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 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 enable 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 low sequence complexity and 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 physio-chemical properties they did not perturb the structure, conformational stability nor 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.

Unique Functional and Structural Properties of the LRRK2 Protein ATP-binding Pocket

“…Although the solubility of the isolated human LRRK2 kinase domain can be enhanced dramatically using the recently described entropic bristle approaches to protect the protein from self-association (40), this modified protein has no kinase activity despite intensive biochemical testing. Targeting zebrafish LRRK2 expression during embryonic development results in dopaminergic neurodegeneration and is hypothesized as a disease model relevant to Parkinson disease (45).…”

The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments

“…Electrostatic repulsion may keep proteins apart while they are folding. A similar fusion tag developed independently also gave improved solubility for expressed proteins (25). We modified the FATT tag vector to give pHFATTC, as shown in Fig.…”

The intrinsic disorder alphabet. III. Dual personality of serine

“…82 In biotechnology, EBDs were proposed to be used as protein solubility enhancers, and the translational fusion of a wide variety of natural and artificial EBDs represents an effective solubilizing tool. 128 Among the more successful solubilizers were artificial EBDs containing the most disorder-promoting residues (Glu, Pro, Gln, and Ser) in the proportion Glu:Pro:Gln:Ser D 2:2:1:1; i.e., sequences containing >16% serine residues, as well as EBDs with a Asp:Glu:Pro:Gln:Ser:Gly D 1:2:2:1:2:1 composition (i.e., EBDs containing >22% serine residues), where a larger subset of disorder-promoting residues was added to avoid potential issues with expression problems associated with high levels of sequence redundancy. 128 The aforementioned intrinsically disordered solubilizers, entropic bristles, are a member of a family of protein tags with a wide spectrum of biotechnological applications.…”