Name: purification of endogenous drosophila transient receptor potential channels
Uploaded: 2021-12-28T14:00:00
Description: Watch this Scientific Journal Video about Purification of Endogenous Drosophila Transient Receptor Potential Channels at JoVE.com

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.

1. Purification of GST-tagged TRP and His-tagged NORPA fragments
<ol>
	<li>Purify GST-tagged TRP 1261-1275 fragment
	<ol>
		<li>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 &#176;C. Then, amplify the 10 mL of seeding culture in 1 L of LB medium a...

<ol>
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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 (KD = 0.3 &#181;M)18. INAD PDZ45 tandem interacts with NORPA 863-1095 fragment with an extremely high binding affinity (KD = 30 nM). These findings provide a solid biochemical basis to design the affinity purification plus competition strategy, which enables t...

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 C&#946;/No receptor potential A (PLC&#946;/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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacterial strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3) Competent Cells</td>
			<td>Novagen</td>
			<td>69450</td>
			<td>Protein overexpression</td>
		</tr>
		<tr>
			<td>Experiment models</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D.melanogaster: W1118 strain</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>BDSC:3605</td>
			<td>Drosophila head preparation</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20/30/40 mesh stainless steel sieves</td>
			<td>Jiufeng metal mesh company</td>
			<td>GB/T6003.1</td>
			<td>Drosophila head preparation</td>
		</tr>
		<tr>
			<td>30% Acrylamide-N,N&#8242;-Methylenebisacrylamide(29:1)</td>
			<td>Lablead</td>
			<td>A3291</td>
			<td>SDS-PAGE gel preparation</td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Invitrogen</td>
			<td>HC2005</td>
			<td>SDS-PAGE gel preparation</td>
		</tr>
		<tr>
			<td>Cocktail protease inhibitor</td>
			<td>Roche</td>
			<td>05892953001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Coomassie brilliant blue R-250</td>
			<td>Sangon Biotech</td>
			<td>A100472-0025</td>
			<td>SDS-PAGE gel staining</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)</td>
			<td>Sangon Biotech</td>
			<td>A620058-0100</td>
			<td>Size-exclusion column buffer preparation</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt (EDTA)</td>
			<td>Sangon Biotech</td>
			<td>A500838-0500</td>
			<td>Size-exclusion column buffer preparation</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sangon Biotech</td>
			<td>A610235-0005</td>
			<td>SDS-PAGE buffer preparation</td>
		</tr>
		<tr>
			<td>Glutathione Sepharose 4 Fast Flow beads</td>
			<td>Cytiva</td>
			<td>17513202</td>
			<td>Affinity chromatography</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sangon Biotech</td>
			<td>A500529-0001</td>
			<td>Elution buffer preparation for Ni-column</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td>Sangon Biotech</td>
			<td>A600168-0025</td>
			<td>Induction of protein overexpression</td>
		</tr>
		<tr>
			<td>LB Broth Powder</td>
			<td>Sangon Biotech</td>
			<td>A507002-0250</td>
			<td>E.coli. cell culture</td>
		</tr>
		<tr>
			<td>L-Glutathione reduced (GSH)</td>
			<td>Sigma-aldrich</td>
			<td>G4251-100G</td>
			<td>Elution buffer preparation for Glutathione beads</td>
		</tr>
		<tr>
			<td>Ni-Sepharose excel beads</td>
			<td>Cytiva</td>
			<td>17371202</td>
			<td>Affinity chromatography</td>
		</tr>
		<tr>
			<td>N-Dodecyl beta-D-maltoside (DDM)</td>
			<td>Sangon Biotech</td>
			<td>A610424-001</td>
			<td>Detergent for protein purification</td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-aldrich</td>
			<td>T9281-100ML</td>
			<td>SDS-PAGE gel preparation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sangon Biotech</td>
			<td>E607008-0500</td>
			<td>Homogenization buffer for E.coli. cell</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Lablead</td>
			<td>P0754-25G</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Prestained protein marker</td>
			<td>Thermo Scientific</td>
			<td>26619/26616</td>
			<td>Prestained protein ladder</td>
		</tr>
		<tr>
			<td>Size exclusion column (preparation grade)</td>
			<td>Cytiva</td>
			<td>28989336</td>
			<td>HiLoad 26/60 Superdex 200 PG column</td>
		</tr>
		<tr>
			<td>Size exclusion column (analytical grade)</td>
			<td>Cytiva</td>
			<td>29091596</td>
			<td>Superose 6 Increase 10/300 GL column</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sangon Biotech</td>
			<td>A501218-0001</td>
			<td>Protein purification buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sangon Biotech</td>
			<td>A500228-0001</td>
			<td>SDS-PAGE gel/buffer preparation</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-aldrich</td>
			<td>T1503-10KG</td>
			<td>Protein purification buffer preparation</td>
		</tr>
		<tr>
			<td>Ultrafiltration spin column</td>
			<td>Millipore</td>
			<td>UFC901096/801096</td>
			<td>Protein concentration</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>DENVER</td>
			<td>APX-60</td>
			<td>Metage of Drosophila head</td>
		</tr>
		<tr>
			<td>Desk-top high-speed refrigerated centrifuge for 15mL and 50mL conical centrifugation tubes</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Protein concentration</td>
		</tr>
		<tr>
			<td>Desk-top high-speed refrigerated centrifuge 1.5mL centrifugation tubes</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td>Centrifugation of Drosophila head lysate after homogenization</td>
		</tr>
		<tr>
			<td>Empty gravity flow column (Inner Diameter=1.0cm)</td>
			<td>Bio-Rad</td>
			<td>738-0015</td>
			<td>TRP protein purification</td>
		</tr>
		<tr>
			<td>Empty gravity flow column (Inner Diameter=2.5cm)</td>
			<td>Bio-Rad</td>
			<td>738-0017</td>
			<td>Bait and competitor protein purification from E.coli.</td>
		</tr>
		<tr>
			<td>Gel Documentation System</td>
			<td>Bio-Rad</td>
			<td>Universal Hood II Gel Doc XR System</td>
			<td>SDS-PAGE imaging</td>
		</tr>
		<tr>
			<td>High-speed refrigerated centrifuge</td>
			<td>Beckman coulter</td>
			<td>Avanti J-26 XP</td>
			<td>Centrifugation of E.coli. cells/cell lysate</td>
		</tr>
		<tr>
			<td>High pressure homogenizer</td>
			<td>UNION-BIOTECH</td>
			<td>UH-05</td>
			<td>Homogenization of E.coli. cells</td>
		</tr>
		<tr>
			<td>Liquid nitrogen tank</td>
			<td>Taylor-Wharton</td>
			<td>CX-100</td>
			<td>Drosophila head preparation</td>
		</tr>
		<tr>
			<td>Protein purification system</td>
			<td>Cytiva</td>
			<td>AKTA purifier</td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>Refrigerator (-80&#176;C)</td>
			<td>Thermo</td>
			<td>900GP</td>
			<td>Drosophila head preparation</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>MAPADA</td>
			<td>UV-1200</td>
			<td>OD600 measurement of E.coli. cells</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop 2000c</td>
			<td>Determination of protein concentration</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman coulter</td>
			<td>Optima XPN-100 Ultracentrifuge</td>
			<td>Ultracentrifugation</td>
		</tr>
	</tbody>
</table>

purification of endogenous drosophila transient receptor potential channels

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 C&#946;/No receptor potential A (PLC&#946;/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.

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 ...

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Purification of Endogenous Drosophila Transient Receptor Potential Channels

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.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

Research

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Biochemistry

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...