Name: טיהור של מכלולי Native לחקר מבני בשיטת תג טנדם זיקה (וידאו) | JoVE Journal
Uploaded: 2016-07-27T15:00:00.000+00:00
Duration: 10 min 36 s
Description: חקור שיטת תג טנדם זיקה לטיהור מכלולים נטיביים מ-S. cerevisiae לחקר מבני. הפרוטוקול מבטיח הומוגניות הרכב ושמירה על שלמות מבנית לניתוח ביופיזיקלי.

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The authors are grateful for the support and advice of Nikolaus Grigorieff. We thank Anna Loveland, Axel Brilot, Chen Xu, and Mike Rigney for helpful discussions and EM guidance. This work was funded by the National Science Foundation, Award No. 1157892. The Brandeis EM facility is supported by National Institutes of Health grant P01 GM62580.

Note: The following protocol was devised for purification of a complex from 4 L of cell culture, approximately 40 g wet weight of cells. Once prepared, all buffers should be stored at 4 &#176;C and used within a month of their preparation. Reducing agent and protease inhibitors are only added to buffers just prior to use.
1. Preparation of Whole Cell Extract for Tandem Affinity Purification&#160;
<ol>
	<li>Growth of S. cerevisiae Cells
	<ol>
		<li>Streak the desired TAP tagged S. cerevisiae strain from storage (-80 &#176;C) onto a yeast peptone dextrose (YPD) plate. Incubate for 3 - 5 days (30 &#176;C), until yeast cells appear white and fluffy.</li>
		<li>Inoculate a streak of approximately 2 mm2 of the fresh cells from the plate into 10 ml YPD media in a 50 ml conical polypropylene centrifuge tube. Shake the tube at 180 revolutions per min (rpm) overnight (30m&#176;C).</li>
		<li>Inoculate 2 ml of the overnight culture per liter of YPD media in a 2 L Erlenmeyer or conical flask. Shake at 180 rpm (30 &#176;C). Monitor cell growth at an optical density of 600 nm (OD600) until late log phase, OD600 of 1.8 to 2, typically 20 - 24 hr.</li>
	</ol>
	</li>
	<li>Harvesting S. cerevisiae Cells
	<ol>
		<li>Harvest cells by centrifugation at 5,400 x g for 15 min (4 &#176;C).</li>
		<li>Quickly decant the supernatant and keep cells at 4 &#176;C or on ice.</li>
		<li>Suspend each liter of cell pellet with 2 ml chilled Lysis Buffer (10 mM Tris-HCl, pH 8.0; 300 mM NaCl; 10 mM KCl; 0.2 mM EDTA, pH 8.0; 5 mM imidazole, pH 8.0; 10% glycerol; 0.1% v/v NP-40). Suspend cells by swirling and/or repeat pipetting using a 10 ml serological pipet. 
		Note: The importance of NP-40 as it pertains to complex quality and post-purification analysis is discussed below.</li>
		<li>Transfer the cell suspension into an empty 50 ml conical polypropylene centrifuge tube kept on ice. Wash each centrifuge bottle with an additional 2 ml of chilled Lysis Buffer and add to the cell suspension in the 50 ml tube.</li>
		<li>Determine the wet weight of cell pellet by weighing the tube and subtracting the weight of the empty tube as well as the added Lysis Buffer. 
		Note: A liter of cell culture having an OD600 of 1.8 is approximately 10 g.</li>
		<li>Create a liquid nitrogen bath in a 50 ml conical polypropylene centrifuge tube. Draw suspended cells into a 5 ml syringe. Connect a 16 G needle to the syringe. Freeze the cell suspension by passing it through the syringe/needle to generate cell &#34;droplets.&#34; Rate of freezing should be approximately 1 min/ml.</li>
		<li>Store frozen cell droplets (-80 &#176;C) or proceed to lysis.</li>
	</ol>
	</li>
	<li>Lysing Cells and Lysate Clarification
	<ol>
		<li>Lyse the yeast cells using a coffee grinder. Lyse cells eight to nine times with 25 sec grinding bursts, while shaking the coffee grinder. Prior to use, pre-chill the coffee grinder with liquid nitrogen. Every two rounds of grinding, add a shallow layer of liquid nitrogen to the grinder and allow it to evaporate.</li>
		<li>Stir with a liquid nitrogen chilled spatula as needed to keep cells from clumping. Take caution to prevent thawing of cells, which can result in cell clumping. The cells should appear as a fine white powder following lysis. A maximum cell pellet from 4 L of yeast culture (~ 40 g) can be ground at a time. 
		Note: Observations regarding method of lysis are discussed below.</li>
		<li>Assess the degree of cell lysis using a hemocytometer or alternative cell counting method. To provide an adequate analysis, take the following samples: (1) before lysis; (2) after four rounds of lysis; and (3) following lysis. To check these samples, take a small amount of cells with a liquid nitrogen chilled spatula and place in a 1.5 ml tube.</li>
		<li>Once cells are thawed, pipette 5 &#181;l of cells and mix with 495 &#181;l water. If 40 - 60% lysis of cells is observed, proceed to next step in the protocol. 
		Note: Observations regarding effect of long periods of lysis are discussed below.</li>
		<li>Once adequate lysis is observed, store lysed cells (-80 &#176;C) or proceed to next step.</li>
		<li>Prepare two 50 ml conical polypropylene centrifuge tubes each with 15 ml Lysis Buffer, including 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and a commercially available cocktail of protease inhibitors. 
		Note: If significant proteolysis is observed, consider use of a protease deficient strain.</li>
		<li>Use a liquid nitrogen chilled spatula to scoop the frozen lysed cell powder into the prepared 50 ml tubes. Add the cell powder/solid incrementally to the Lysis Buffer, containing both a reductant and protease inhibitor(s).</li>
		<li>Rotate the 50 ml tubes gently at RT so as to thaw/dissolve the cells, but also to prevent bubbles from forming. As the cell powder/solid dissolves, add additional cell solid/powder.</li>
		<li>Fill each of the two 50 ml tubes until each has approximately 20 g of cells or all cells added. Continue thawing/dissolving until there are no frozen cell clumps observed in the 50 ml tubes. This should take approximately 50 min.</li>
		<li>Centrifuge the suspended cell lysate for 20 min at 25,000 x g (4 &#176;C). Well-lysed cells may exhibit a small amount of black precipitate.</li>
		<li>Transfer the 50 ml of supernatant to polycarbonate ultra-centrifuge tubes (26.3 ml tubes used) and add 10 &#181;l 200 mM PMSF to each full tube.</li>
		<li>Centrifuge for 1 hr at 100,000 x g (4&#176;C). Four layers should be visible, from bottom to top: (1) a hard clear pellet, containing ribosomal complexes; (2) a soft lipid-rich pellet; (3) a large clear yellow-tinged layer containing most of the cell&#39;s soluble proteins and complexes; and (4) a &#39;scaly&#39; top layer consisting of lipids.</li>
		<li>Perform all subsequent steps in a cold room (4 &#176;C). Remove as much of the top &#39;scaly&#39; lipid layer as possible using a 1 ml pipet and discard. Use a 10 ml serological pipet to recover most of the yellow, clear layer.</li>
		<li>Recover the last few ml of this layer using a 1 ml pipette to avoid disturbing the bottom two layers. From 40 g wet cell pellet, approximately 48 ml of the middle layer is typically recovered and 8 ml of lipid layer removed/discarded. 
		Note: Column purification flow is greatly hindered by the presence of lipids, which also may reduce the quality of the complex, discussed below.</li>
	</ol>
	</li>
</ol>
2. Column Purification step 1: IgG Chromatography
<ol>
	<li>Resin Equilibration and Binding
	<ol>
		<li>Prepare a 300 &#181;l IgG Sepharose slurry for 40 g of cell pellet. Cut the end of a 1 ml pipet tip to facilitate pipetting the slurry. Wash/equilibrate the IgG resin three times, each time with 5 ml IgGD150 Buffer (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 150 mM KCl; 1 mM MgCl2; 5 mM imidazole, pH 8; 0.1% v/v NP-40; 1 mM DTT; a protease inhibitor tablet per 50 ml volume).
		<ol>
			<li>Spin to pellet at 160 x g (4&#176;C) between the washes and remove supernatant.</li>
		</ol>
		</li>
		<li>Rotate the IgG resin with the middle phase supernatant and two mini protease inhibitor tablets per 40 g of cells, using an appropriate rotator, for 2 hr (4 &#176;C). This is the IgG batch solution.</li>
		<li>Prepare two 10 ml poly-prep columns for use by cutting the end of the column to produce a flat cut. To expedite the packing and washing of the column by gravity flow, load a maximum of 200 &#181;l of packed IgG resin or ~ 25 ml of the IgG batch solution per 10 ml column.</li>
		<li>Pour the IgG batch solution into the column and allow to sediment by gravity. If sedimentation takes longer than 30 min, there most likely was lipid contamination when recovering the middle phase from the centrifuge tubes.</li>
		<li>Wash the packed column with four successive 10 ml volumes of IgGD150 Buffer.</li>
	</ol>
	</li>
	<li>On Column TEV Cleavage 
	<ol>
		<li>Seal the bottom of column and add 1 ml of IgGD150 Buffer and 100 &#181;l of TEV protease (0.6 mg/ml stock, mutant variant S219V of TEV produced in-house). Seal the top of the column and mix for 20 min (18 &#176;C) using a thermal mixer, shaking at 750 rpm. Resuspend resin and mix again for 20 min, repeat once more for a total of 1 hr incubation. 
		Note: It is critical to keep time of incubation to a minimum.</li>
		<li>Return the column to 4 &#176;C and elute the protein/complex by gravity. Elute the dead-volume with an additional 200 &#956;l of IgGD150 Buffer.</li>
	</ol>
	</li>
</ol>
3. Column Purification Step 2: Calmodulin Affinity Chromatography
<ol>
	<li>Resin Equilibration and Binding 
	<ol>
		<li>Prepare 200 &#181;l of Calmodulin affinity resin per 40 g of cell pellet. Wash the resin three times, each time with 5 ml Calmodulin Binding Buffer (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 10 mM KCl; 1 mM MgCl2; 5 mM imidazole, pH 8; 2 mM CaCl2; 0.1% v/v NP-40; 10 mM &#946;-mercaptoethanol), centrifuging at 160 x g (4 &#176;C) to pellet for each wash.</li>
		<li>To each 1 ml IgG eluate, add 3 volumes of Calmodulin Binding Buffer. To keep the calcium level constant, add CaCl2 and &#946;-mercaptoethanol to account for the lack of these constituents in the IgG elution. Final concentrations should be 2 mM CaCl2 and 10 mM &#946;-mercaptoethanol.</li>
		<li>Add the sample to the Calmodulin resin and bind for 1 hr (4 &#176;C) using a tube rotator.</li>
	</ol>
	</li>
	<li>Packing and Eluting from Calmodulin Resin
	<ol>
		<li>Prepare a 2 ml poly-prep column per 100 &#181;l Calmodulin slurry by cutting the end of the column to create a flat opening. Load no more than 100 &#181;l Calmodulin slurry per column to minimize the amount of resin the sample comes in contact with during elution.</li>
		<li>Pack the column by gravity and wash the resin in the column three times with 5 ml Calmodulin Binding Buffer.</li>
		<li>Elute protein with addition of 200 &#181;l Calmodulin Elution Buffer (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 10 mM KCl; 1 mM MgCl2; 5 mM imidazole, pH 8.0; 4 mM EGTA, pH 8.0; 0.08% v/v NP-40; 10 mM &#946;-mercaptoethanol). Repeat six times the addition of 200 &#181;l of Calmodulin Elution Buffer.</li>
		<li>For elutions 1 to 3, apply the volume to the column and collect the eluate immediately. For elution 4, seal the bottom of the column and incubate with elution buffer for 2.5 min and then unseal and allow to elute by gravity. For elution 5, incubate for 5 min and then unseal and elute. For elution 6, incubate for 10 min and then unseal and elute. 
		Note: The integrity of the complex may vary between elutions/fractions (see representative results section).</li>
		<li>Dialyze elutions 2 to 6 individually overnight using an appropriate micro dialysis method. Dialyze the fractions against Dialysis Buffer (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 10 mM KCl; 1 mM MgCl2; 5 mM imidazole, pH 8; 10 mM &#946;-mercaptoethanol). 
		Note: NP-40 does not pass appreciably, if at all, through the dialysis membrane.</li>
	</ol>
	</li>
</ol>
4. Post-Purification Analyses to Assess Complex Quality
<ol>
	<li>Native Gel and Silver Staining
	<ol>
		<li>Prepare a 4 - 16% precast native gel for analysis of the complex as per the instructions from the manufacturer. Use an appropriate molecular weight protein standard and load 15 &#181;l of each of the dialyzed Calmodulin elution/fractions onto the gel.</li>
		<li>Electrophorese the gel at 150 V for approximately 3 hr (4 &#176;C).</li>
		<li>Silver stain the gel to assess the quality of each of the six elutions. Alternatively, use equally sensitive commercial fluorescent stain(s). At this stage, the concentration of the complex is most likely below the level of detection for Coomassie blue staining.</li>
	</ol>
	</li>
	<li>Additional Gel Analysis Methods
	<ol>
		<li>To detect protein by Western blotting, resolve the complex by SDS or native PAGE and then probe the gel for the Calmodulin Binding Peptide (CBP) epitope using a TAP tag antibody as per the instructions from the manufacturer.</li>
		<li>Using a specific fluorescent stain(s), confirm the presence and integrity of protein and/or nucleic acid in a TAP purified complex. Take care that no spectral overlap is detected between these stains.</li>
	</ol>
	</li>
	<li>Negative Stain Electron Microscopy Visualization
	<ol>
		<li>Use continuous carbon grids glow-discharged at -15 mA for 30 sec, within 30 min of complex application.</li>
		<li>Apply 3 &#181;l of TAP purified complex (typically at a concentration of 1 nM) to grid. Incubate for a minute. Blot from the side of the grid to remove excess buffer.</li>
		<li>Wash twice with 20 &#181;l drops of deionized water. Side blot between washes.</li>
		<li>Apply grid to a 50 &#181;l negative stain drop and incubate for a min. Staining with uranyl acetate (2%) or uranyl formate (0.75%) is recommended. 
		Caution: Uranyl salts and solutions are weakly radioactive and contain heavy metal. These should be handled with care and all material that comes in contact with these solutions should be disposed of following institutional recommended waste guidelines.</li>
		<li>Apply grid to a fresh 50 &#181;l negative stain drop, without blotting. Incubate for a min, then side blot, and finally air dry and visualize.</li>
	</ol>
	</li>
	<li>Cryo Electron Microscopy (cryo-EM) Visualization
	<ol>
		<li>Perform cryo-EM with TAP purified complex, though optimization may be necessary, according to manufacturer&#39;s protocol. 
		Note: Presence of detergent at high concentrations may impede progress and is discussed below.</li>
	</ol>
	</li>
</ol>
5. Complex storage
<ol>
	<li>Preparing Complex for Storage
	<ol>
		<li>Concentrate the complex, if needed, using a centrifugal filter having an appropriate molecular weight cut-off for the complex. Spin at 14,000 x g for 1 min increments until desired concentration is reached. 
		Note: NP-40 concentration will increase in concentration during concentrating, as it does not pass through the filter nor dialysis membrane.</li>
		<li>Flash freeze the complex in 30 &#181;l aliquots in liquid nitrogen or dry ice cooled ethanol bath and then store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Optional Post-Purification Removal of Detergent</li>
</ol>
Note: The presence of a detergent such as NP-40 and at a concentration above that of its critical micelle concentration may hinder or inhibit progress for some applications. The consequences of its removal from the protocol as well as replacement with other additives or co-solvents are discussed below. If no NP-40 is desired, an option is to use a commercial application for removal, for example Bio-beads.
<ol style="margin-left: 40px;">
	<li>Pre-equilibrate commercially available detergent absorbing beads in Dialysis Buffer. Mix five beads per 30 &#956;l of complex (usually at 10 nM concentration).</li>
	<li>Incubate equilibrated beads from Step 5.2.1 with TAP purified complex for 15 min on ice. Use complex within a few hours following the removal of detergent.</li>
</ol>

<ol><li>Gavin, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+organization+of+the+yeast+proteome+by+systematic+analysis+of+protein+complexes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Functional organization of the yeast proteome by systematic analysis of protein complexes.</a> Nature. 415 (6868), 141-147 (2002).</li>
<li>Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., Seraphin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nature Biotechnology. 17 (10), 1030-1032 (1999).</li>
<li>Puig, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+tandem+affinity+purification+%28TAP%29+method%3A+A+general+procedure+of+protein+complex+purification%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">The tandem affinity purification (TAP) method: A general procedure of protein complex purification.</a> Methods. 24 (3), 218-229 (2001).</li>
<li>Vaillancourt, P., Zheng, C. F., Hoang, D. Q., Breister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Affinity+purification+of+recombinant+proteins+fused+to+calmodulin+or+to+calmodulin-binding+peptides%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Affinity purification of recombinant proteins fused to calmodulin or to calmodulin-binding peptides.</a> Methods Enzymol. 326, 340-362 (2000).</li>
<li>Braisted, A. C., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimizing+a+binding+domain+from+protein+A%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Minimizing a binding domain from protein A.</a> Proc Natl Acad Sci U S A. 93 (12), 5688-5692 (1996).</li>
<li>Kapust, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tobacco+etch+virus+protease%3A+mechanism+of+autolysis+and+rational+design+of+stable+mutants+with+wild-type+catalytic+proficiency%5BTitle%5D)+AND+%22Protein+Eng%22%5BJournal%5D)">Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.</a> Protein Eng. 14 (12), 993-1000 (2001).</li>
<li>Nallamsetty, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+site-specific+processing+of+fusion+proteins+by+tobacco+vein+mottling+virus+protease+in+vivo+and+in+vitro%5BTitle%5D)+AND+%22Protein+Expr+Purif%22%5BJournal%5D)">Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro.</a> Protein Expr Purif. 38 (1), 108-115 (2004).</li>
<li>Ghaemmaghami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+analysis+of+protein+expression+in+yeast%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li>
<li>Howson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction%2C+verification+and+experimental+use+of+two+epitope-tagged+collections+of+budding+yeast+strains%5BTitle%5D)+AND+%22Comp+Funct+Genomics%22%5BJournal%5D)">Construction, verification and experimental use of two epitope-tagged collections of budding yeast strains.</a> Comp Funct Genomics. 6 (1-2), 2-16 (2005).</li>
<li>Cox, D. M., Du, M., Guo, X., Siu, K. W., McDermott, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tandem+affinity+purification+of+protein+complexes+from+mammalian+cells%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Tandem affinity purification of protein complexes from mammalian cells.</a> Biotechniques. 33 (2), 267-268 (2002).</li>
<li>Gully, D., Moinier, D., Loiseau, L., Bouveret, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+partners+of+acyl+carrier+protein+detected+in+Escherichia+coli+by+tandem+affinity+purification%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">New partners of acyl carrier protein detected in Escherichia coli by tandem affinity purification.</a> FEBS Lett. 548 (1-3), 90-96 (2003).</li>
<li>Tharun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+analysis+of+the+decapping+activator+Lsm1p-7p-Pat1p+complex+from+yeast%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Purification and analysis of the decapping activator Lsm1p-7p-Pat1p complex from yeast.</a> Methods Enzymol. 448, 41-55 (2008).</li>
<li>Xu, X., Song, Y., Li, Y., Chang, J., Zhang, H., An, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+tandem+affinity+purification+method%3A+an+efficient+system+for+protein+complex+purification+and+protein+interaction+identification%5BTitle%5D)+AND+%22Protein+Expr+Purif%22%5BJournal%5D)">The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.</a> Protein Expr Purif. 72 (2), 149-156 (2010).</li>
<li>Cheng, Y., Grigorieff, N., Penczek, P. A., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Primer+to+Single-Particle+Cryo-Electron+Microscopy%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A Primer to Single-Particle Cryo-Electron Microscopy.</a> Cell. 161 (3), 438-449 (2015).</li>
<li>Gottschalk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comprehensive+biochemical+and+genetic+analysis+of+the+yeast+U1+snRNP+reveals+five+novel+proteins%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins.</a> RNA. 4 (4), 374-393 (1998).</li>
<li>Neubauer, G., Gottschalk, A., Fabrizio, P., Seraphin, B., Luhrmann, R., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+the+proteins+of+the+yeast+U1+small+nuclear+ribonucleoprotein+complex+by+mass+spectrometry%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry.</a> Proc Natl Acad Sci U S A. 94 (2), 385-390 (1997).</li>
<li>Lucast, L. J., Batey, R. T., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+purification+of+a+stable+form+of+recombinant+tobacco+etch+virus+protease%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Large-scale purification of a stable form of recombinant tobacco etch virus protease.</a> Biotechniques. 30 (3), 544-546 (2001).</li>
</ol>

The authors have nothing to disclose.

The TAP method utilizes two tags that balance a need for tight and selective binding to an affinity resin with a desire to maintain near physiological solution conditions. This balance serves to preserve stable interaction(s) of the tagged protein with interacting factor(s) for post-purification characterization. In addition, individual TAP tagged ORFs are available from a commercial source, so that one can obtain any yeast strain with a tagged protein for any yeast complex. Preserving the integrity of a complex and the ...

Many major cellular processes are carried out by large protein and protein-RNA complexes1. A significant bottleneck to conducting biophysical and structural studies of such complexes is obtaining them of a suitable quality (i.e., homogeneity) and at an appropriate concentration. Isolating a complex from a native source has many advantages, including retaining relevant post-transcriptional and/or translational modifications of subunits and insuring proper complex assembly. However, large cellular complexes are often present in a cell at a low copy number and the purification must be highly efficient and occur under near physiological conditions to ensure complex integrity is maintained. Purifying a complex from a eukaryotic source is particularly challenging and can be financially prohibitive. Thus, strategies or methods that are efficient and yield a homogeneous complex are highly desired.
A strategy that has been successful in purifying native complexes from eukaryotic cells for their initial characterization is the Tandem Affinity Purification (TAP) method2,3. The TAP method was initially devised to purify a native protein from the budding yeast (S. cerevisiae) in complex with interacting factor(s)2. The TAP method utilized two tags, each tag fused in tandem to the same protein-coding gene sequence. The tags were selected so as to balance a need for tight and selective binding to an affinity resin with a desire to maintain near physiological solution conditions. This balance serves to preserve stable interaction(s) of the tagged protein with interacting factor(s) for post-purification characterization. The genomically incorporated TAP tag was placed at the end (C-terminal) of a protein-coding gene and consisted of a sequence coding for a Calmodulin Binding Peptide (CBP) followed by Protein A - an addition of just over 20 kDa to the tagged protein. CBP is short, 26 amino acids, and recognized by the ~ 17 kDa protein Calmodulin (CaM) in the presence of calcium with a KD on the order of 10-9 M 4. The Protein A tag is larger, consisting of two repeats of 58 residues with a short linker between the repeats. Each 58 amino acid repeat is recognized by immunoglobulin G (IgG) with a KD ~ 10-8 M 5. Between these two tags was incorporated a recognition site for TEV protease, an endopeptidase from the Tobacco Etch Virus6,7. As illustrated in Figure 1, in the first affinity step of the method the TAP tagged protein is bound to an IgG resin via the Protein A. The tagged protein is eluted by on column cleavage upon the addition of TEV protease, site-specifically cleaving between the two tags. This is a necessary step as the interaction of IgG and Protein A is very strong and can only be adequately perturbed under denaturing solution conditions. Lacking a Protein A tag, the protein is bound to CaM resin in the presence of calcium and eluted from this resin with addition of the metal ion chelator EGTA (ethylene glycol tetraacetic acid) (Figure 1).
Soon after the introduction of the TAP method, it was used in a large-scale study to generate a &#39;map&#39; of complex interactions in S. cerevisiae8. Importantly, as a consequence of this effort an entire yeast-TAP Tagged Open Reading Frame (ORF) library as well as individual TAP tagged ORFs9 are available from a commercial source. Thus, one can obtain any yeast strain with a tagged protein for any yeast complex. The TAP method also spurred modifications or variations of the TAP tag, including: its use for purification of complexes from other eukaryotic as well as bacterial cells10,11; the design of a &#34;split tag&#34;, wherein the Protein A and CBP are placed on different proteins12; and the tags changed, so as to accommodate the need of the investigator, such as sensitivity of the complex to Ca2+ or EGTA13.
Recent advances in both instrumentation and methodology have led to significant advances in the application of electron microscopy (EM) for structure determination, that have led to high, near atomic resolution images of macromolecular complexes14. The resolution obtainable of a complex by EM, however, remains contingent upon the quality of the complex under study. This study has utilized the TAP tag approach to purify from S. cerevisiae the U1 snRNP, an 18-subunit (~ 0.8 MDa) low copy number ribonucleoprotein complex that is part of the spliceosome15,16. A number of steps have been taken to purify this complex such that it is homogeneous and of an adequate concentration. Potential problems encountered at various stages of the purification are described and strategies taken to overcome challenges highlighted. By carefully assessing and optimizing steps in the purification, the U1 snRNP purified is of a quality and at a quantity suitable for negative stain and electron cryo microscopy (cryo-EM) studies. An optimized TAP method protocol for purification of native complexes for structural studies is described herein.

<table><tbody><tr><td>S. cerevisiae TAP tagged strain</td><td>Open Biosystems</td><td>YSC1177</td><td>This is the primary yeast strain used to develop the TAP protocol. Its background is S288C: ATCC 201388: MATa, his3&amp;Delta;1, leu2&amp;Delta;0, met15&amp;Delta;0, ura3&amp;Delta;0, SNU71::TAP::HIS3MX6</td></tr><tr><td>Coffee grinder</td><td>Mr. Coffee</td><td>IDS77</td><td>Used for cell lysis</td></tr><tr><td>Hemocytometer, Bright Line</td><td>Hausser Scientific</td><td>3120</td><td>Used to assess cell lysis</td></tr><tr><td>JA 9.100 centrifuge rotor&amp;nbsp;</td><td>Beckman Coulter, Inc.</td><td></td><td>Used to harvest the yeast cells</td></tr><tr><td>JA 20 fixed-angle centrifuge rotor</td><td>Beckman Coulter, Inc.</td><td></td><td>Used to clear the cell extract of non-soluble cellular material</td></tr><tr><td>Ti 60 fixed-angle centrifuge rotor</td><td>Beckman Coulter, Inc.</td><td></td><td>Used to further clear the soluble cell extract</td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>R5355</td><td>Temperature controlled shaker</td></tr><tr><td>Novex gel system</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td></td><td></td></tr><tr><td>IgG resin</td><td>GE Healthcare</td><td>17-0969-01</td><td>Sepharose 6 fast flow</td></tr><tr><td>Calmodulin resin</td><td>Agilent Technologies, Inc.</td><td>214303</td><td>Affinity resin</td></tr><tr><td>Protease inhibitor cocktail, mini tablets</td><td>Sigma Aldrich</td><td>589297</td><td>Mini cOmplete ultra EDTA-free tablets</td></tr><tr><td>Protease inhibitor cocktail, large tablets</td><td>Sigma Aldrich</td><td>5892953</td><td>cOmplete ultra EDTA-free tablets</td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td></td><td></td><td>Dissolved in isopropanol</td></tr><tr><td>2 ml Bio-spin column</td><td>Bio-Rad Laboratories, Inc.</td><td>7326008</td><td>Used to pack and wash the Calmodulin resin</td></tr><tr><td>10 ml poly-prep column</td><td>Bio-Rad Laboratories, Inc.</td><td>7311550</td><td>Used to pack and wash the IgG resin</td></tr><tr><td>Precast native PAGE Bis-Tris gels</td><td>Life Technologies</td><td>BN1002</td><td>Novex NativePAGE Bis-Tris 4 - 16% precast polyacrylamide gels</td></tr><tr><td>NativeMark protein standard</td><td>Thermo Fisher Scientific</td><td>LC0725</td><td>Unstained protein standard used for native PAGE. Load 7.5 &amp;mu;l for a silver stained gel and 5 &amp;mu;l for a SYPRO Ruby stained gel</td></tr><tr><td>Precast SDS PAGE Bis-Tris gels</td><td>Life Technologies</td><td>NP0321</td><td>Novex Nu-PAGE Bis-Tris 4 - 12% precast polyacrylamide gels&amp;nbsp;</td></tr><tr><td>PageRuler protein standard</td><td>Thermo Fisher Scientific</td><td>26614</td><td>Unstained protein standard used for Western blotting</td></tr><tr><td>SDS running buffer</td><td>Life Technologies</td><td>NP0001</td><td>1x NuPAGE MOPS SDS Buffer</td></tr><tr><td>TAP antibody</td><td>Thermo Fisher Scientific</td><td>CAB1001</td><td>Primary antibody against CBP tag</td></tr><tr><td>Secondary antibody</td><td>Thermo Fisher Scientific</td><td>31341</td><td>Goat anti-rabbit alkaline phosphatase conjugated</td></tr><tr><td>BCIP/NBT</td><td>Thermo Fisher Scientific</td><td>34042</td><td>5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium&amp;nbsp;</td></tr><tr><td>Dialaysis units</td><td>Thermo Fisher Scientific</td><td>88401</td><td>Slide-A-Lyzer mini dialysis units</td></tr><tr><td>Centrifugal filter units, 100kDa MWCO</td><td>EMD Millipore</td><td>UFC5100008</td><td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane</td></tr><tr><td>Detergent absorbing beads</td><td>Bio-Rad Laboratories, Inc.</td><td>1523920</td><td>Bio-bead SM-2 absorbants</td></tr><tr><td>SYBR Green II</td><td>Thermo Fisher Scientific</td><td>S-7564</td><td>Flourescent dye for nucleic acid staining, when detecting with SYPRO Ruby present, use excitation wavelength of 488 nm and emission wavelength of 532 nm</td></tr><tr><td>SYPRO Ruby</td><td>Molecular Probes</td><td>S-12000</td><td>Flourescent dye for protein staining, when detecting with SYBR Green II present, use excitation wavelength of 457 nm and emission wavelength of 670 nm</td></tr><tr><td>Copper grids</td><td>Electron Microscopy Sciences</td><td>G400-CP</td><td></td></tr></tbody></table>

purification of native complexes for structural study using a tandem affinity tag method

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

A modified TAP method was used to purify from S. cerevisiae the U1 snRNP, an 18-subunit ribonucleoprotein complex. An initial TAP purification of the complex following the published protocol2,3 yielded a complex that appeared heterogeneous, migrating as three bands on a silver stained native polyacrylamide gel (Figure 2A). Multiple rounds of optimization of the TAP method, yielded a complex that migrated as primarily a single band on a native gel indic...

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

The overall goal of this protocol is to purify a native macromolecular complex of suitable quality for biophysical studies. This protocol serves not only as a way to purify a complex, but as a detailed guide to assessing the structural quality of the sample during purification. The main advantage of this protocol is that it allows one to purify a native assembly under near physiological buffer conditions simply and rapidly, so as to ensure compositional homogeneity.After centrifugation of S.cerevisiae cells, and decanting the supernatant according to the text protocol, add two milliliters of chilled lysis buffer, and swirl and/or use a 10 milliliter serelogical pipette to suspend the pellet. Transfer the cell suspension into an empty 50 milliliter conical polypropylene centrifuge tube kept on ice. Then use an additional two milliliters of chilled lysis buffer to wash each centrifuge bottle and add the solution to the 50 milliliter tube.After determining the wet weight of the cell pellet, create a liquid nitrogen bath in and around a 50 milliliter conical polypropylene centrifuge tube. Next, draw suspended cells into a five milliliter syringe and connect a 16 gauge needle to the syr

purification-native-complexes-for-structural-study-using-tandem

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JoVE Journal

Biology

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identification of Protein Interacting Partners Using Tandem Affinity Purification

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

This video demonstrates tandem affinity purification — a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

חקור שיטת תג טנדם זיקה לטיהור מכלולים נטיביים מ-S. cerevisiae לחקר מבני. הפרוטוקול מבטיח הומוגניות הרכב ושמירה על שלמות מבנית לניתוח ביופיזיקלי.

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

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