Funding for this work is provided by the National Health and Medical Research Council of Australia (NHMRC project grant 1011352 to M.E.C and M.J.C.; Independent Research Institutes Infrastructure Support Scheme [IRIISS] grant to WEHI) and the Victorian Government (VESKI Innovation Fellowship to M.E.C.; Victorian State Government Operational Infrastructure Support to WEHI). M.E.C. is a Queen Elizabeth II Fellow of the Australian Research Council. E.F.X.B acknowledges support from the Norma Hilda Schuster Scholarship Program at the University of Melbourne.

1. Cloning and Construct Design
Clone the sequences of interest into the pMM-peptide vector (which can be provided on request) using HindIII and BamHI restriction sites (see Figure 1). The double-stranded DNA insert should incorporate, in the following order: a HindIII site; a single methionine codon for CNBr cleavage; the E. coli codon-optimized peptide coding sequence; a stop codon; a BamHI site. All other methionines in the peptide should be eliminated and the dipeptide sequence asp-pro should be avoided as it will also be cleaved in acidic conditions.
A unique cysteine residue should be introduced into each peptide sequence according to the desired positioning of the disulfide crosslink in the final complex, and all other cysteines should be mutated to serine. The plasmid carries a kanamycin resistance cassette for selection.
2. Expression of trpLE-peptide Fusion
<ol>
 <li>Make up and sterile filter M9/Kan minimal growth medium (0.1 mM CaCl2, 2 mM MgSO4, 3 g/L KH2PO4, 7.5 g/L Na2HPO4&middot;2H2O, 0.5 g/L NaCl, 1 g/L NH4Cl, 4 g/L glucose, 50 mg/L kanamycin sulfate). Supplement with Centrum Complete A to Zinc to provide B-vitamins and trace metals: dissolve 1 tablet in 40 ml H2O with mixing for 30 min, centrifuge to remove insoluble material and sterile filter the orange supernatant. Store stock solution at 4 &deg;C in the dark for up to 2 weeks and use 4 ml/L in M9. 
 NOTE: 15N-enriched NH4Cl, 13C-enriched glucose or other stable-isotope-labeled precursors may be included in M9 medium at this stage if desired.</li>
 <li>Inoculate a 100 ml starter culture from freshly transformed BL21(DE3) strain E. coli in M9/Kan medium. Grow overnight at 37 &deg;C with shaking at 200 rpm.</li>
 <li>The next morning, check the OD600 of the starter culture - this should be in the range of 2 or greater. Dilute the 100 ml starter culture into 1 L total volume of Centrum-supplemented M9 medium. Split into two 2 L baffled flasks (500 ml culture in each flask) and grow at 37 &deg;C shaking at 140 rpm until the OD600 reaches 0.6.</li>
 <li>Set the shaker temperature to 18 &deg;C and continue shaking for 1 hr, allowing cultures to cool completely before induction.</li>
 <li>Take a pre-induction sample for SDS-PAGE analysis (equivalent of 50 &mu;l from a culture at OD600 = 1), then add IPTG to 0.1 mM final concentration to induce expression of the trpLE-peptide fusion. Continue growing at 18 &deg;C/140 rpm overnight (16-20 hr) under IPTG induction.</li>
</ol>
3. Inclusion Body Preparation and Nickel Matrix Binding
<ol>
 <li>The final OD600 of overnight expression cultures in minimal medium is variable and should be between 2.5 and 5.0. Take a sample for SDS-PAGE analysis (equivalent of 50 &mu;l from a culture at OD600 = 1) and collect cells by centrifugation for 20 min at 5,000 x g.</li>
 <li>Resuspend cell pellet from 1 L culture in 50 ml lysis buffer (50 mM Tris-HCl pH 7.5, 20 mM &beta;-ME, 0.2 M NaCl). Cell suspensions can be stored frozen for later processing.</li>
 <li>Lyse cells by sonication on ice at maximum output for 1 min and rest on ice for 5 min. We use a Misonix Sonicator 3000 (max output 600 watts) with a &frac12;-inch high-intensity replaceable tip. Repeat sonication/cooling for three cycles.</li>
 <li>Collect insoluble inclusion body (IB) material by centrifugation for 15 min at 20,000 x g and discard the supernatant. A loose, thin layer of lighter-colored cell debris may be visible on top of the dense IB pellet; this can be washed away using water or buffer with gentle agitation. Steps 3.3 and 3.4 may be repeated to improve the purity of the IB fraction if desired. Samples of pellet and supernatant material should be taken for SDS-PAGE analysis to confirm trpLE-TM fusion localisation to inclusion bodies.</li>
 <li>Dissolve IB pellets in guanidine solution (6 M guanidine HCl, 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1% Triton X-100, 5 mM &beta;-ME), using 25-50 ml per liter of culture processed. This step will require several hours with agitation and occasional mild sonication.</li>
 <li>Clear the IB lysate by centrifugation for 1 hr at 75,000-100,000 x g and 20 &deg;C. Decant the supernatant and filter through a 0.2 &mu;m membrane.</li>
 <li>Combine the cleared IB lysate, in a 50 ml conical tube or a larger vessel that can be closed securely, with nickel affinity resin that has been washed with water and equilibrated to guanidine solution. Use 3-4 ml settled resin per liter of culture processed for fusions that express to a similar degree as trpLE-DAP12TM shown here (Figure 3, lane 2). Batch bind by incubating overnight at room temperature with gentle mixing.</li>
</ol>
4. On-column Oxidative Crosslinking
<ol>
 <li>Load the IB lysate/nickel resin suspension into an empty gravity flow column with porous bed support, adding continuously until the entire volume flows through. Collect and put aside the flowthrough, which may still contain unbound fusion protein.</li>
 <li>Wash the nickel resin by gravity flow with 5 bed volumes of urea solution (8 M urea, 50 mM Tris-HCl pH 8.0, 200 mM NaCl) containing 5 mM &beta;-ME.</li>
 <li>Wash the nickel resin again with 5 bed volumes of urea solution without &beta;-ME.</li>
 <li>Wash the nickel resin a third time with 5 bed volumes of urea solution that has been supplemented with 20 &mu;M CuSO4 and 2 mM oxidized glutathione. After this wash, close the column outlet and incubate 30 min at room temperature to allow maximal disulfide bond formation.</li>
</ol>
5. TFA Elution and Quantitation
CAUTION: Trifluoroacetic acid (TFA) causes severe burns on contact with skin and fumes are highly irritating. Concentrated TFA should be used only in an approved chemical fume hood with eye protection and non-latex gloves. Check the chemical compatibility of all materials used; polypropylene is compatible with all steps of this protocol.
<ol>
 <li>Wash out the oxidizing urea solution with 10 bed volumes of water and dry the column bed by applying a vacuum line to the column outlet.</li>
 <li>Close the column outlet and add 1.5 bed volumes of neat (99-100%) TFA. Stir the nickel resin with a small spatula to ensure even exposure to acid and incubate for 5 min. Open the column outlet and collect the acid eluate, pressurizing gently with a syringe or compressed air line if necessary to push out all of the liquid.</li>
 <li>Repeat the acid elution step with another 1.5 bed volumes of neat TFA. Work quickly at this step to avoid complete dissolution of the beaded agarose matrix. Protein yield can be determined at this point according to the Beer-Lambert equation and the theoretical molar extinction coefficient of the trpLE-peptide sequence. Dilute a sample of the TFA eluate (1:20 to 1:50) in trifluoroethanol (TFE) and measure the absorbance at 280 nm, using TFA/TFE at the same dilution as a blank.</li>
</ol>
6. CNBr Digestion
CAUTION: Cyanogen bromide (CNBr) is extremely toxic and should be handled only in an approved chemical fume hood. PPE including safety glasses, lab coat and non-latex gloves are absolutely required. CNBr is reactive to moisture and should be brought to room temperature before opening. Contact your institutional safety office for instructions on neutralization/disposal of CNBr-contaminated solutions and materials.
<ol>
 <li>Dilute the acid eluate to 80% final TFA concentration by dropwise addition of water while mixing gently. If a precipitate forms, add back a small volume (0.5 to 1 ml) of neat TFA until the solution clarifies, then re-correct to 80% with water. Take a 5 &mu;l pre-digest sample for SDS-PAGE analysis, freezing in liquid nitrogen and lyophilizing the sample immediately.</li>
 <li>Add CNBr crystals to approximately 0.2 g/ml, taking care to weigh the toxic chemical safely in closed, pre-weighed tubes or using a balance inside the chemical fume hood. Mix gently until completely dissolved. Flush and seal the reaction vessel under inert gas (nitrogen or argon stream) and incubate 3-4 hr at room temperature, protected from light.</li>
 <li>Take a 5 &mu;l post-digest sample for SDS-PAGE analysis and freeze and lyophilize immediately. Transfer the digest reaction to a regenerated cellulose dialysis cassette or tubing (cellulose acetate will dissolve in concentrated acid) with a molecular weight cutoff of 3.5 kDa or less, according to the size of the expected peptide complex. Dialyze overnight to 4 L of water in the chemical fume hood to reduce the TFA concentration and decompose any unreacted CNBr. Leave room in the dialysis cassette or tubing for a significant increase in volume (as much as two- to three-fold) during overnight dialysis.</li>
 <li>Remove the dialyzed reaction solution with suspended precipitate from the dialysis cassette or tubing (most of the protein will precipitate when the TFA concentration is reduced) and transfer the suspension to 50 ml conical tubes. Freeze and lyophilize to remove water and traces of CNBr/TFA. This step is likely to require several days. 
 CAUTION: Both the dialyzed digest reaction and the dialysis water should continue to be treated as toxic and handled/disposed with appropriate precautions.</li>
 <li>Samples of cultures from pre-induction and harvest stages, inclusion body supernatant and pellet, TFA eluate and CNBr-digested material may be analyzed by SDS-PAGE. Prepare samples by heating to 95 &deg;C in Invitrogen NuPAGE LDS sample buffer. TFA eluate and digest samples should be prepared in both reducing and non-reducing conditions to facilitate product identification and evaluation of oxidative crosslinking.</li>
 <li>Separate the samples on a 12% NuPAGE bis-tris pre-cast acrylamide gel using MES-SDS running buffer for optimal resolution of the smallest digest products.</li>
</ol>
7. Reversed-phase HPLC Purification of Disulfide-crosslinked Peptide Complexes
<ol>
 <li>Dissolve up to 100 mg of lyophilized digest products into 2-3 ml neat formic acid and load onto a preparative-scale (21.2 x 150 mm) Agilent ZORBAX SB-300 C3 PrepHT column in solvent A (typically 10-20% acetonitrile or 5-10% isopropanol in water with 0.1% TFA).</li>
 <li>Elute the digest products in a gradient of solvent B (acetonitrile or isopropanol with 0.1% TFA) over at least 5 column volumes. See DISCUSSION for suggestions on the selection of optimal gradient conditions for different peptide sequences.</li>
 <li>Take 50-100 &mu;l samples from each HPLC fraction for SDS-PAGE analysis and freeze and lyophilize immediately. Removal of HPLC solvents is rapid and should be complete in 1-2 hr.</li>
 <li>Prepare lyophilized HPLC samples for SDS-PAGE by heating to 95 &deg;C in Invitrogen NuPAGE LDS sample buffer with no reducing agent. Cool and split each sample evenly into 2 microcentrifuge tubes, adding 100 mM DTT to one of them and re-heating briefly.</li>
 <li>Separate the reduced and non-reduced HPLC samples on a 12% NuPAGE bis-tris pre-cast acrylamide gel with MES-SDS running buffer as above. Small, hydrophobic peptides often do not take up Coomassie stain well and may not run at their expected molecular weights. However, comparison of reduced and non-reduced samples should facilitate identification of the crosslinked peptide products and assessment of purity.</li>
 <li>Analyze the candidate peptide-containing fractions by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (see DISCUSSION) to identify the species of interest and confirm its expected mass.</li>
 <li>Combine, freeze and lyophilize HPLC fractions containing the pure, crosslinked TM peptide complex and take a dry weight of the final product to determine yield. Fractions with high isopropanol content may not remain frozen. If peptide fractions dry down to a film rather than a fluffy lyophilized product, re-dissolve the film completely in a small volume of pure hexafluoroisopropanol (HFIP), freeze and lyophilize again. The HFIP-treated product should be a small, white cone that is easily tipped out and weighed.</li>
</ol>

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	<li>Call, M. E., Chou, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+view+into+the+blind+spot:+solution+NMR+provides+new+insights+into+signal+transduction+across+the+lipid+bilayer.">A view into the blind spot: solution NMR provides new insights into signal transduction across the lipid bilayer.</a> Structure. 18, 1559-1569 (2010).</li><li>Bocharov, E. V., 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=Spatial+structure+of+the+dimeric+transmembrane+domain+of+the+growth+factor+receptor+ErbB2+presumably+corresponding+to+the+receptor+active+state.">Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state.</a> The Journal of Biological Chemistry. 283, 6950-6956 (2008).</li><li>Mineev, K. S., 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=Spatial+structure+of+the+transmembrane+domain+heterodimer+of+ErbB1+and+ErbB2+receptor+tyrosine+kinases.">Spatial structure of the transmembrane domain heterodimer of ErbB1 and ErbB2 receptor tyrosine kinases.</a> J. Mol. Biol. 400, 231-243 (2010).</li><li>Mineev, K. S., 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=Spatial+structure+and+dimer--monomer+equilibrium+of+the+ErbB3+transmembrane+domain+in+DPC+micelles.">Spatial structure and dimer--monomer equilibrium of the ErbB3 transmembrane domain in DPC micelles.</a> Biochim. Biophys Acta. 1808, 2081-2088 (2011).</li><li>Lau, T. L., Kim, C., Ginsberg, M. H., Ulmer, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+integrin+alphaIIbbeta3+transmembrane+complex+explains+integrin+transmembrane+signalling.">The structure of the integrin alphaIIbbeta3 transmembrane complex explains integrin transmembrane signalling.</a> The EMBO Journal. 28, 1351-1361 (2009).</li><li>Zhu, J., 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=The+structure+of+a+receptor+with+two+associating+transmembrane+domains+on+the+cell+surface:+integrin+alphaIIbbeta3.">The structure of a receptor with two associating transmembrane domains on the cell surface: integrin alphaIIbbeta3.</a> Molecular Cell. 34, 234-249 (2009).</li><li>Call, M. 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E. x. p. r. e. s. s. i. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=purification,+and+isotope+labeling+of+cannabinoid+CB2+receptor+fragment,+CB2(180-233).">purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180-233).</a> Protein Expr. Purif. 38, 61-68 (2004).</li><li>Miozzari, G. F., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+of+the+leader+region+of+the+Escherichia+coli+tryptophan+operon.">Translation of the leader region of the Escherichia coli tryptophan operon.</a> J. Bacteriol. 133, 1457-1466 (1978).</li><li>North, C. L., Blacklow, S. 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The authors declare that they have no competing financial interests.

Expression of trpLE-TM fusions. In our experience, trpLE-TM fusions are poorly expressed in rich culture medium at 37 &deg;C, and the culture conditions described here have proven successful for many different sequences containing from one to four TM domains with yields ranging from 50 to 150 mg/L of pure, intact fusion. Fusions encoding three- or four-TM GPCR fragments (human CCR5 TM1-TM3 and TM4-TM7, respectively) or a core catalytic fragment of human signal peptide peptidase (four TM domains; see ref 15<...

This protocol details a procedure we have developed to produce disulfide-stabilized complexes of transmembrane (TM) peptides for structural studies using solution NMR. The procedure takes advantage of the robust expression afforded by the &Delta;trpLE1413 fusion system (see below) and allows TM peptide complexes of defined composition to be generated using sophisticated stable-isotope labeling techniques for modern multi-dimensional NMR experiments. We have employed these techniques to determine several NMR structures that revealed important new information about how lymphocyte-activating immunoreceptors are assembled from multiple membrane protein subunits through interactions among their TM domains (recently reviewed in 1). These techniques are applicable to many other membrane protein systems as well as a wide range of downstream analytical methods in addition to solution NMR. While the example given here utilizes native cysteine residues to create a complex that mimics the naturally disulfide-bonded protein, the design is equally well suited for creating engineered disulfide bonds to stabilize complexes that are normally held together by weaker, non-covalent interactions such as homo- and hetero-dimeric TM complexes of epidermal growth factor receptor (EGFR)-family proteins2-4 or heterodimeric &alpha;&beta; integrin complexes5,6.
Extremely hydrophobic peptide sequences such as those derived from the lipid bilayer-spanning portions of TM proteins are exceedingly difficult subjects for biochemical and biophysical studies. In addition to being very challenging to manipulate using standard protein and peptide chemistry techniques, they are often quite toxic to cells and are therefore difficult to produce recombinantly. We7,8 and others9-11 have had significant success expressing such difficult peptide sequences in bacteria as in-frame carboxy-terminal fusions to a modified version of the &Delta;trpLE1413 sequence derived from the E. coli trp operon12. The ~13 kDa trpLE polypeptide encoded by this sequence can be produced at high levels under a T7 promoter and is entirely localized to inclusion bodies where problems relating to toxicity and/or instability are circumvented. Modification of the sequence by addition of an amino-terminal 9-histidine tag13 and elimination of internal methionine and cysteine residues from the trpLE sequence14 allowed trpLE-peptide fusions to be purified by metal-ion affinity chromatography and digested using cyanogen bromide (CNBr) to release the desired peptide sequence. We have successfully used this system to express more than a dozen different sequences as trpLE fusions representing membrane protein fragments that contain as many as four TM domains (7,8 and unpublished results; see also DISCUSSION section).
The key innovation in this protocol is the identification of conditions under which the unstructured and very poorly soluble trpLE-TM fusions can be efficiently disulfide-crosslinked in the context of a streamlined workflow. Several aspects of high-yield expression, handling and purification of peptide products have also been thoroughly optimized, and the recommendations presented here are equally relevant for production of non-disulfide-crosslinked (monomeric) TM peptide products.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cyanogen bromide</td>
			<td>ALDRICH</td>
			<td>P.No- C91492,CAS-506-68-3</td>
			<td>HAZARDOUS SUBSTANCE. DANGEROUS GOODS. Very toxic by inhalation, in contact with skin and if swallowed. Contact with acids liberates very toxic gas. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>P.CODE-1000984387, CAS Number 76-05-1</td>
			<td>HAZARDOUS SUBSTANCE. DANGEROUS GOODS., Causes severe burns. Harmful by inhalation. Harmful to aquatic organisms, may cause long-term adverse effects in the aquatic environment.</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>P.No M7154, CAS Number 60-24-2</td>
			<td>HAZARDOUS SUBSTANCE. DANGEROUS GOODS. Toxic by inhalation, in contact with skin and if swallowed. Irritating to skin. Risk of serious damage to eyes. May cause sensitization by skin contact. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.</td>
		</tr>
		<tr>
			<td>1,1,1,3,3,3-Hexafluoro-2-propanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>Product Number 52512, CAS-No. 920-66-1</td>
			<td>HAZARDOUS SUBSTANCE. DANGEROUS GOODS. Harmful by inhalation and if swallowed. Causes burns.</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Merck KGaA</td>
			<td>K41186564</td>
			<td>Flammable liquid and vapour. Causes severe skin burns and eye damage.</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>UNIVAR, AJAX FINECHEM</td>
			<td>Product Number, 817, CAS-No 57-13-6</td>
			<td>When heated, decomposes to carbon dioxide and ammonia; if burned, emits small amounts of nitrogen oxides. Can cause redness and irritation of skin and eyes.</td>
		</tr>
		<tr>
			<td>GUANIDINE HYDROCHLORIDE</td>
			<td>Amresco</td>
			<td>P.No-M110, CAS Number: 50-01-1</td>
			<td>Harmful if swallowed, Causes serious eye irritation,Causes skin irritation, Acute Toxicity Oral, Skin Irritant, Eye Irritant.</td>
		</tr>
		<tr>
			<td>TRITON X-100</td>
			<td>SIGMA</td>
			<td>Product Number- T8532 CAS No: 9002-93-1</td>
			<td>Triton X-100 is a nonionic detergent, 100% active ingredient, which is often used in biochemical applications to solubilize proteins. Triton X-100 has no antimicrobial properties and considered a comparatively mild non-denaturing detergent</td>
		</tr>
		<tr>
			<td>His-Select Nickel-Affinity gel</td>
			<td>SIGMA-ALDRICH</td>
			<td>Catalog Num- P6611</td>
			<td>IS-Select Nickel Affinity Gel is an immobilized metal- ion affinity chromatography (IMAC) product. The HIS-Select Nickel Affinity gel is a proprietary quadridentate chelate on beaded agarose charged with nickel that is designed to specifically bind histidine containing proteins.</td>
		</tr>
		<tr>
			<td>(-)-Glutathione, oxidized</td>
			<td>SIGMA-ALDRICH</td>
			<td>Catalog num 150568</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Misonix S-3000 sonicator</td>
			<td>QSONICA</td>
			<td>S-3000 (discontinued)</td>
			<td>Max power output 600 watts. 1/2-inch replaceable-tip probe takes 1/2-inch high-intensity, high-volume tips and a range of high-intensity, low-volume tips. Closest models currently available from this company are Q500 and Q700.</td>
		</tr>
		<tr>
			<td>RP-HPLC: BioLogic DuoFlow chromatography system, Software Version 5.3</td>
			<td>Bio-Rad Laboratories</td>
			<td>Catalog Num 760-0047, Config No: AU500571, Serial No: 484BR3705</td>
			<td>Peptides binds to reverse phase HPLC columns in high aqueous mobile phase and are eluted from RP HPLC columns with high organic mobile phase. In RP HPLC peptides are separated based on their hydrophobic character. Peptides can be separated by running a linear gradient of the organic solvent.</td>
		</tr>
		<tr>
			<td>Prep HT C3 ZORBAX 300SB-Analytical HPLC Column, 21.2 x 150 mm, 5 &mu;m particle size</td>
			<td>Agilent</td>
			<td>Product No: 895150-909</td>
			<td>Reversed-phase HPLC colum</td>
		</tr>
		<tr>
			<td>NuPAGE 12% Bis-Tris Gels</td>
			<td>Life Technologies</td>
			<td>NP0341BOX</td>
			<td>Pre cast gels for protein electrophoresis</td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO</td>
			<td>Thermo Scientific</td>
			<td>Product No: 87724</td>
			<td>Sample dialysis</td>
		</tr>
	</tbody>
</table>

production of disulfide-stabilized transmembrane peptide complexes for structural studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

The level of expression achieved for trpLE fusions is variable and heavily dependent on the amino-acid sequence of the attached peptide. Figure 3 shows the SDS-PAGE analysis of pre-induction (lane 1) and time-of-harvest (lane 2) samples from a culture that yielded approximately 120 mg of pure, intact trpLE-DAP12TM fusion from 1 liter of culture and 4 ml nickel matrix. All of the trpLE-DAP12TM fusion was localized to the inclusion body pellet (lane 4) as opposed to supernatant (lane 3).
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Watch this Scientific Journal Video about Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies at JoVE.com

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

production-disulfide-stabilized-transmembrane-peptide-complexes-for

Research

JoVE Journal

Chemistry

14.0K Views.  The Walter and Eliza Hall Institute of Medical Research. Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.