Purification of Endogenous Drosophila Transient Receptor Potential Channels

Summary

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Abstract

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca²⁺)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase Cβ/No receptor potential A (PLCβ/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Introduction

Phototransduction is a process where absorbed photons are converted into electrical codes of neurons. It exclusively relays opsins and the following G protein-coupled signaling cascade in both vertebrates and invertebrates. In Drosophila, by using its five PDZ domains, scaffold protein inactivation-no-after-potential D (INAD) organizes a supramolecular signaling complex, which consists of a transient receptor potential (TRP) channel, phospholipase Cβ/No receptor potential A (PLCβ/NORPA), and eye-specific protein kinase C (ePKC)¹. The formation of this supramolecular signaling complex guarantees the correct subcellular localization, high efficiency, and specificity of Drosophila phototransduction machinery. In this complex, light-sensitive TRP channels act as downstream effectors of NORPA and mediate calcium influx and the depolarization of photoreceptors. Previous studies showed that the opening of the Drosophila TRP channel is mediated by protons, disruption of the local lipid environment, or mechanical force^2,3,4. The Drosophila TRP channel also interacts with calmodulin⁵ and is modulated by calcium by both positive and negative feedback^6,7,8.

So far, electrophysiology studies on the gating mechanism of Drosophila TRP and TRP-like (TRPL) channels were based on excised membrane patches, whole-cell recordings from dissociated wild-type Drosophila photoreceptors, and hetero-expressed channels in S2, SF9, or HEK cells^{2,9,10,11,12,13}, but not on purified channels. The structural information of the full-length Drosophila TRP channel also remains unclear. In order to study the electrophysiological properties of purified protein in a reconstituted membrane environment and to gain structural information of the full-length Drosophila TRP channel, obtaining purified full-length TRP channels is the necessary first step, similar to the methodologies used in mammalian TRP channel studies^14,15,16,17.

Recently, based on the assembling mechanism of INAD protein complex^18,19,20, an affinity purification plus competition strategy was first developed to purify the TRP channel from Drosophila head homogenates by streptavidin beads⁵. Considering the low capacity and expensive cost of streptavidin beads, an improved purification protocol is introduced here that uses His-tagged bait protein and corresponding low-cost Ni-beads with much higher capacity. The proposed method will help to study the gating mechanism of the TRP channel from structural angles and to measure the electrophysiological properties of the TRP channel with purified proteins.

Protocol

1. Purification of GST-tagged TRP and His-tagged NORPA fragments

1. Purify GST-tagged TRP 1261-1275 fragment
   1. Transform the pGEX 4T-1 TRP 1261-1275 plasmid¹⁰ into Escherichia coli (E. coli) BL21 (DE3) cells using the CaCl₂ heat-shock transformation method²¹. Inoculate a single colony in 10 mL of Luria Bertani (LB) medium and grow overnight at 37 °C. Then, amplify the 10 mL of seeding culture in 1 L of LB medium at 37 °C.
   2. After the optical density (OD₆₀₀) of the cells reaches 0.5, cool down the cells to 16 °C and add 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; final concentration) to induce the overexpression of the target protein and incubate at 16 °C for 18 h.
   3. After overexpression, pellet 1 L of cultured cells by centrifugation at 3,993 × g for 20 min and resuspend in 40 mL of phosphate-buffered saline (PBS) buffer.
   4. Load the resuspended cells in a high-pressure homogenizer pre-cooled at 4 °C. Slowly increase the homogenizer pressure to 800 bar. Open the inlet tap and let the resuspended cells circularly pass through a valve with very narrow slits.
      NOTE: The cells are homogenized by the high shearing forces caused by a large pressure drop and cavitation.
   5. Load 5 mL of glutathione beads to a gravity flow column and wash the beads with 50 mL of PBS buffer for a total of three times.
   6. Centrifuge the cell lysate from the high-pressure homogenizer at 48,384 x g. Add the supernatant of the centrifuged cell lysate (40 mL) to the equilibrated glutathione beads in the gravity flow column and incubate for 30 min at 4 °C. Resuspend the glutathione beads every 10 min.
   7. After 30 min of incubation, open the column outlet tap to separate the beads and flow-through fraction. Discard the flow-through fraction. Rinse the remaining glutathione beads twice with 50 mL of PBS buffer.
   8. Add 15 mL of elution buffer to the glutathione beads and incubate for 30 min. Resuspend the beads every 10 min.
   9. After 30 min of incubation, elute the GST-tagged TRP 1261-1275 fragment in a 50 mL conical tube and load in a size-exclusion column (preparation grade), which is equilibrated using 50 mM Tris (pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT) buffer.
   10. Keep the elution flow rate of the size-exclusion column to 3 mL/min. Collect the eluted proteins at the rate of 5 mL/tube.
   11. Identify the peak of the target protein in the size-exclusion column by analyzing the UV absorption signals at 280 nm and verify by SDS-PAGE gel analysis (electrophoresis parameters: 150 V for the stacking gel; 200 V for the resolving gel). Stain the gel with Coomassie blue R250.
   12. Concentrate the purified GST-tagged TRP 1261-1275 fragment from the size-exclusion column to 1 mL using a 15 mL ultrafiltration spin column centrifuged at 3,000 x g at 4 °C in a desk-top refrigerated centrifuge.
   13. Determine the concentration of concentrated protein using the Beer-Lambert Law. Measure the UV absorption of GST-tagged TRP 1261-1275 fragment at 280 nm using a spectrophotometer.
   14. Obtain the extinction coefficient at 280 nm by importing the protein sequences into the Protparam program (https://web.expasy.org/protparam/). Typically, 1 L culture of GST-tagged TRP 1261-1275 yields 1 mL of 600 μM of protein (6 x 10^-7 mol). See Table 1 for materials needed.
2. Purification of His-tagged NORPA 863-1095 fragment
   1. Transform the pETM.3C NORPA 863-1095 plasmid²⁰ into E. coli BL21 (DE3) cells using the CaCl₂ heat-shock transformation method²¹. Inoculate a single colony in 10 mL of LB medium and grow overnight at 37 °C. Then, amplify the 10 mL seeding culture in 1 L of LB medium at 37 °C.
   2. After the OD₆₀₀ of the cells reaches 0.5, cool down the cells to 16 °C and add 0.1 mM IPTG (final concentration) to induce the overexpression of target protein and incubate at 16 °C for 18 h.
   3. After overexpression, pellet 1 L of cultured cells by centrifugation at 3,993 x g for 20 min and resuspend in 40 mL of binding buffer. Next, lyse the resuspended cells in a high-pressure homogenizer at 4 °C as described in step 1.1.4.
   4. Load 5 mL of Ni-beads into a gravity flow column and wash three times with 50 mL of binding buffer.
   5. Centrifuge the cell lysate from the high-pressure homogenizer at 48,384 x g. Add the supernatant of the centrifuged cell lysate to the equilibrated Ni-beads in the gravity flow column and incubate for 30 min at 4 °C. Resuspend the Ni-beads every 10 min.
   6. After 30 min of incubation, open the column outlet tap to separate the beads and flow-through fraction. Discard the flow-through fraction and wash the remaining Ni-beads twice with 50 mL of wash buffer.
   7. Add 15 mL of elution buffer to the Ni-beads and incubate for 30 min. Resuspend the Ni-beads every 10 min.
   8. After 30 min of incubation, collect the eluted His-tagged NORPA 863-1095 fragment in a 50 mL conical tube and load into a size-exclusion column (preparation grade), which is equilibrated using 50 mM Tris (pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT).
   9. Keep the elution flow rate of the size-exclusion column to 3 mL/min. Collect the eluted protein at the rate of 5 mL/tube.
   10. Identify the peak of the target protein in the size-exclusion column by analyzing the UV absorption signals at 280 nm and verify by SDS-PAGE gel analysis (electrophoresis parameters: 150 V for the stacking gel; 200 V for the resolving gel). Stain the gel using Coomassie blue R250.
   11. Concentrate the purified His-tagged NORPA 863-1095 fragment from the size-exclusion column to 1 mL using a 15 mL ultrafiltration spin column centrifuged at 3,000 x g at 4 °C in a desk-top refrigerated centrifuge.
   12. Determine the concentration of concentrated protein using the Beer-Lambert Law. Measure the UV absorption of His-tagged NORPA 863-1095 fragment at 280 nm using a spectrophotometer.
   13. Obtain the extinction coefficient at 280 nm by importing the protein sequences into the Protparam program (https://web.expasy.org/protparam/). Typically, 1 L culture of His-tagged NORPA 863-1095 fragment yields 1 mL of 600 μM of protein (6 x 10^-7 mol). See Table 2 for materials needed.

2. Preparation of Drosophila heads

1. Collect adult flies in 50 mL conical centrifugation tubes using the CO₂ anaesthetization method^22,23; immediately freeze in liquid nitrogen for 10 min and store in a -80°C freezer.
2. After collecting a sufficient number of flies, vigorously shake the frozen 50 mL conical tubes by hand to separate the flies' legs, heads, wings, and bodies. Transfer the mixture to three sequentially stacked pre-cooled stainless-steel sieves (20/30/40 mesh size, respectively) and shake the sieves.
3. Next, since the heads cannot pass through the 40-mesh sieve, use a brush to sweep the fly heads off the 40-mesh sieve, transfer them into 50 mL conical tubes, and store them at −80 °C.
4. Continuously collect the flies and their heads and store them in a -80 °C freezer until they reach the required amount needed for experimentation (0.5 g). Typically, to collect 0.5 g of heads, 35 mL of flies in a 50 mL conical tube are needed. See Table 3 for materials needed.

3. Drosophila TRP channel purification

1. Weigh a total of 0.5 g of heads and completely homogenize in liquid nitrogen using a pre-cooled mortar-pestle. Dissolve the homogenized heads in 10x v/w lysis buffer (5 mL), incubate in a shaker at 4 °C for 20 min, and then centrifuge at 20,817 x g for 20 min at 4 °C.
2. Collect the spin-down supernatant ("20817 g S", Figure 4) and further centrifuge it at 100,000 x g for 60 min at 4 °C. Use the spin-down supernatant ("100,000 g S", Figure 4) for the following pull-down assay.
3. Add 1 mL of Ni-beads into the gravity flow column and wash the beads with 10 mL of double-distilled H₂O (ddH₂O) at 4 °C for a total of three times. Equilibrate the beads with 10 column volumes of lysis buffer three times at 4 °C.
4. Add 500 µL of 600 µM purified His-tagged NORPA 863-1095 protein (3 x 10^-7 mol) into the Ni-column and incubate for 30 min at 4 °C. Resuspend the beads every 10 min.
5. Open the column outlet tap to separate the beads and flow-through fraction. Take the flow-through fraction for SDS-PAGE analysis (NORPA F, Figure 4). In this section, the bait proteins are immobilized on the Ni-beads.
6. Wash the Ni-beads with 10 column volumes of lysis buffer (10 mL) at 4 °C and keep the washing fraction for SDS-PAGE analysis (Wash 1, Figure 4A). Repeat the above steps and keep the sample for SDS-PAGE analysis (Wash 2, Figure 4A). In this section, the excessive bait proteins on the Ni-beads are removed.
7. Add the supernatant of Drosophila head homogenate after 100,000 x g centrifugation into the Ni-column at 4 °C, where the His-tagged NORPA 863-1095 fragment has been immobilized.
8. Incubate the supernatant with the Ni-beads at 4 °C for 30 min. Resuspend the beads every 10 min. Then, open the column outlet tap to separate the beads and flow-through fraction.
9. Collect the supernatant for SDS-PAGE analysis (Dro head lysis F, Figure 4A). In this section, the INAD protein complexes (INAD/TRP/ePKC) in the head homogenates are captured by the immobilized NORPA 863-1095 fragments on the Ni-beads.
10. Wash the Ni-beads with 10 column volumes of lysis buffer (10 mL) at 4 °C and keep the supernatant from gravity precipitation for SDS-PAGE analysis (Wash 3, Figure 4A). Repeat the above steps and collect the supernatant for SDS-PAGE analysis (Wash 4, Figure 4A). In this section, the unbound proteins on the Ni-beads are removed.
11. Add 500 µL of 600 µM of GST-tagged TRP 1261-1275 protein (3 x 10^-7 mol) into the Ni-beads and incubate for 20 min at 4 °C. Resuspend the beads every 10 min.
12. Collect the eluted fraction from the gravity column (TRP E1, Figure 4B), which contains the endogenous Drosophila TRP channel. Repeat the above stepsand collect the elution fraction (TRP E2, Figure 4B). In this step, by using the GST-tagged TRP 1261-1275 fragments as the competitor, the TRP channels are eluted from the captured INAD protein complexes (INAD/TRP/ePKC) on the Ni-beads.
13. Wash the Ni-beads with 10 column volumes of binding buffer (10 mL; Table 1) at 4 °C and collect the washing fraction for SDS-PAGE analysis (Wash 5, Figure 4B).
14. Add 500 µL of elution buffer (Table 1) into the Ni-beads and incubate for 20 min at 4 °C. Collect the elution fraction from the gravity flow column (NORPA E1, Figure 4B). Repeat the above steps, and collect the elution fraction (NORPA E2, Figure 4B).
15. Using the elution buffer, elute the His-tagged NORPA 863-1095 fragment accompanied with the INAD/ePKC protein complexes. Next, resuspend the Ni-beads in 500 µL of binding buffer.
16. Take the resuspended Ni-beads to run the SDS-PAGE (stained by Coomassie blue R250) to analyze the efficiency of the elution and evaluate whether the elute buffer works (beads, Figure 4B). See Table 4 for materials needed.

4. Size-exclusion column purification of Drosophila TRP channel

1. Install a size-exclusion column (analytical grade) on the protein purification system. Equilibrate the column with the column buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT, 0.75 mM DDM), which is filtered by a 0.45 µm filter.
2. Concentrate the TRP E1 and E2 fraction from step 3.14 using a 4 mL ultrafiltration spin column, centrifuged at 3,000 x g at 4 °C in a refrigerated centrifuge.
3. Rinse the sample loop with the column buffer and load the sample into the sample loop. Inject the sample into the size-exclusion column and elute the proteins with a proper flow rate (0.5 mL/min).
4. Identify the peak of the target protein by absorption at 280 nm and run an SDS-PAGE gel to detect the purified endogenous Drosophila TRP channel (Figure 5). See Table 5 for materials needed.

Results

In this article, a protein purification method is demonstrated to purify endogenous Drosophila TRP channel (Figure 1).

First, recombinant protein expression and purification are applied to obtain the bait and competitor proteins. Then, a GST-tagged TRP 1261-1275 fragment is expressed in E. coli BL21 (DE3) cells in LB medium and purified using glutathione beads and a size-exclusion column (Figure 2). The samples were ...

Discussion

INAD, which contains five PDZ domains, is the core organizer of Drosophila phototransduction machinery. Previous studies showed that INAD PDZ3 binds to the TRP channel C-terminal tail with exquisite specificity (K_D = 0.3 µM)¹⁸. INAD PDZ45 tandem interacts with NORPA 863-1095 fragment with an extremely high binding affinity (K_D = 30 nM). These findings provide a solid biochemical basis to design the affinity purification plus competition strategy, which enables t...

Materials

Name	Company	Catalog Number	Comments
Bacterial strains
BL21(DE3) Competent Cells	Novagen	69450	Protein overexpression
Experiment models
D.melanogaster: W1118 strain	Bloomington Drosophila Stock Center	BDSC:3605	Drosophila head preparation
Material
20/30/40 mesh stainless steel sieves	Jiufeng metal mesh company	GB/T6003.1	Drosophila head preparation
30% Acrylamide-N,N′-Methylenebisacrylamide(29:1)	Lablead	A3291	SDS-PAGE gel preparation
Ammonium Persulfate	Invitrogen	HC2005	SDS-PAGE gel preparation
Cocktail protease inhibitor	Roche	05892953001	Protease inhibitor
Coomassie brilliant blue R-250	Sangon Biotech	A100472-0025	SDS-PAGE gel staining
DL-Dithiothreitol (DTT)	Sangon Biotech	A620058-0100	Size-exclusion column buffer preparation
Ethylenediaminetetraacetic acid disodium salt (EDTA)	Sangon Biotech	A500838-0500	Size-exclusion column buffer preparation
Glycine	Sangon Biotech	A610235-0005	SDS-PAGE buffer preparation
Glutathione Sepharose 4 Fast Flow beads	Cytiva	17513202	Affinity chromatography
Imidazole	Sangon Biotech	A500529-0001	Elution buffer preparation for Ni-column
Isopropyl-beta-D-thiogalactopyranoside (IPTG)	Sangon Biotech	A600168-0025	Induction of protein overexpression
LB Broth Powder	Sangon Biotech	A507002-0250	E.coli. cell culture
L-Glutathione reduced (GSH)	Sigma-aldrich	G4251-100G	Elution buffer preparation for Glutathione beads
Ni-Sepharose excel beads	Cytiva	17371202	Affinity chromatography
N-Dodecyl beta-D-maltoside (DDM)	Sangon Biotech	A610424-001	Detergent for protein purification
N,N,N',N'-Tetramethylethylenediamine (TEMED)	Sigma-aldrich	T9281-100ML	SDS-PAGE gel preparation
PBS	Sangon Biotech	E607008-0500	Homogenization buffer for E.coli. cell
PMSF	Lablead	P0754-25G	Protease inhibitor
Prestained protein marker	Thermo Scientific	26619/26616	Prestained protein ladder
Size exclusion column (preparation grade)	Cytiva	28989336	HiLoad 26/60 Superdex 200 PG column
Size exclusion column (analytical grade)	Cytiva	29091596	Superose 6 Increase 10/300 GL column
Sodium chloride	Sangon Biotech	A501218-0001	Protein purification buffer preparation
Sodium dodecyl sulfate (SDS)	Sangon Biotech	A500228-0001	SDS-PAGE gel/buffer preparation
Tris base	Sigma-aldrich	T1503-10KG	Protein purification buffer preparation
Ultrafiltration spin column	Millipore	UFC901096/801096	Protein concentration
Equipment
Analytical Balance	DENVER	APX-60	Metage of Drosophila head
Desk-top high-speed refrigerated centrifuge for 15mL and 50mL conical centrifugation tubes	Eppendorf	5810R	Protein concentration
Desk-top high-speed refrigerated centrifuge 1.5mL centrifugation tubes	Eppendorf	5417R	Centrifugation of Drosophila head lysate after homogenization
Empty gravity flow column (Inner Diameter=1.0cm)	Bio-Rad	738-0015	TRP protein purification
Empty gravity flow column (Inner Diameter=2.5cm)	Bio-Rad	738-0017	Bait and competitor protein purification from E.coli.
Gel Documentation System	Bio-Rad	Universal Hood II Gel Doc XR System	SDS-PAGE imaging
High-speed refrigerated centrifuge	Beckman coulter	Avanti J-26 XP	Centrifugation of E.coli. cells/cell lysate
High pressure homogenizer	UNION-BIOTECH	UH-05	Homogenization of E.coli. cells
Liquid nitrogen tank	Taylor-Wharton	CX-100	Drosophila head preparation
Protein purification system	Cytiva	AKTA purifier	Protein purification
Refrigerator (-80°C)	Thermo	900GP	Drosophila head preparation
Spectrophotometer	MAPADA	UV-1200	OD600 measurement of E.coli. cells
Spectrophotometer	Thermo Scientific	NanoDrop 2000c	Determination of protein concentration
Ultracentrifuge	Beckman coulter	Optima XPN-100 Ultracentrifuge	Ultracentrifugation