Production and purification of TRPV2 and TRPV5 for structural and functional studies

Abstract

The transient receptor potential (TRP) vanilloid 2 (TRPV2) and TRP vanilloid 5 (TRPV5) cation channels play an important role in various physiological and pathophysiological processes. The heterologous expression and purification of these channels is critical for functional and structural characterization of these important proteins. Full-length rat TRPV2 and rabbit TRPV5 can both be expressed in Saccharomyces cerevisiae and affinity purified using the 1D4 epitope and antibody to yield pure, functional channels. Further, these channels can be reconstituted into lipid nanodiscs for a more functionally relevant environment. Presented here are protocols for the expression of full-length rat TRPV2 and rabbit TRPV5 in Saccharomyces cerevisiae, their affinity purification, and their reconstitution into nanodiscs for structural and functional studies.

Keywords: 1D4 antibody; Affinity protein purification; Nanodiscs; Saccharomyces cerevisiae protein expression; TRPV2; TRPV5; Transient receptor potential channel.

Fig. 1

Timeline for the production of rTRPV2 and rbTRPV5.

Fig. 2

Expression and affinity purification of rbTRPV5 and rTRPV2. Western blots showing (A) the expression of rbTRPV5 and rTRPV2 in S. cerevisiae and (B) the affinity purification of rbTRPV5. (A) Lane 1: undiluted TRPV5 lysate; Lane 2: 10-fold diluted TRPV5 lysate; Lane 3: undiluted TRPV2 lysate; Lane 4: 10-fold diluted TRPV2 lysate. These are standard relative expression levels for rbTRPV5 and rTRPV2. The bands for both rbTRPV5 and rTRPV2 are indicated by a star. (B) Lane 1: Solubilization mixture; Lane 2: Supernatant after ultracentrifugation; Lane 3: Pellet after ultracentrifugation; Lane 4: Flow through from 1D4 beads; Lane 5: Wash of 1D4 beads; Lane 6: Pooled elutions 1–10. rbTRPV5, indicated by a star, is entirely solubilized and there is little protein left unbound in the flow through. The higher molecular weight bands (indicated by arrows) in the final pooled sample are TRPV5 dimers, trimers and tetramers.

Fig. 3

Nanodisc reconstitution and SEC polishing of rTRPV2. (A) SEC A₂₈₀ traces of nanodisc-reconstituted rTRPV2 (black), detergent-solubilized rTRPV2 (gray), and empty reconstituted MSP2N2 (dashed line). Fractions of nanodisc-reconstituted rTRPV2 run on SDS-PAGE are marked with corresponding lane numbers. The peak for the void volume is marked by a star. (B) SDS-PAGE of nanodisc reconstitution of rTRPV2. Lane 1: Concentrated rTRPV2 eluted from 1D4 beads; Lane 2: TRPV2 nanodisc reconstitution mixture before SEC; Lane 3: final, concentrated sample of TRPV2 reconstituted in nanodiscs after SEC; Lanes 4–9: SEC fractions as marked in (A). MSP2N2 (double star) co-migrates with rTRPV2 (single star) in the main peak (6–7) and additionally elutes by itself in a smaller peak (9) that corresponds to the control trace for empty nanodiscs.