion partners on total and soluble expression yield.

The inconsistency of the data from these comparative studies only solidifies the statement that tools and optimal conditions for each protein remain empirical and that no technology or reagent is a panacea. Nevertheless, it is likely that in the future generalities about specific fusion tags may be made (e.g., for entire protein classes). As the comparative studies suggest, gene fusion tag systems range dramatically in efficiency.

YIELD AND ACTIVITY FACTORS

Factors that influence yield and biological activity include: a) the affinity purification scheme; b) enhancement of recombinant protein expression; c) protein folding and enhanced solubility; d) protection from degradation; e) size of the fusion tag; and f) the specificity, efficiency, and site of cleavage. Herein lies a further discussion of these factors.

Affinity Purification Scheme

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

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

Size of the Fusion Tag

The size of the fusion tag plays a crucial role in the overall yield of the purified protein. As seen in Table 4, the yield of the purified target protein is dictated by the yield of the fusion protein: the larger the fusion tag, the lesser the overall yield. Split SUMO, which is 47 amino acids long, is only 19% of the fusion when Split SUMO is fused to a target protein that is 200 amino acids in length (47/247). In contrast, NusA, which is 495 amino acids long, is 71% of the fusion with the same target protein (495/695). Therefore, if expression yielded one gram of fusion protein for both Split SUMO–Target and NusA–Target, the yield of the purified target after cleavage would be 0.810 g and 0.288 g, for Split SUMO and NusA, respectively.

Table 4. The size of the fusion tag influences the yield of the purified protein.

Specificity, Efficiency, and Site of Cleavage

The quality, quantity, and activity of the purified protein are influenced by the specificity, efficiency, and site of cleavage. Cleavage of the fusion usually is necessary because the fusion interferes with the structural or functional properties of the recombinant protein.57 Fusions can be cleaved by either chemical or enzymatic strategies.58,59 These methods include the use of engineered cleavage sites that are recognized by the proteases and are positioned between the fusion tag and the protein target. Proteases that have been employed to cleave fusion tags include tobacco etch virus (Tev) protease,60 factor Xa, thrombin protease,59 and the SUMO protease.12–16 Problems associated with proteolytic cleavage of fusion tags are low yield, precipitation of the protein of interest, labor-intensive optimization of cleavage conditions, expense of proteases, failure to recover active, structurally intact protein,61 and the generation of non-native N-terminal amino acids (not the case with SUMO and ubiquitin fusions12–16 ). As a result, choosing the right chemical or enzymatic strategy is crucial for achieving active protein of high quality and quantity.

Table 5. Abbreviations

JOHN HALL is vice president of business development at LifeSensors, Inc., Malvern, PA, 610.644.8845 x 305, hall@lifesensors.com

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