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.
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.
