Solute carriers or SLCs are responsible for uptaking nutrients and are involved in human diseases. However, there are few drugs targeting SLCs because they're poorly studied. We developed efficient approaches to clone, express and purify SLCs.
Our workflow yields pure proteins, allowing biochemical and structural studies, and ultimately provides insights for drug discovery campaigns. With the numerous roles in biological processes, disease and drug delivery, SLCs have increasingly received attention and become attractive therapeutical targets. The private public resolute IMI projects generated open Nexus resources and data to enable novel discoveries in SLC function, physiology, pathology and therapeutic targeting.
Traditionally, SLCs were challenging to study as they are integral membrane proteins with various substrates and diverse transport mechanisms. Recently cryo-EM, the Bacmid expression system and new certivisation reagents have led to a step change in layer catheterization. Pure protein samples are needed for biochemical and structural studies.
Yet human Solute carriers often have a poor yield when over expressed and unstable when purified. Therefore, in the past, each target required years to develop individual structures to produce the protein sample for downstream applications. Our method is optimized for the parallel and cost efficient study of multiple targets or constructs of a single target.
Therefore, it balances experimental flexibility, cost, time and lab resources carefully. Plus, we offer open access to all our protocols and resources in this article and in the resolute web portal. To begin, add 150 nanograms of Solute carrier or SLC clone and 100 nanograms of the vector in a 96 well plate, make the reaction volume to eight microliters with 10 millimolar Tris solution pH eight.
Then add two microliters of recombinant enzyme mix and incubate for one hour at room temperature. After incubation, add one microliter of proteinase K and incubate for 30 minutes at 37 degrees Celsius. Add four microliters of reaction mixture to 50 microliters of chemically competent E-coli MAC one cells, and transform the cells using the heat shock method.
After transformation, plate the bacterial suspension onto an lb agar plate. Identify colonies harboring the PHTBV 1.1 vector with the SLC gene insert using appropriate primers and standard protocols for Colony PCR. Begin by thawing the frozen solute carriers or SLC cell pellets in a water bath at room temperature.
Prepare solubilization buffer by combining 135 milliliters of base buffer and three protease inhibitor cocktail tablets. Once the tablets are dissolved, re-suspend the pellet in the solubilization buffer. Then add DNAs and transfer the suspension into an ice cold down homogenizer.
Homogenize the solution by moving the plunger up and down approximately 20 times, keeping the homogenizer on ice. Next, add detergent stock solution to 1%final concentration. Transfer the solubilization mixture to a 50 milliliter conical tube and rotate slowly at four degrees Celsius.
After one hour, centrifuge the mixture and collect the supernatant. Equilibrate a four to six milliliter bed volume of strep tactin resin with the base buffer. Then add equilibrated resin to the solubilized supernatant and rotate for two hours at four degrees Celsius.
Pour the solution onto a gravity flow column and allow the solution to flow through. Wash the resin with 30 times the bed volume of strep wash buffer in three equal steps. Next, add three to five milliliters of illusion buffer and incubate for 15 minutes, before collecting the elute.
Measure protein concentration by UV absorbent spectroscopy. Combine the desired illusion fractions and add three C protease. Rotate the tube slowly overnight at four degrees Celsius.
The next day, equilibrate two to four milliliters of bed volume of cobalt metal affinity resin with SEC buffer. Add the equated cobalt metal affinity resin to the overnight three C reaction mixture and rotate at four degrees Celsius. After one hour, pour the solution into a gravity flow column and collect the flow through.
Concentrate the flow through in a 100 kilodalton cutoff centrifugal filter by spinning at 3000 G at four degrees Celsius. Equilibrate a based SEC column with SEC buffer. Inject the sample into the sample loop and run the SEC program with a flow rate such that column pressure is below the column manufacturer's specifications.
Using a fraction collector, collect 300 microliter fractions over the entire SEC run. After pooling peak fractions, measure the absorbance at 280 nanometers. Then concentrate the samples to the required volume in a 100 kilodalton cutoff filter.
Initial small scale SLC protein expression was analyzed by SDS-PAGE Electrophoresis. Fluorescence microscopy of a GFP tagged SLC confirmed the protein localization specific to the plasma membrane with significant intracellular accumulation. Chemically and structurally homogeneous protein yielded a single mono disperse A2 80 peak during size exclusion chromatography.
