Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression

Abstract

Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have a low level of sequence complexity and a high net charge but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently used fusions such as maltose-binding protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physiochemical properties, they did not perturb the structure, conformational stability, or function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically disordered fusions and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.

Figure 1. Evaluation of intrinsic disorder in the members of the Arabidopsis thaliana dehydrin family

A. PONDR^® VLXT analysis of ERD10. B. CH-plot analysis of: dehydrins (squares) ERD10 (grey), ERD14 (blue), COR47 (yellow), Rab18 (green), Xero1 (cyan), LTI30 (red); ordered proteins from DISPROT (light grey triangles), disordered proteins from DISPROT (light grey diamonds). The black delineates the boundary above which proteins which most extended, disordered proteins locate. C. Compositional profiling of dehydrins: Disprot (black), ERD10 (grey), ERD14 (blue), COR47 (yellow), Rab18 (green), Xero1 (cyan), LTI30 (red).

Figure 2. Analyzing the solubilization capabilities of dehydrins

A. SDS-PAGE analysis of CTLA4 solubility alone or fused to ERD10, ERD14, LTI30 dehydrins. B. The ability of ERD10 fusion (black bars) to enhance the percentage of soluble protein yield compared to the 6x–His fusion control (grey bars) for 10 insoluble proteins.

Figure 3. Soluble protein expression comparison of EB sequences with commonly used fusions

A bar graph showing the soluble expression performance of EB60A, EB60B, EB144 and EB250 (dark bars) with Trx, GST, NusA and MBP fusions (light bars) marketed to enhance soluble protein expression. B. Western blot analysis of the protein TIMP2 hybridized to the various fusion partners (anti-6x His blot, G-18 from Santa Cruz Biotechnologies).

Figure 4. Effect of fusion of various EBs on conformational stability of GFP and transferase activity of GST

A. Coomassie gel image showing the enrichment of GFP after purification on a NiNTA column. Lanes contain the following samples: 1. Molecular weight standard (Invitrogen); 2. 6xHis-GFP (29.3 kD); 3. 6xHis-MBP-GFP (69.5 kD); 4. 6xHis-EB60A–GFP (36.1 kD); 5. 6xHis-EB60B–GFP (36.0 kD); 6. 6xHis-EB144-GFP (44.4 kD); 7. 6xHis-EB250-GFP (55.5 kD). B. The fluorescence unfolding curve of GFP constructs fused to: 6xHis (♦), MBP (■), EB60A (▲), EB60B (▼), EB144 (right-facing triangle), EB250 (left-facing triangle). C. A table reporting the transferase activity of purified GST-fusions toward the synthetic CDNB substrate. Please note the following: (*) the molecular weight is based on the dimer weight of GST; (#) the GST transferase activity assay was measured in units of µmol_CDNB •min⁻¹ •pmol_GST⁻¹ to correct for differences in the molecular weight of the various fusions.

Figure 5. Removal of the EB fusions via proteolytic cleavage using enterokinase (EK)

A. Coomassie gel image visualizing the cleavage of the 6x–MBP fusion from GFP at 0, 2, 4, 8 and 24 hour time points. B. Coomassie gel image visualizing the cleavage of the 6x–EB60A fusion from GFP at 0, 2, 4, 8 and 24 hour time points. C. Coomassie gel image visualizing the cleavage of the 6x–EB250 fusion from GFP at 0, 2, 4, 8 and 24 hour time points.