Synthetic intrinsically disordered protein fusion tags that enhance protein solubility

Introduction:: The overexpression of many recombinant proteins result in low yields of soluble functionally active protein.[1] One solution to this problem is to use a fusion partner to enhance the solubility of a target protein. To explore the hypothesis that polypeptide tags derived from intrinsically disordered proteins (IDPs) can promote the soluble expression of recombinant proteins, we report the de novo design of small (< 20kDa) and highly soluble synthetic IDPs—termed SynIDPs hereafter—that confer solubility to a fusion partner with minimal effect on the bioactivity of the fused protein.


Materials and Methods:: To identify highly soluble SynIDPs, we created a pooled gene-library utilizing a novel DNA synthesis technology. The steps for creating the DNA library and screening for solubility include rolling circle amplification (RCA), next generation sequencing (NGS), colony filtration (CoFi), colony PCR and recombinant protein expression in E. coli. SynIDPs were characterized by SDS-PAGe, matrix assisted laser desorption ionization time-of-flight (MALDI-TEF) MS, circular dichroism (CD) and small angle X-ray scattering (SAXS).


Results, Conclusions, and Discussions:: We have developed a novel pooled library approach that uses RCA to create large and complex gene libraries of repetitive motifs. Using this approach, we identified three new, small (~15 kDa) and highly soluble SynIDPs. These SynIDPs have high proline and glycine content, which ensure a lack of secondary structure and high solvation. These properties are driven by a combination of proline’s fixed φ dihedral angle and glycine’s conformational entropy.[2, 3]

To demonstrate the utility of these three SynIDPs as fusion tags, they were fused to three proteins that are notoriously difficult to produce in E. coli due to their formation of insoluble inclusion bodies: (1) murine terminal deoxynucleotidyl transferase (mTdT); (2) a modular fusion protein of a dimeric affibody domain (Z2) that targets epidermal growth factor receptor (EGFR) that is fused to a P. aeruginosa exotoxin A (Z2-LO10); and (3) tobacco etch virus (TEV) protease. We demonstrate that the fusion of these SynIDPs enables the soluble expression of all three proteins in E. coli. Finally, we show that the bioactivity of each protein is maintained even without cleaving off the SynIDP tags.

We note that this is a new and interesting application of IDPs, as to our knowledge there is only one previous paper that explored and used native IDPs as tags to promote soluble protein expression.[4] However, these native IDPs were not optimized to serve as fusion tags, may interfere with the expression, folding and bioactivity of their fused protein partners because of their native function can be toxic to cells, if overexpressed. To the best of our knowledge, this is also the first time that mTdT has been soluble expressed using a fusion tag, and importantly the SynIDP-mTdT fusion retains bioactivity without the need to cleave the tag. This finding highlights the utility of SynIDPs as tags, as they are small, unstructured proteins that minimally interfere with biological activity. The three proteins span a range of molecular weights from 25 to 59 kDa and isoelectric points from 5.2 to 8, indicating they may be useful to promote the soluble expression of other proteins in E.coli as well.


Acknowledgements (Optional): :

References (Optional): : 1. Walls, D. and S.T. Loughran, Tagging recombinant proteins to enhance solubility and aid purification. Protein chromatography: Methods and protocols, 2011: p. 151-175.

2. Rauscher, S. and R. Pomès, The liquid structure of elastin. Elife, 2017. 6: p. e26526.

3. Muiznieks, L.D., A.S. Weiss, and F.W. Keeley, Structural disorder and dynamics of elastin. Biochemistry and Cell Biology, 2010. 88(2): p. 239-250.

4. Santner, A.A., et al., Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression. Biochemistry, 2012. 51(37): p. 7250-7262.