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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G., Hawley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A laboratory handbook. Second Edition. 80 (2), (2005).</li><li>Nicolas, G., Sillans, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+and+latent+effects+of+carbon+dioxide+on+insects.">Immediate and latent effects of carbon dioxide on insects.</a> Annual Review of Entomology. 34 (1), 97-116 (1989).</li><li>Arachea, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent+selection+for+enhanced+extraction+of+membrane+proteins.">Detergent selection for enhanced extraction of membrane proteins.</a> Protein Expression and Purification. 86 (1), 12-20 (2012).</li><li>Hellmich, U. 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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 ...

Watch this Scientific Journal Video about Purification of Endogenous Drosophila Transient Receptor Potential Channels at JoVE.com

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, ...

In this protocol, we demonstrate the purification process to obtain purified, full-length TRP channels, which is the necessary first step to study electrophysiological properties, and gain structural information of the full-length Drosophila TRP channel. Based on the sampling mechanism of the INAD protein complex and using low cost nickel beads with much higher capacity, sufficient amount of TRP channels can be purified from fly heads. Demonstrating the procedure will be Yuyang Liu, a technician from our lab.To begin, transform the pGEX 4T-1 TRP 1261-1265 plasmid into E.coli BL21 cells, using the calcium chloride heat shock transformation method. Inoculate a single colony in 10 milliliters of Luria Bertani medium, and grow overnight at 37 degrees Celsius. Add the 10 milliliters of seeding culture into one liter of Luria Bertani medium and incubate at 37 degrees Celsius.After the optical density of the cells reaches 0.5, cool the cells to 16 degree Celsius, and add 0.1 millimolar isopropyl-beta-D-1-thiogalactopyranoside. After overexpression, pellet one liter of cultured cells by centrifugation, and resuspend in 40 milliliters of phosphate-buffered saline. Load the resuspended cells into a high pressure homogenizer, pre-cooled to four degrees Celsius.Slowly increase the homogenizer pressure to 800 bar. Open the inlet tab and let the resuspended cells circularly pass through a valve with narrow slits. Load five milliliters of glutathione beads into a gravity flow column, and wash the beads with 50 milliliters of phosphate-buffered saline buffer, for a total of three times.Centrifuge the cell lysate from the high pressure homogenizer at 48, 384 times G.Add around 40 milliliters of the supernatant of the centrifuged cell lysate to the equilibrated glutathione beads in the gravity flow column, and incubate for 30 minutes at 4 degrees Celsius. Resuspend the glutathione beads every 10 minutes. After 30 minutes of incubation, open the column outlet tab to separate the beads and flow-through fraction.Discard the flow-through fraction, and rinse the remaining glutathione beads twice, with 50 milliliters of phosphate-buffered saline buffer. Add 15 milliliters of elution buffer to the glutathione beads, and resuspend the beads every 10 minutes. Elute the GST-tagged TRP 1261-1275 fragment into a 50 mL conical tube, and load it into a size exclusion column.Collect five milliliters of the eluted proteins per tube, and concentrate the purified fragments to one milliliter by centrifugation in an ultrafiltration spin column in a refrigerated centrifuge. To purify the His-tagged NORPA 863-1095 fragment, follow the previously demonstrated procedure using nickel beads. Collect adult flies in 50 milliliter conical centrifuge tubes, and freeze them in liquid nitrogen for 10 minutes.Vigorously shake the frozen tubes by hand and transfer the mixture to three, sequentially stacked, pre-cooled stainless-steel sieves. Shake the sieves, and sweep the fly heads off the 40 mesh sieve using a brush. Transfer them into five milliliter conical tubes and store them at minus 80 degrees Celsius.Homogenize 0.5 grams of heads in liquid nitrogen using a pre-cooled mortar and pestle. Dissolve the homogenized heads in five milliliters of 10X lysis buffer, followed by centrifugation at 20, 817 times G at four degrees Celsius for 20 minutes. The spin down supernatant is further centrifuged 100, 000 times G at four degrees Celsius for 60 minutes.Add one milliliter of nickel beads into the gravity flow column, and wash the beads three times with 10 milliliters of double distilled water at four degrees Celsius. Equilibrate the beads with 10 column volumes of lysis buffer, three times at four degrees Celsius. Add 500 microliters of 600 micromolar purified His-tagged NORPA 863-1095 protein into the nickel column, and incubate the column.Resuspend the beads every 10 minutes. Open the column outlet tap to separate the beads and flow-through fraction, and wash the nickel-beads with 10 milliliters of cold lysis buffer. Then, add cold supernatant of Drosophila head homogenate into the nickel column.After incubation, wash the beads with 10 milliliters of lysis buffer and add 500 microliters of 600 micromolar GST-tagged TRP 1261-1275 protein into the beads. Collect the eluted fraction from the gravity column. Wash the nickel beads with 10 milliliters of binding buffer.Then, add elution buffer and collect the elution fraction from the gravity flow column. Concentrate the fractions eluted from Drosophila TRP channel purification, using a four milliliter ultrafiltration spin column. Inject the sample into the size exclusion column, and elute the proteins with a reasonable flow rate.GST-tagged TRP 1261-1275 fragment was expressed in E.coli cells and purified using glutathione beads and a size exclusion column. Similarly, the His-tagged NORPA 863-1095 fragment was expressed and purified using nickel beads and size exclusion column. The eluted TRP channel is separated from the excessive GST-tagged TRP 1261-1275 peptide, by size exclusion chromatography.As a byproduct, the INAD, ePKC, NORPA 863-1095 complexes are obtained after TRP 1261-1275 peptide competition. The most important thing to remember when attempting this protocol, is homogenize the fly heads in liquid nitrogen and resolve the homogenate in buffer containing a detergent called DDM. Following this procedure, we can also purify the whole INAD protein complex which can be used to study its assembly and regulation mechanisms.A potential future application of this method will be to study the structural information of the endogenous Drosophila TRP channel using Cryo-EM techniques. In addition, measuring the electrophysiological properties of purified endogenous Drosophila TRP channels in artificial bilayer lipid membrane is also feasible.

purification-endogenous-drosophila-transient-receptor-potential

Research

JoVE Journal

Biochemistry

1.6K visualizações.  Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center. Neste protocolo, demonstramos o processo de purificação para obter canais TRP purificados e completos, que é o primeiro passo necessário para estudar propriedades eletrofisiológicas, e obter informações estruturais do canal TRP Drosophila de comprimento completo. Com base no mecanismo de amostragem do complexo proteico INAD e usando contas de níquel de baixo custo com capacidade muito maior, a quantidade suficiente de canais TRP pode ser purificada a partir de cabeças de mosca. Demon...