This protocol produces covalently stabilized complexes of transmembrane peptides for structural studies aimed at understanding how membrane proteins interact with one another through their lipid embedded domains. First, express a hist tagged fusion protein containing the transmembrane sequence of interest in e coli. Then extract the protein from the inclusion body fraction using guine and detergent using a nickel affinity matrix.
Concentrate the hist tagged fusion protein and di sulfide cross-link it to the diametric form by washing the column in a solution containing oxidizing agents. Next, elute the cross-linked fusion protein in Tri Fluor acetic acid and treat with cyanogen bromide to release the fusion partner proceed to isolate the cross-linked peptide product from the digest mixture by reversed phase HPLC. Ultimately, the identity and purity of the products are verified by SDS page and mass spectrometry analyses.
The main advantage of this technique is that pure sociometric complexes are produced for structural studies using techniques like solution or solid state NMR. Even when two transmembrane domains are known to interact, peptide solubility and complex formation are often at odds under experimental conditions. Beginning with a well-defined covalently stabilized complex can significantly broaden the range of experimental conditions that can be tested for high quality data collection.
Generally, individuals new to this method will struggle with the difficulty of handling these extremely hydrophobic protein fragments. This protocol and the advice provided in the accompanying manuscript are designed to provide some simple solution to some of the most common challenges in the prediction and purification process. Palate, a one liter culture of bacteria expressing the tripe peptide fusion and resuspend in 50 milliliters of lysis buffer.
Then perform three cycles of sonication at maximum output for one minute and cool on ice for five minutes. Harvest the insoluble inclusion body material by centrifugation at 20, 000 G for 15 minutes decant and put aside the supernatant. The sonication and centrifugation steps may be repeated if an improved purity of the IB fraction is required.
Reserve samples of the pellet and the supernatant material for SDS page analysis to confirm the trip. TM fusion localization to inclusion bodies now dissolve the inclusion body pellets in guine solution using between 25 and 50 milliliters per liter of culture. Processed mix gently at room temperature for several hours with occasional sunation.
Next, clear the inclusion body lysate by centrifugation at 75, 000 to 100, 000 times G for one hour. At room temperature, filter the supernat through 0.2 micron membrane in a 50 milliliter conical tube. Combine the cleared inclusion body lysate with nickel affinity resin that has been equilibrated to guine solution to batch bind.
Mix the suspension gently overnight at room temperature. Load the inclusion body lysate nickel resin suspension into an empty gravity flow column with porous bed support until the entire volume flows through. Collect and put aside the flow through which may still contain unbound fusion protein.
Then wash the nickel resin by gravity. Flow with five bed volumes of urea solution containing five millimolar beam mercaptoethanol. Perform a second wash without beam mercaptoethanol for the third wash.
Use urea solution supplemented with copper sulfate and oxidized glutathione. Then close the column outlet and incubate 30 minutes at room temperature to allow maximal di sulfide bond formation. Wash out the oxidizing urea solution with 10 bed volumes of water.
Then apply a vacuum line to dry the column bed and close the column outlet. Now in a chemical safety hood, add 1.5 bed volumes of neat TFA, stir the nickel resin with a small spatula to ensure even exposure to acid and incubate for five minutes. Collect the acid eluate pressurizing gently with a compressed airline if necessary to push out all of the liquid.
Repeat the acid dilution step with another 1.5 bed volumes of neat TFA To measure the protein yield, make dilution of the TFA EIT in tri fluoro ethanol and measure the absorbence at 280 nanometers against A-T-F-A-T-E. Dilute the acid elu to 80%final TFA concentration by dropwise. Addition of water while mixing gently.
If you precipitate forms, add back a small amount of TFA until the solution clarifies. Then recorrect to 80%with water. Reserve a five microliter predigests sample for SDS page analysis and freeze it immediately in liquid nitrogen.
Next, weigh cyanogen bromide crystals for approximately 0.2 grams per milliliter of sample. Taking care to weigh the toxic chemical safely. Add the cyanogen bromide to the sample in a safety hood and mix gently until completely dissolved.
Now flush the reaction vessel within inert gas seal and incubate for three to four hours at room temperature protected from light. Reserve a five microliter post digests sample for SDS page analysis, freezing it immediately in liquid nitrogen and lyophilize it together with the predigests sample. Then transfer the digest reaction to a regenerated cellulose dialysis cassette, allowing for a significant increase in volume.
Dialyze the sample overnight against four liters of water in the chemical femme hood. The next morning, remove the dialyzed reaction solution with suspended protein precipitate from the dialysis cassette and transfer the suspension to a 50 milliliter conical tube. Then freeze and lyophilize the sample for several days to remove the water and any traces of cyanogen, bromide and TFA dissolve up to 100 milligrams of Lyophilized digest products into three milliliters of neat formic acid mixing until the solution fully clears.
Load the sample onto a preparative scale zoax SB 300 C3 column in a flow of solvent A using a five milliliter sample loop. Then elute the digest products in a gradient of solvent B, over at least five column volumes. Analyze the HPLC fractions by SDS page and MALDI TOF mass spectrometry to identify the species of interest and confirm its expected mass lyophilize.
The HPLC fractions containing the cross-linked peptide product. If the product dries down to a film rather than a powder, it can be re dissolved in a small volume of HFIP frozen in liquid and lyophilized. Again, the result will be a fluffy white cone that is easily tipped out and weighed A DNA sequence encoding the transmembrane and flagging regions of the immuno receptor transmembrane signaling molecule.
DAP 12 was cloned into the PMM peptide vector using Hindi three and BAM H one restriction sites. The resulting bacterial expression construct produces a his tagged trip fusion protein with unique internal methionine and cystine residues for cyanogen bromide cleavage and oxidative cross-linking. The level of expression achieved for tripe fusions varies depending on the amino acid sequence of the attached peptide.
This preparation yielded approximately 120 milligrams of pure intact fusion protein from one liter of culture and four milliliters of nickel matrix. After cell lysis, the fusion protein was localized to the inclusion body pellet. Approximately 70%of the nickel purified tripe DAP 12 TM fusion was disulfide cross-linked to the DME form.
Although the DAP 12 TM peptide products are not readily identifiable in total CYANOGEN bromide digest samples comparison of the intact fusion and trip fragment bands in the reduced digest sample indicates that digestion of the fusion was approximately 60%complete in this typical separation of digest products. On a preparative scale C3 column using reverse phase HPLC, the bound products alluded in a two stage gradient of solvent B.The diss sulfide cross-linked peptide product is identifiable in the SDS page analysis of the major HPLC fractions under non reducing and reducing conditions. Maldi tof mass spectrometry analysis confirms the identity of the deme peptide in peak four with the major product at 6729.2 Daltons.
Don't forget that working with cyanogen bromide organic solvents and concentrated acids can be extremely hazardous. Appropriate protective gear should be wor while performing these procedures and cyanogen bromide should never be handled outside the approved chemical safety hood. This technique has provided the key study material for three different solution n mr structures of transmembrane peptide complexes demonstrating how multi subunit immune receptors assemble through protein interactions within the lymphocyte membrane.
We believe the application of this technique will prove equally valuable for other membrane protein systems.
