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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-T <t>50</t> <t>initiator</t> 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 <t>1X</t> 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-T 50 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. (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.
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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-T <t>50</t> <t>initiator</t> 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 <t>1X</t> 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-T 50 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. (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.
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terminal deoxynucleotidyl transferase (tdt) including 10× tdt reaction buffer  (Promega)
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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-T <t>50</t> <t>initiator</t> 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 <t>1X</t> 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-T 50 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. (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.
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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-T 50 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-T 50 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. (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.

Journal: Nature Communications

Article Title: Synthetic intrinsically disordered protein fusion tags that enhance protein solubility

doi: 10.1038/s41467-024-47519-7

Figure Lengend Snippet: 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-T 50 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-T 50 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. (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.

Article Snippet: A 10 μL polymerization reaction was prepared containing 0.5 μM (1X) and 1 μM (2X) final concentrations of each of the purified mTdT variants, 250 μM of the monomer-deoxythymidine triphosphate (dTTP), 0.5 μM of initiator- Cy5-(dT) 50 , and 1X TdT reaction buffer (Promega).

Techniques: Western Blot, Purification, SDS Page, Activity Assay, Negative Control, Positive Control, Expressing, Fluorescence