Synthetic intrinsically disordered protein fusion tags that enhance protein solubility

Abstract

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein.

Fig. 1. The steps for identification of highly soluble SynIDPs.

A Generation of SynIDP gene library. A pool of 1020 ssDNA 72-nt long oligonucleotides was circularized by CircLigase and amplified using RCA. Substitution of dCTP with 10% 5-methyl-dCTP randomly incorporates 5mC into the RCA product, resulting in various DNA size products upon digestion by SexAI (see Supplementary Fig. 1). The products are separated by electrophoresis. Fragments of the desired size (360–576 bp) are then gel purified and ligated into a plasmid. B Restriction digestion of RCA products with 0% 5mCTP (left) vs. 10% 5mCTP (right) using the restriction enzyme SexAI shows that digests occur in multiples of 72 bp when 5mC is incorporated. Desired size (360–576 bp, marked with arrow) DNA are then gel purified (C). Illumina Miseq analysis of cloned library of plasmid DNA verifies the presence of 865 unique motifs (See Supplementary Fig. 2 for enlarged version). Source data are provided as a Source Data file (D). CoFi protein expression (see Supplementary Fig. 3) allows identification of E. coli colonies that express soluble protein by an anti-His₆ western blot (left panel). Colonies were identified manually on a greyscale image (middle panel) and a black and white pixel-thresholded image overlaying the green channel (right panel), was printed and aligned underneath the plate of colonies as a visual aid for colony picking. Images represent a subset of the total set of agar plates that were analyzed (see Supplementary Fig). E Dot blot of insoluble and soluble lysate fractions to determine target protein solubility. The dot blot membrane was subjected to His₆-tag antibody detection. The distinct dots marked by arrows represent an example target SynIDP—[GTHGTP]₂₄—that was determined to be soluble. Solubility was determined by the intensity of the soluble fraction relative to the intensity of the insoluble fraction in both dot blot and PAGE gels (see Supplementary Figs. 6, 7).

Fig. 2. Characterization of the physical and molecular properties of SynIDPs demonstrate that they are unstructured proteins.

A SDS-PAGE of purified SynIDPs. All SynIDPs run slightly higher than their expected molecular weight, which is characteristic of many IDPs. B CD spectra of the SynIDPs shows a random coil structure, which is deduced from the characteristic negative peak at 197 nm and a positive peak at 215 nm. C Kratky plots [I(q)·q² as a function of q] of the SynIDP reveal a characteristic of flexible disordered chain for SynIDP-1 and SynIDP-2 while demonstrating extended chain conformation for SynIDP-3. D Scattering profiles [I(q) vs. q] of SynIDPs in a log-log plot. Black lines represent Fit of the PEV model to the SAXS data of SynIDP-1 and SynIDP-2. Curves were offset along Y-axis for better visibility. Source data are provided as a Source Data files.

Fig. 3. Fusion to SynIDPs rescues soluble and functional expression of mTdT.

A Western blot of insoluble and soluble fractions of mTdT and SynIDP-1/2/3-mTdT using anti-TdT antibody. B Purified SynIDP-1/2/3-mTdT after IMAC and SEC visualized on SDS-PAGE. C mTdT activity assay showing elongation of Cy5-poly-T₅₀ initiator on TBE-Urea PAGE gels. Low Range ssRNA ladder was used to quantify nucleotide addition. From left to right; ladder, initiator (negative control), Promega-TdT (positive control), SynIDP-1-mTdT 1X and 2X, SynIDP-2-mTdT 1X and 2X, SynIDP-3-mTdT 1X and 2X. D Insoluble (Ins) and soluble (S) fractions of mTdT, SynIDP-1-mTdT, SUMO-mTdT and MBP-mTdT visualized on SDS-PAGE. Large increase in soluble expression of mTdT are observed when fused to SynIDP-1 compared to fusion with SUMO or MBP. Arrows indicate the desired protein product. E TdT activity assay showing elongation of Cy5-poly-T₅₀ initiator on TBE-Urea PAGE gels. Low Range ssRNA ladder was used to quantify nucleotide addition. From left to right; cleaved mTdT, SynIDP-1-mTdT, SUMO-mTdT, MBP-mTdT, Ladder. Image was processed from Supplementary Fig. 14D (see experimental). F ImageJ analysis of fluorescence intensity as measurement for dispersity and degree of polymerization of mTdT variants’ reaction products. The peak at a distance of ~100 pixels stems from the dye front and is not a real product. SynIDP-1-mTdT outperforms the other variants by displaying lower dispersity and a higher degree of polymerization. The calibration of nt size to the distance on the gel (shown in the line above) was obtained by similar image analysis of the ladder. Source data are provided as a Source Data file.

Fig. 4. Fusion to SynIDPs rescues soluble and functional expression of Z2-LO10 and TEV proteins.

A Purified SynIDP-1/2/3-Z2-LO10 after IMAC and SEC. B Log-fold dilutions of SynIDP-Z2-LO10 and SynIDP controls (see Supplementary Fig. 15C) were incubated with CT-2A-EGFRviii, an EGFR positive murine glioma cell line for 48 h and tested for viability by an MTS assay. n = 3 replicates, error bars represent SD. C Purified SynIDP-1/2/3-TEV after His-purification and SEC. D MALDI-TOF MS of products of SynIDP-2-TEV reaction with ELP-tev-FGF21 substrate at 37 °C for 30 min confirms proteolytic activity. The absence of the intact substrate is evident by the lack of a peak at m/z 45,459. The cleaved FGF-21 (m/z of 20,038) and the ELP-tev (m/z of 25,439) can be seen in the MS spectra. The peak at m/z ~ 37,000 matches the expected Mw of SynIDP-2-TEV. Mass spectra for the control and products of SynIDP-1/3-TEV reaction with ELP-tev-FGF21 are shown in SI (see Supplementary Fig. 18). Source data are provided as a Source Data file.