Recombinant Expression in Pichia pastoris System of Three Potent Kv1.3 Channel Blockers: Vm24, Anuroctoxin, and Ts6

Figures

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  Figure 1. Graphical representation of recombinant plasmid pPICZαA-Vm24, -AnTx, and -Ts6.

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  Figure 2. Tricine SDS-PAGE analysis of the different clones expressing the recombinant peptides. MM: molecular marker (kDa). The figure shows the 16% tricine SDS-PAGE analysis of the expression of recombinant Vm24 (a), AnTx (b), and Ts6 (c) by the clones growing at 2000 µg/mL zeocin (numbers above the lines refer to the clone numbers). The red circle indicates the selected clones that showed the highest protein expression.

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  Figure 3. RP-HPLC purification of Vm24, AnTx, and Ts6. (a) RP-HPLC chromatograms of the recombinant peptides. Chromatograms show the fractions that are biologically active on the hKv1.3 channel. Acetonitrile % of elution and retention time for recombinant peptides were: Vm24 23.4%/31.8 min, AnTx 21.2%/32.7 min, and Ts6 25.9%/36.8 min. The inset in each chromatogram represents an ESI-QTOF-MS spectrum showing the monoisotopic mass of the respective fraction. (b) Sixteen percent tricine SDS-PAGE of the purified peptides. MM: molecular marker (kDa), line 1: Vm24, line 2: Ts6, and line 3: AnTx.

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  Figure 4. Inhibition of hKv1.3 currents by tagged recombinant Vm24, AnTx, and Ts6. These peptides are indicated in the figure as Tag-Vm24, Tag-AnTx, Tag-Ts6. Whole-cell currents through hKv1.3 were evoked from activated human peripheral T lymphocytes by depolarization to +50 mV from a holding potential −120 mV for 15 ms duration. Test pulses were applied every 15 s. Representative traces show the K+ current before the application of toxin (control) and after reaching the equilibrium block in the presence of Tag-Vm24 (blue), Tag-AnTx (green), and Tag-Ts6 (purple).

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  Figure 5. Blocking kinetics of recombinant Vm24, AnTx, and Ts6 on hKv1.3. Whole-cell currents through hKv1.3 were evoked from activated human peripheral T lymphocytes by depolarization to +50 mV from a holding potential −120 mV for 15 ms duration. Test pulses were applied every 15 s. Time courses of the development and recovery of the hKv1.3 current inhibition are shown for each peptide. Normalized peak K+ currents (I/I0) were determined and plotted as a function of time. Down and up arrows indicate the start and the end of perfusion with the recombinant peptide containing solution or toxin-free solution, respectively. Data points in blue, green, or purple shades represent the addition application of recombinant Tag-Vm24 (panel (a,b)), AnTx (panel (c,d)), and Ts6 (panel (e,f)), respectively, in their tagged or untagged version, as indicated.

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  Figure 6. Concentration-dependent block of hKv1.3 by recombinant Vm24, AnTx, and Ts6. A Hill equation (see Materials and Methods for details) was fitted to the averaged remaining current fraction (RCF) values calculated at different toxin concentrations as indicated. The best fit resulted in the Kd values shown at the top of each graph. Hill coefficients were H = 0.92 for Tag-Vm24 (panel (a)), H = 0.91 for Tag-AnTx, H = 0.95 for Untag-AnTx (panel (b)), H = 1.06 for Tag-Ts6, and H = 1.03 for Untag-Ts6 (panel (c)). Solid lines represent tagged peptide, dashed lines represent untagged peptides. Error bar represents SEM and n = 3–5.

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  Figure 7. Structural analysis of Vm24, Ts6, and AnTx. (a) Sequence alignment of Vm24, Ts6, and AnTx. Conserved Cys between the three peptides are highlighted in yellow; the functional dyad is highlighted in blue and pink. Conserved disulfide bridges are shown in red; the extra disulfide bridges are shown in green. (b) Structural alignment between Vm24 and Ts6. Vm24 structure is shown in yellow (PDB:2K9O); Ts6 structure is shown in green (PDB:1C56); functional dyads are shown in the structural alignment as sticks, lysine (blue) and tyrosine (pink). (c) Disulfide pattern of Vm24 and Ts6. Conserved disulfide bridges are shown in red sticks; the other disulfide bridges are shown in green. The red arrow points to the disulfide bridge found in the N-terminus of Ts6.