Purification of Endogenous Drosophila Transient Receptor Potential Channels

Published: December 28, 2021

doi:

Jia Liu*¹, Yuyang Liu*¹, Weidi Chen, Yuzhen Ding, Xiaoru Lan, Wei Liu

¹Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute,Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center

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 force²^,³^,⁴. The Drosophila TRP channel also interacts with calmodulin⁵ and is modulated by calcium by both positive and negative feedback⁶^,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 cells²^,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 studies¹⁴^,¹⁵^,¹⁶^,¹⁷.

Recently, based on the assembling mechanism of INAD protein complex¹⁸^,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 Purify GST-tagged TRP 1261-1275 fragment 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 a…

Representative 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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 31870746), Shenzhen Basic Research Grants (JCYJ20200109140414636), and Natural Science Foundation of Guangdong Province, China (No. 2021A1515010796) to W. L. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Materials

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

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Liu, J., Liu, Y., Chen, W., Ding, Y., Lan, X., Liu, W. Purification of Endogenous Drosophila Transient Receptor Potential Channels. J. Vis. Exp. (178), e63260, doi:10.3791/63260 (2021).