This work was supported by a National Science Foundation Grant NSF-MCB1243645 (JZT), any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

NOTE: An overview of the generation of inducible LAP-tagged stable cell lines for any protein of interest is illustrated in Figure 1 and the overview of LAP-tagged protein expression, purification and preparation for mass proteomic analyses is illustrated in Figure 3.
1. Cloning the Open Reading Frame (ORF) of the Gene of Interest into the LAP-tag Vector
<ol>
	<li>Cloning the ORF of the Gene of Interest into the Shuttle Vector.
	<ol>
		<li>Use polymerase chain reaction (PCR) to amplify the ORF of interest with the appropriate attB1 and attB2 sites within the primers for either N-terminal fusion or C-terminal fusion. See Table 1 for primer sequences and Table 2 for PCR conditions.</li>
		<li>Gel purify the PCR products by resolving them on a 1% agarose gel. Excise the amplified band that is the correct size from the gel and extract it from the gel slice using a DNA gel extraction kit as per manufacturer&#39;s instructions.</li>
		<li>Incubate the purified attB containing PCR products with an attP containing shuttle vector and the recombinase that allows the PCR products to recombine into the vector as per manufacturer&#39;s instructions (see Materials Table). 
		NOTE: Empty shuttle vectors and LAP-tagging vectors contain the ccdB gene and have to be propagated in ccdB resistant bacterial cells (see Materials Table). However the ccdB gene is recombined out when an ORF is inserted into these vectors, hence use standard DH5&#945; E. coli cells when propagating vectors with cloned ORFs.</li>
		<li>Transform DH5&#945; E. coli cells with 1 &#956;l of the reaction product and plate the transformed cells onto a Luria broth (LB) agar plate with 50 mg/ml Kanamycin13.</li>
		<li>Select the Kanamycin resistant colonies.</li>
		<li>Grow the selected colonies in LB media with 50 mg/ml Kanamycin, make a DNA mini-prep and confirm gene integration by DNA sequencing using the sequencing primers listed in Table 3.</li>
	</ol>
	</li>
	<li>Transferring the Gene of Interest from the Shuttle Vector into the LAP-tag Vector.
	<ol>
		<li>Incubate the shuttle vector containing the sequence verified gene of interest ORF with the LAP-tag vector (pGLAP1 for N-terminal fusion) and the recombinase that mediates the transfer of the gene of interest from the shuttle vector into the LAP-tag vector as per manufacturer&#39;s instructions (see Materials). 
		NOTE: A series of LAP/TAP vectors that can be used based on the desired promoter, epitope-tag, and N or C-terminal tagging can be found in Table 4.</li>
		<li>Transform DH5&#945; E. coli cells with 1 &#956;l of the reaction product and plate the transformed cells onto an LB agar plate with 100 mg/ml Ampicillin13.</li>
		<li>Select the Ampicillin resistant colonies.</li>
		<li>Grow the selected colonies in LB media with 100 mg/ml Ampicillin, make a DNA mini-prep, and confirm gene integration by DNA sequencing using the sequencing primers listed in Table 5.</li>
	</ol>
	</li>
</ol>
2. Generation of an Inducible Stable Cell Line that Expresses the LAP-tagged Gene of Interest
<ol>
	<li>Select the cell line best suited for the project of interest. Alternatively, create a host cell line from any existing cell line that constitutively expresses the TetR and contains a FRT site that allows the LAP-tagged gene of interest to be stably integrated into the genome (see Materials). 
	NOTE: This protocol uses a HEK293 cell line that contains the TetR and an FRT site, which is grown in -Tet DMEM/F12 media [made with 10% fetal bovine serum (FBS) that is devoid of Tetracycline (-Tet)] 4.</li>
	<li>Determine the minimum concentration of Hygromycin required to kill the host cell line within 1 to 2 weeks after drug addition. The concentration can vary between host cell lines, with most ranging between 100 &#181;g/ml and 800 &#181;g/ml. 
	NOTE: HEK293 cells grown in -Tet DMEM/F12 media with 100 &#181;g/ml Hygromycin at 37 &#176;C and 5% CO2 will die within 1-2 weeks.</li>
	<li>Co-transfect the vector that expresses the flippase recombinase (mediates the integration of the LAP-tagged gene of interest into the FRT site within the genome of the cell) with the LAP-tagged vector into HEK293 cells using a transfection reagent as per manufacturer&#39;s instructions. Use a ratio of 4:1 of recombinase vector to LAP vector14. 
	NOTE: The optimal ratio is dependent on the host cell line and method of transfection, and may require a titration. A ratio of 4:1 works well for most cell lines. Include a mock-transfected plate as a negative control.</li>
	<li>One day post-transfection, replace the -Tet DMEM/F12 media with fresh media.</li>
	<li>Two days post-transfection, split cells to 25% confluence. Allow cells ~5 hr to attach, then add Hygromycin-containing -Tet DMEM/F12 media at the concentration predetermined in step 2.2. For HEK293 cells use 100 &#181;g/ml Hygromycin. 
	NOTE: The FRT containing HEK293 cell line also contains the TetR that is associated with Blasticidin resistance, therefore 5 &#181;g/ml Blasticidin is used during the stable cell line selection process to select for the TetR and to minimize leaky expression.</li>
	<li>Replace Hygromycin-containing -Tet DMEM/F12 media as needed until distinct cell foci appear that resemble opaque spots against the transparent plate.</li>
	<li>Add 20 &#956;l of trypsin on top of each cell foci and pipet up and down 2 times with a 200 &#956;l pipet tip. Place cells in a 24 well plate and expand the cells by continual growth in Hygromycin-containing -Tet DMEM/F12 media.</li>
	<li>Screen cells for inducible LAP-tagged protein expression by fixed-cell or live-cell fluorescence microscopy and/or Western blotting for the GFP tag within the LAP-tag15.</li>
</ol>
3. Purification of LAP-tagged Protein Complexes
NOTE: The following LAP-tagged protein purification protocol details recommendations on conditions and volumes used for a typical LAP-tagged protein purification based on previous experience. However, caution should be exercised to ensure that empirical optimization is carried out for any protein complex and protein expression level of interest to provide the best results.
<ol>
	<li>Cell Growth and Cell Harvesting.
	<ol>
		<li>Expand the validated LAP-tagged cell line for TAP isolation of protein complexes, by continually passaging all HEK293 cells into larger plates and/or roller bottles in -Tet DMEM/F12 media at 37 &#176;C and 5% CO2.</li>
		<li>For Tet/Dox inducible cell lines, induce for 10-15 hr at a concentration of 0.2 &#181;g/ml Tet/Dox when the cells reach ~70% confluency, before harvesting cells. 
		NOTE: The concentration and induction time should be determined for each protein, a titration of 0.1-1 &#181;g/ml Tet/Dox for 10-15 hr is recommended.</li>
		<li>Harvest cells by agitation or trypsinization and pellet cells at 875 x g for 5-10 min.</li>
	</ol>
	</li>
	<li>Coupling Anti-GFP Antibody to Protein A Beads
	<ol>
		<li>Use 40 &#181;g of antibody for immunoprecipitation from a lysate prepared from a 0.5 ml cell pellet, packed cell volume (PCV). 
		NOTE: The amount of antibody needed will depend on the abundance of the LAP-tagged protein, among other factors, and will require optimization. A titration of 10-40 &#181;g is recommended.</li>
		<li>Equilibrate 160 &#181;l packed volume (PV) Protein A beads into PBST (PBS + 0.1% Tween-20) in a 1.5 ml tube. Wash 3 times with 1 ml of PBST. 
		NOTE: All washes herein are carried out by centrifuging the beads at 5,000 x g for 10 sec.</li>
		<li>Resuspend beads in 500 &#181;l PBST and add 80 &#181;g of affinity-purified rabbit anti-GFP antibody to each tube of 160 &#181;l beads. Mix for 1 hr at room temperature (RT).</li>
		<li>Wash beads 2 times with 1 ml of PBST. Then, wash beads 2 times with 1 ml of 0.2 M sodium borate, pH 9 (20 ml 0.2 M sodium borate + 15 ml 0.2 M boric acid). After the final wash, add 900 &#181;l of the 0.2 M sodium borate, pH 9 to bring the final volume to 1 ml.</li>
		<li>Add 100 &#181;l of 220 mM dimethylpimelimidate (DMP) to a final concentration of 20 mM. Rotate the tubes gently at RT for 30 min. For 220 mM DMP, resuspend contents of one 50 mg bottle in 877 &#181;l of 0.2 M sodium borate, pH 9 and add immediately to the bead suspension.</li>
		<li>After incubation with DMP, wash beads 1 time with 1 ml of 0.2 M ethanolamine, 0.2 M NaCl pH 8.5 to inactivate the residual crosslinker. Resuspend beads in 1 ml of the same buffer and rotate for 1 hr at RT. Pellet beads and resuspend beads in 500 &#181;l of 0.2 M ethanolamine, 0.2 M NaCl pH 8.5. Beads are stable for several months at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of Buffers for Cell Lysis and Complex Purification
	<ol>
		<li>Prepare LAPX buffers (where X is the desired salt concentration [mM KCl] of the LAP buffer; 300 mM for cell lysis, 200 mM for most bead washes, and 100 mM for washing beads prior to eluting proteins) by making a pH 7.4 solution containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), X mM KCl, 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM MgCl2, and 10% glycerol. 
		NOTE: The components of this buffer are used to approximate the environment in living cells. HEPES is used as a buffer in the pH rage of 7.2-8.2. KCl is a salt used to maintain the ionic strength of the buffer. EGTA is a chelating agent that binds calcium ions to reduce the levels of calcium compared to magnesium. Glycerol and MgCl2 are used to improve the stability of proteins.</li>
		<li>Prepare LAPXN buffer by adding 0.05% nonyl phenoxypolyethoxylethanol to the LAPX buffer. 
		NOTE: Nonyl phenoxypolyethoxylethanol is a mild detergent that solubilizes proteins, but preserves protein-protein interactions, thus a higher concentration is used during the extraction process and it is then lowered during the binding and washing steps.</li>
	</ol>
	</li>
	<li>Preparation of Cell Lysates
	<ol>
		<li>Resuspend 500 &#181;l of PCV into 2.5 ml of LAP300 with 0.5 mM dithiothreitol (DTT) and protease inhibitors. Add 90 &#181;l of 10% nonyl phenoxypolyethoxylethanol (0.3% final) and mix by inversion.</li>
		<li>Place on ice for 10 min. Centrifuge at 21,000 x g for 10 min. Collect this low speed supernatant (LSS). Take a 10 &#181;l sample of the LSS for gel analysis.</li>
		<li>Transfer LSS to a TLA100.3 tube and spin at 100,000 x g for 1 hr at 4 &#176;C. Collect this high speed supernatant (HSS), in a tube and place on ice. Take a 10 &#181;l sample of the HSS for gel analysis. 
		NOTE: Avoid taking the top most lipid layer and the bottom most cell debris layer. The lipid layer should be removed by vacuum aspiration prior to collecting the HSS.</li>
	</ol>
	</li>
	<li>First Affinity Capture: Binding to Anti-GFP Beads
	<ol>
		<li>Pre-elute antibody coupled beads (use 160 &#181;l of beads per 0.5 ml cell pellet (PCV)) by washing them 3 times with 1 ml of elution buffer [3.5 M MgCl2 with 20 mM Tris, pH 7.4] to get rid of uncoupled antibodies and reduce background. Do quickly. Do not leave beads in high salt for a long time.</li>
		<li>Wash beads 3 times with 1 ml of LAP200N. Mix HSS extract with antibody beads for 1 hr at 4 &#176;C. Centrifuge at 21,000 x g for 10 min. Take a 10 &#181;l sample of the supernatant (i.e., the flow through (FT)) for gel analysis.</li>
		<li>Wash beads 3 times with 1 ml of LAP200N with 0.5 mM DTT and protease inhibitors. Wash beads 2 times (5 min each) with 1 ml of LAP200N with 0.5 mM DTT and protease inhibitors. Wash quickly 2 times with 1 ml of LAP200N with 0.5 mM DTT and no protease inhibitors before adding the tobacco etch virus (TEV) protease.</li>
	</ol>
	</li>
	<li>TEV Cleavage
	<ol>
		<li>Dilute 10 &#181;g TEV protease in 1 ml of LAP200N and rotate tubes at 4 &#176;C overnight. 
		NOTE: This step can be optimized for any LAP-tagged protein to be completed in a few hours by adjusting the concentration of TEV protease, which can aid the preservation of LAP-tagged protein complexes.</li>
		<li>Pellet beads and transfer supernatant to a fresh tube. Rinse beads twice with 160 &#181;l LAP200N with 0.5 mM DTT and protease inhibitors (triple concentration) to remove any residual protein. Take a 10 &#181;l sample of the supernatant for gel analysis.</li>
	</ol>
	</li>
	<li>Second Affinity Capture: Binding to S Protein Agarose
	<ol>
		<li>Wash 1 tube of 80 &#181;l S protein agarose slurry (40 &#181;l packed resin) 3 times with 1 ml of LAP200N. 
		NOTE: S protein binding to the S-tag will reconstitute an active RNase and an alternative second epitope tag should be considered for RNA containing protein complexes.</li>
		<li>Add TEV eluted supernatant to S protein agarose beads and rock for 3 hr at 4 &#176;C. Pellet beads and wash 3 times with 1 ml of LAP200N with 0.5 mM DTT and protease inhibitors. Wash beads 2 times with 1 ml of LAP100.</li>
	</ol>
	</li>
	<li>Protein Elution
	<ol>
		<li>Elute proteins off S protein agarose by adding 50 &#181;l of 4x Laemmli sample buffer and heat at 97 &#176;C for 10 min. 
		NOTE: Proteins can also be eluted from the beads with elution buffer (3.5 M MgCl2 with 20 mM Tris, pH 7.4).</li>
	</ol>
	</li>
</ol>
4. Identify Interacting Proteins by Mass Spectrometry Analysis
<ol>
	<li>Test the quality of the purification by analyzing the collected samples by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), silver staining the gel (see Materials Table) and by immunoblotting the eluates and probing with anti-GFP antibodies to ensure that the LAP-tagged purification worked16, see Figure 4.</li>
	<li>To identify stoichiometric and substoichiometric co-purifying species, take the final elution sample and separate it by SDS-PAGE. Stain the gel with a mass spectrometry compatible protein stain. Excise the most prominent bands and the space in between them from the gel and process them for analysis by mass spectrometry separately16. 
	NOTE: There are numerous approaches for separating the final purification eluates and preparing them for mass spectrometry5. For example, LAP-purified complexes can be eluted from S-protein beads by using high salt (3.5 M MgCl2) and the entire protein population en masse can be analyzed by mass spectrometry17. Alternatively, final eluates can be separated for 1 mm by SDS-PAGE and a single 1 mm band can be excised and analyzed. This clears the complex mixture of any beads or particulate matter that will interfere with the analysis.</li>
</ol>

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<li>Gupta, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Dynamic+Protein+Interaction+Landscape+of+the+Human+Centrosome-Cilium+Interface%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface.</a> Cell. 163, 1484-1499 (2015).</li>
</ol>

The authors have nothing to disclose.

The outlined protocol describes the cloning of genes of interest into the LAP-tagging vector, the generation of inducible LAP-tagged stable cell lines, and the purification of LAP-tagged protein complexes for proteomic analyses. With respect to other LAP/TAP-tagging approaches, this protocol has been streamlined to be compatible with high-throughput approaches to map protein localization and protein-protein interactions within any cellular pathway. This approach has been widely applied to the functional characterization of proteins critical for cell cycle progression, mitotic spindle assembly, spindle pole homeostasis, and ciliogenesis to name a few and has aided the understanding of how misregulation of these proteins can lead to human diseases15,16,19,20. For example, our group recently utilized this system to define the function and regulation of the STARD9 mitotic kinesin (a candidate cancer target) in spindle assembly15,21, to define a new molecular link between the Tctex1d2 dynein light chain and short rib polydactyly syndromes (SRPS)19, and to define a new molecular link to understanding how mutation of the Mid2 ubiquitin ligase can lead to X-linked intellectual disabilities16. Other laboratories have also successfully applied this method, including one that determined that Tctn1, a regulator of mouse Hedgehog signaling, was a part of a ciliopathy-associated protein complex that regulated ciliary membrane composition and ciliogenesis in a tissue-dependent manner22,23. Therefore, this protocol can be broadly applied to the dissection of any cellular pathway.
A critical step in this protocol is the selection of LAP-tagged stable cell lines that are Hygromycin resistant. Special care should be taken to ensure that all cells in the control plate are dead before selecting foci in the experimental plate for amplification. Hygromycin can also be added during routine cell culturing of LAP-tagged stable cell lines to further ensure that all cells maintain the LAP-tagged gene of interest at the FRT site. We caution that not all LAP-tagged proteins will be functional and that it is important to have assays in place that can be used to test protein function. Examples of assays used to test protein function include the rescue of siRNA-induced phenotypes and in vitro activity assays. To address any potential problems with the addition of a large LAP-tag, we have previously generated TAP-tag vectors compatible with this system that contain smaller tags, like FLAG, which are less likely to inhibit the function and localization of the protein of interest4. In addition, LAP-tagging vectors exist for generating C-terminal LAP-tagged proteins or C-terminal TAP-tagged proteins that are compatible with this system, which can be used in cases where a LAP/TAP tag is not tolerated at the N-terminus of a protein. Additionally, the salt and detergent concentrations of the purification buffers (LAPXN) can be modified to increase or decrease the purification stringency if none or too many interactions are observed. Similarly, the tandem affinity purification procedure is more stringent than single purification procedures and weak interactors may be lost, thus a single purification scheme can be used when few or no interactors are identified.
It is important to note that other GFP epitope tagging approaches exist that allow large scale GFP protein tagging for protein localization and purification studies24,25. These include the BAC TransgenOmics approach that utilizes bacterial artificial chromosomes to express GFP-tagged genes of interest from their native environment that contains all the regulatory elements, which mimics endogenous gene expression24. More recently, CAS9/single-guided RNA (sgRNA) ribonucleoprotein complexes (RNPs) have been used to endogenously tag genes of interest with a split-GFP system that allows the expression of GFP-tagged genes from their endogenous genomic loci25. Although both of these approaches enable the expression of tagged proteins under endogenous conditions, compared to the LAP-tagging protocol described here, they do not allow for inducible and tunable expression of the tagged genes of interest. Additionally, they have yet to be applied to tandem epitope tagging for TAP. It is also important to note that other tagging systems can also be modified to become compatible with the system described here for generating inducible epitope-tagged stable cell lines. For example, proximity-dependent biotin identification (BioID) has garnered considerable attention due to its ability to define spatial and temporal relationships among interacting proteins26. This technique exploits protein fusions to a promiscuous strain of the Escherichia coli biotin ligase BirA, which biotinylates any protein within a ~10 nm radius of the enzyme. The biotinylated proteins are then affinity purified using biotin-affinity capture and analyzed for composition by mass spectrometry. BirA will biotinylate any protein in close proximity, even transiently, which makes it especially suited for detecting weaker interacting partners within a complex27. Additionally, the purification scheme does not necessitate that endogenous protein-protein interactions remain intact and can be carried out under denaturing conditions, thus reducing the rate of false positives. Within our current protocol, the substitution of the pGLAP1 vector by a BirA-tagging vector could transform this system from identifying protein-protein interactions based on affinity to detecting them based on proximity. Such a system would be highly advantageous for detecting transient protein interactions as is the case between many enzyme-substrate interactions and for mapping the spatiotemporal protein-protein interactions within defined structures as has been carried out for the centrosome and cilia26,28.

To investigate the cellular function of an uncharacterized protein it is important to determine its in vivo spatiotemporal subcellular localization and its interacting protein partners. Traditionally, single and tandem epitope tags fused to the N or C-terminus of a protein of interest have been used to facilitate protein localization and protein interaction studies. For example, the tandem affinity purification (TAP) technology has enabled the isolation of native protein complexes, even those that are in low abundance, in both yeast and mammalian cell lines1,2. The localization and affinity purification (LAP) technology, is a more recent development that modifies the TAP procedure to include a localization component through the introduction of the green fluorescent protein (GFP) as one of the epitope tags3. This approach has given researchers a deeper understanding of a protein's subcellular localization in living cells while also retaining the ability to perform TAP complex purifications to map protein-protein interaction networks. 
However, there are many issues associated with the use of TAP/LAP technologies that has hampered their widespread use in mammalian cells. For example, the length of time that is necessary to generate a stable cell line expressing a TAP/LAP tagged protein of interest; which typically relies on cloning the gene of interest into a viral vector and selecting single cell stable integrants with the desired expression level. Additionally, many cellular pathways are sensitive to constitutive protein overexpression (even at low levels) and can arrest cells or trigger cell death over time making the generation of a TAP/LAP stable cell line impossible. These and other constraints have impeded LAP/TAP methodologies from becoming high-throughput systems for protein localization and protein complex elucidation. Therefore, there has been considerable interest in the development of an inducible high-throughput LAP-tagging system for mammalian cells that takes advantage of current innovations in cloning and cell line technologies. 
Here we present a protocol for generating stable cell lines with Doxycycline/Tetracycline (Dox/Tet) inducible LAP-tagged proteins of interest that applies advances in both cloning and mammalian cell line technologies. This approach streamlines the acquisition of data with regards to LAP-tagged protein subcellular localization, protein complex purification and identification of interacting proteins4. Although affinity proteomics utilizes a wide range of techniques for protein complex elucidation5, our approach is beneficial for expediting the identification of these complexes and their native interaction networks and is amenable to high-throughput protein tagging that is necessary to investigate complex biological pathways that contain a multitude of protein constituents. Key to this approach are advancements in cloning strategies that enable high fidelity and expedited cloning of target genes into an array of vectors for gene expression in vitro, in various organisms like bacteria and baculovirus, and in mammalian cells6,7. Additionally, the ORFeome collaboration has cloned thousands of sequence validated open reading frames in vectors that incorporate these advances in cloning, which are available to the scientific community8-11. In our system, the pGLAP1 LAP-tagging vector enables the simultaneous cloning of a large number of clones, which facilitates high-throughput LAP-tagging. This expedited cloning procedure is coupled to a streamlined approach for generating cell lines with LAP-tagged genes of interest inserted at a single pre-determined genomic locus. This makes use of cell lines that contain a single flippase recognition target (FRT) site within their genome, which is the site of integration for LAP-tagged genes. These cell lines also express the tetracycline repressor (TetR) that binds to Tet operators (TetO2) upstream of the LAP-tagged genes and silences their expression in the absence of Dox/Tet. This allows for Dox/Tet inducible expression of the LAP-tagged protein at any given time. Having the capability of inducible LAP-tagged protein expression is critical, since many cellular pathways are sensitive to the levels of critical proteins governing the pathway and can arrest cell growth or trigger cell death when these proteins are constitutively overexpressed, even at low levels, making the generation of non-inducible LAP-tagged stable cell lines impossible12.

<table><tbody><tr><td>Flp-In T-REx Core Kit</td><td>Invitrogen</td><td>K6500-01</td><td>Kit for generating cell lines that contain an FRT site and TrtR expression</td></tr><tr><td>PETG, 5x</td><td>Nunc, Inc.&amp;nbsp;</td><td>73520-734</td><td>Roller bottle for growing cells</td></tr><tr><td>PETG, 2.5x</td><td>Nunc, Inc.&amp;nbsp;</td><td>73520-420</td><td>Roller bottle for growing cells</td></tr><tr><td>Cell stackers</td><td>Corning CellSTACK</td><td>3271</td><td>Cell stacker for growing cells</td></tr><tr><td>500 ml conical centrifuge tubes</td><td>Corning&amp;nbsp;</td><td>431123</td><td>Tubes for harvesting cells</td></tr><tr><td>Anti-GFP antibody</td><td>Invitrogen</td><td>A11122</td><td>Rabbit anti GFP antibody</td></tr><tr><td>Affiprep Protein A beads</td><td>Biorad</td><td>156-0006</td><td>Used as a matrix for conjugating anti-GFP antibodies</td></tr><tr><td>Dimethylpimelimidate (DMP)</td><td>ThermoFisher Scientific</td><td>21667</td><td>Used for conjugating anti-GFP antibodies to Protein A beads</td></tr><tr><td>TLA100.3 tubes</td><td>Beckman</td><td>349622</td><td>Tubes for centrifuging protein lysates during the clearing step</td></tr><tr><td>TEV protease</td><td>Invitrogen</td><td>12575-015</td><td>Used for cleaving the GFP tag off of N-terminal LAP-tagged proteins</td></tr><tr><td>Precession Protease</td><td>GE Healthcare</td><td>27-0843-01</td><td>Used for cleaving the GFP tag off of C-terminal LAP-tagged proteins</td></tr><tr><td>S-protein agarose</td><td>Novagen</td><td>69704</td><td>Used as a second affinity matrix during the purification of LAP-tagged protein complexes</td></tr><tr><td>QIAquick DNA gel extraction kit</td><td>Qiagen</td><td>28704/28706</td><td>For use in purifying PCR products from an agarose gel</td></tr><tr><td>BP clonase II</td><td>Invitrogen</td><td>11789020</td><td>Used for cloning ORF PCR products into the pDONR221 shuttle vector</td></tr><tr><td>LR clonase II</td><td>Invitrogen</td><td>11791020</td><td>Used for cloning the ORF of the gene of interest into the pGLAP1 LAP-tagging vector</td></tr><tr><td>&lt;em&gt;ccdB&lt;/em&gt; Survival 2 T1&lt;sup&gt;R&lt;/sup&gt; &lt;em&gt;E. coli&lt;/em&gt;</td><td>Invitrogen</td><td>A10460</td><td>Used for propgating shuttle vectors and pGLAP empty vectors</td></tr><tr><td>Fugene 6</td><td>Promega</td><td>E2691</td><td>Transfection reagent for transfecting vectors into human cells</td></tr><tr><td>Tetracycline</td><td>Invitrogen</td><td>Q100-19</td><td>Drug for inducing Dox/Tet inducible protein expression&amp;nbsp;</td></tr><tr><td>Doxycycline</td><td>Clontech</td><td>631311</td><td>Drug for inducing Dox/Tet inducible protein expression&amp;nbsp;</td></tr><tr><td>Hygromycin B</td><td>Invitrogen</td><td>10687010</td><td>Drug for selecting stable LAP-tagged integrants</td></tr><tr><td>Kanamycin</td><td>Corning</td><td>61-176-RG</td><td>Drug for selecting Kanamycin resistant bacterial colonies</td></tr><tr><td>Ampicillin</td><td>Fisher</td><td>BP1760-5</td><td>Drug for selecting Ampicillin resistant bacterial colonies</td></tr><tr><td>4-20% Tris Glycine SDS-PAGE gels</td><td>Biorad</td><td>4561094</td><td>Used for separating protein samples and final LAP-tag purification eluates</td></tr><tr><td>Silver Stain Plus Kit</td><td>Biorad</td><td>1610449</td><td>Used for silver staining the eluates of LAP-tagged pufications and samples collected throughout the purification process&amp;nbsp;</td></tr><tr><td>Coomassie Blue stain</td><td>Invitrogen</td><td>LC6060</td><td>Used for staining SDS-PAGE gels to visulize LAP-tagged purifications and cutting out protein bands, mass spectrometry compatible</td></tr><tr><td>Shuttle vector pDONR221</td><td>Invitrogen</td><td>12536017</td><td>Shuttle vector for cloning the ORFs of genes of interest</td></tr><tr><td>Flippase expressing vector pOG44</td><td>Invitrogen</td><td>V600520</td><td>Vector that expresses the Flippase recombinase for integrating LAP-tagged genes into the genome of FRT site containing cell lines</td></tr><tr><td>Platinum Taq DNA Polymerase</td><td>ThermoFisher Scientific</td><td>10966018</td><td>Used for PCR amplification of the ORFs of genes of interest</td></tr><tr><td>4x Laemmli sample buffer</td><td>Biorad</td><td>1610747</td><td>Sample buffer for eluting purified LAP-tagged protein complexes from the bead matrix</td></tr><tr><td>Luria broth (LB) media</td><td>Fisher</td><td>BP9723-2</td><td>Used for growing DH5&amp;alpha; bacteria</td></tr><tr><td>DNA miniprep kit</td><td>Promega</td><td>A1222</td><td>Used for making DNA plasmid minipreps</td></tr><tr><td>DMEM/F12 media</td><td>Hyclone</td><td>SH30023.01</td><td>For growing Hek293 human cells</td></tr><tr><td>FBS lacking Tet</td><td>Altanta Biologicals</td><td>S10350</td><td>Used for making -Tet DMEM/F12 media for generating and growing inducible LAP-tagged stable cell lines</td></tr><tr><td>Trypsin</td><td>Hyclone</td><td>SH30042.01</td><td>For lifting Hek293 cell foci from plates</td></tr><tr><td>Protease inhibitor tablets</td><td>Roche</td><td>11836170001</td><td>Used for making protocol buffers, EDTA-free</td></tr><tr><td>10% nonyl phenoxypolyethoxylethanol&amp;nbsp;</td><td>Roche</td><td>11332473001</td><td>Used for making protocol buffers</td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CM</td><td>Used for making protocol buffers</td></tr><tr><td>Tween-20</td><td>Fisher</td><td>BP337-500</td><td>Used for making protocol buffers</td></tr><tr><td>Sodium Borate</td><td>Fisher</td><td>S249-500</td><td>Used for making protocol buffers</td></tr><tr><td>Boric Acid</td><td>Fisher</td><td>A78-500</td><td>Used for making protocol buffers</td></tr><tr><td>Ethanolamine</td><td>Calbiochem</td><td>34115</td><td>Used for making protocol buffers</td></tr><tr><td>NaCl</td><td>Fisher</td><td>P217-3</td><td>Used for making protocol buffers</td></tr><tr><td>KCl</td><td>Fisher</td><td>BP358-10</td><td>Used for making protocol buffers</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Fisher</td><td>BP172-25</td><td>Used for making protocol buffers</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>M33-500</td><td>Used for making protocol buffers</td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP152-5</td><td>Used for making protocol buffers</td></tr></tbody></table>

inducible lap-tagged stable cell lines for investigating protein function, spatiotemporal localization and protein interaction networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

To highlight the utility of this system, the open reading frame (ORF) of the Tau microtubule binding protein was cloned into the shuttle vector by amplifying the Tau ORF with primers containing attB1 and attB2 sites (Table 1) and incubating the PCR products with the shuttle vector and a recombinase that mediates the insertion of the PCR products into the shuttle vector. The reaction products were used to transform DH5&#945; bacteria13 and plasmid DNA from Kanamycin resistant colonies was sequenced to ensure Tau insertion. A sequence validated shuttle-Tau vector was then used to transfer the Tau ORF into the pGLAP1 vector, which fused Tau in frame with the LAP (EGFP-TEV-S-Protein) tag, by incubating the shuttle-Tau vector with the pGLAP1 vector and the recombinase that mediates the transfer of the ORF from the shuttle vector to pGLAP1. The reaction products were used to transform DH5&#945; bacteria13 and plasmid DNA from Ampicillin resistant colonies was sequenced to ensure that the LAP-Tau fusion was in frame. Sequence validated pGLAP1-LAP-Tau was then co-transfected with a vector that expresses the flippase recombination enzyme into HEK293 cells that contained a single flippase recognition target (FRT) site within their genome, which is the site of integration for LAP-tagged genes14. This cell line also expressed the TetR that binds to Tet operators upstream of the LAP-tagged genes and silences their expression in the absence of Tet/Dox. Stable integrants were selected with -Tet DMEM/F12 media with 100 &#181;g/ml Hygromycin for 5 days. Individual Hygromycin resistant cell foci were harvested by adding 20 &#181;l of trypsin on top and pipetting up and down 2 times. Cells were placed in a 24 well plate and expanded by continual growth in -Tet DMEM/F12 media.
To verify that the Hygromycin resistant cells were capable of expressing LAP-Tau, HEK293 LAP-Tau cells were induced with 0.1 &#181;g/ml Dox for 15 hr and protein extracts were prepared from non-induced and Dox-induced cells. These extracts were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted for GFP and Tubulin as loading control. As seen in Figure 4A, LAP-Tau (visualized with anti-GFP antibodies) was only expressed in the presence of Dox. To validate that LAP-Tau was properly localized to the mitotic microtubule spindle during mitosis, as had been previously shown for endogenous Tau18, HEK293 LAP-Tau cells were induced with 0.1 &#181;g/ml Dox for 15 hr and cells were fixed with 4% paraformaldehyde and co-stained for DNA (Hoechst 33342) and microtubules (anti-Tubulin antibodies). Consistently, LAP-Tau was localized to the mitotic spindle during metaphase of mitosis (Figure 4B). To verify that LAP-Tau and its interacting proteins could be purified with this system, HEK293 LAP-Tau cells were grown in roller bottles to ~70% confluency, induced with 0.1 &#181;g/ml Dox for 15 hr, harvested by agitation, lysed with LAP300 buffer, and LAP-Tau was purified using the above protocol. Eluates from the LAP-Tau purification were resolved by SDS-PAGE and the gel was silver stained. Figure 4C shows the LAP-Tau purification, marked with an asterisk is LAP-Tau and several other bands indicative of Tau interacting proteins can be seen.
<img alt="Figure 1" src="/files/ftp_upload/54870/54870fig1.jpg" /> 
Figure 1: Overview of the Generation of LAP-tagged Inducible Stable Cell Lines for any Protein of Interest. The open reading frame (ORF) of genes of interest are amplified with attB1 and attB2 sites flanking the 5&#39; and 3&#39; end sequences, respectively (primer sequences are given in Table 1) and cloned into the shuttle vector. Sequence verified shuttle vectors with the gene of interest are then used to transfer the gene of interest into the pGLAP1 vector. The sequence verified pGLAP1 vector with the gene of interest is then co-transfected with the vector containing the flippase recombinase into the desired cell line that contains a single flippase recognition target (FRT) site within their genome, which is the site of integration for LAP-tagged genes. These cell lines also express the Tet repressor (TetR) that binds to Tet operators (TetO2) upstream of the LAP-tagged genes and silences their expression in the absence of Tet/Dox. The LAP-tagged gene of interest is then recombined into the FRT site and stable integrants are selected with Hygromycin. <a href="http://cloudflare.jove.com/files/ftp_upload/54870/54870fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54870/54870fig2.jpg" /> 
Figure 2: Overview of the Applications for LAP-tagged Stable Cell Lines. LAP-tagged inducible stable cell lines are induced to express the LAP-tagged protein of interest by Dox addition and can be synchronized at various stages of the cell cycle or can be stimulated with chemicals or ligands to activate any desired signaling pathway. The subcellular localization of the LAP-tagged protein of interest can be analyzed by live cell or fixed cell imaging. LAP-tagged proteins can also be tandem affinity purified and their interacting proteins can be identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Finally, Cytoscape can be used to generate a protein-protein interaction network of the bait protein. Dox indicates Doxycycline, IP indicates immunoprecipitate, EGFP indicates enhanced green fluorescent protein, Tev indicates TEV protease cleavage site, and S indicates S-tag. <a href="http://cloudflare.jove.com/files/ftp_upload/54870/54870fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54870/54870fig3.jpg" /> 
Figure 3: Overview of LAP-tagged Protein Expression, Purification and Preparation for Mass Spectrometry. The protocol has 9 steps: 1) growth and induction of LAP-tagged protein expression, 2) cell harvesting and lysis, 3) the preparation of lysates, 4) the binding of lysates to anti-GFP beads, 5) TEV protease cleavage of the GFP-tag, 6) the binding of lysates to S-protein beads, 7) the elution of the bait protein and interacting proteins, and 8-9) the preparation of samples for mass spectrometry-based proteomic analyses. <a href="http://cloudflare.jove.com/files/ftp_upload/54870/54870fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54870/54870fig4.jpg" /> 
Figure 4: Verification of LAP-Tau expression. (A) Western blot (WB) analysis of protein samples from non-induced and Dox induced LAP-Tau HEK293 cells probed with anti-GFP and anti-Tubulin antibodies to detect the LAP-tagged Tau protein and the Tubulin loading control, respectively. Note that LAP-Tau is only expressed when the cells are induced with Dox. (B) Mitotic cells expressing LAP-Tau were fixed and co-stained for DNA (Hoechst 33342) and Tubulin (Tub) with anti-tubulin antibodies and the subcellular localization of LAP-tau was analyzed by fluorescence microcopy. Note that LAP-Tau localizes to the mitotic spindle and spindle poles during mitosis. (C) Silver stained gel of the LAP-Tau purification. MW indicates molecular weight, CL indicates cleared lysates, and E indicates final eluates. Samples were run on a 4-20% SDS-PAGE and the gel was silver stained to visualize the purified proteins. Note that a band corresponding to LAP-Tau is marked with an asterisk and several other bands corresponding to co-purifying proteins can be seen. <a href="http://cloudflare.jove.com/files/ftp_upload/54870/54870fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>N-terminal fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Forward</td>
			<td>5&#39;-GGGGACAAGT TTGTACAAAAAAGCAGGCTTCATG-(&#62;18gsn)-3&#8217;</td>
		</tr>
		<tr>
			<td>Reverse</td>
			<td>5&#39;-GGGGACCACTTTGTACAAGAAAGCTGGGTT TTATCA-(&#62;18gsn)-3&#8217;</td>
		</tr>
		<tr>
			<td>C-terminal fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Forward</td>
			<td>5&#39;-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACC-(&#62;18gsn)-3&#8217;</td>
		</tr>
		<tr>
			<td>Reverse</td>
			<td>5&#39;-GGGGACCACTTTGTACAAGAAAGCTGGGTG-(&#62;18gsn)-3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 1: Forward and Reverse Primers for Amplifying ORFs or Interest for Insertion into the Shuttle Vector. The attB sites are highlighted in bold letters, gsn denotes that more than 18 gene specific nucleotides are added to the primer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Step</td>
			<td>Temperature</td>
			<td>Time</td>
		</tr>
		<tr>
			<td colspan="2">Initial denaturation</td>
			<td>94 &#176;C</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td rowspan="3">PCR Amplification Cycles (35)</td>
			<td>Denature</td>
			<td>94 &#176;C</td>
			<td>30 sec</td>
		</tr>
		<tr>
			<td>Anneal</td>
			<td>55 &#176;C (depending on the primer Tm)</td>
			<td>30 sec</td>
		</tr>
		<tr>
			<td>Extend</td>
			<td>72 &#176;C</td>
			<td>1 min/kb</td>
		</tr>
		<tr>
			<td colspan="2">Hold</td>
			<td>4 &#176;C</td>
			<td>indefinitely</td>
		</tr>
	</tbody>
</table>
Table 2: PCR Conditions for Amplification of the ORFs of Interest.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vector</td>
			<td>Forward Sequencing Primer</td>
			<td>Reverse Sequencing Primer</td>
		</tr>
		<tr>
			<td>Shuttle Vector</td>
			<td>5&#8217;-TGTAAAACGACGGCCAGT-3&#8217;</td>
			<td>5&#8217;-CAGGAAACAGCTATGAC-3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 3: Forward and Reverse Sequencing Primers for the Shuttle Vector.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ID</td>
			<td>Structure</td>
			<td>Parental&#160;</td>
			<td>Promoter</td>
			<td>Bac Res</td>
			<td>Mam Res</td>
			<td>Tet reg?</td>
		</tr>
		<tr>
			<td>pGLAP1</td>
			<td>N-term EGFP-TEV-S peptide</td>
			<td>pcDNA5/FRT/TO</td>
			<td>CMV</td>
			<td>Amp</td>
			<td>Hyg</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>pGLAP2</td>
			<td>N-term Flag-TEV-S peptide</td>
			<td>pcDNA5/FRT/TO</td>
			<td>CMV</td>
			<td>Amp</td>
			<td>Hyg</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>pGLAP3</td>
			<td>N-term EGFP-TEV-S peptide; C-term V5</td>
			<td>pEF5/FRT-V5</td>
			<td>EF1a</td>
			<td>Amp</td>
			<td>Hyg</td>
			<td>No</td>
		</tr>
		<tr>
			<td>pGLAP4</td>
			<td>N-term Flag-TEV-S peptide; ; C-term V5</td>
			<td>pEF5/FRT-V5</td>
			<td>EF1a</td>
			<td>Amp</td>
			<td>Hyg</td>
			<td>No</td>
		</tr>
		<tr>
			<td>pGLAP5</td>
			<td>C-term S peptide-PreProt x2-EGFP</td>
			<td>pEF5/FRT-V5</td>
			<td>EF1a</td>
			<td>Amp</td>
			<td>Hyg</td>
			<td>No</td>
		</tr>
	</tbody>
</table>
Table 4: List of Available LAP/TAP Vectors with Variable Promoters, Epitope-tags, and Dox Inducible Expression Capabilities for N or C-terminal Protein Tagging. Vectors are commercially available. Bac Res indicates bacterial resistance marker, Mam Res indicates mammalian cell resistance marker, Tet reg? indicates whether expression is Tet/Dox regulatable.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vector</td>
			<td>Forward Sequencing Primer</td>
			<td>Reverse Sequencing Primer</td>
		</tr>
		<tr>
			<td>pGLAP1</td>
			<td>5&#8217;-ATCACTCTCGGCATGGACGAGCTGTACAAG-3&#8217;</td>
			<td>5&#8217;-TGGCTGGCAACTAGAAGGCACAGTCGAGGC-3&#8217;</td>
		</tr>
		<tr>
			<td>pGLAP2</td>
			<td>5&#8217;-CGAACGCCAGCACATGGACAGGG-3&#8217;</td>
			<td>5&#8217;-TGGCTGGCAACTAGAAGGCACAGTCGAGGC-3&#8217;</td>
		</tr>
		<tr>
			<td>pGLAP3</td>
			<td>5&#8217;-AGAAACCGCTGCTGCTAA-3&#8217;</td>
			<td>5&#8217;-TAGAAGGCACAGTCGAGG-3&#8217;</td>
		</tr>
		<tr>
			<td>pGLAP4</td>
			<td>5&#8217;-AGACCCAAGCTGGCTAGGTAAGC-3&#8217;</td>
			<td>5&#8217;-TAGAAGGCACAGTCGAGG-3&#8217;</td>
		</tr>
		<tr>
			<td>pGLAP5</td>
			<td>5&#8217;-CGTAATACGACTCACTATAG-3&#8217;</td>
			<td>5&#8217;-TCCAGGGTCAAGGAAGGCACGG-3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 5: Forward and Reverse Sequencing Primers for pGLAP Vectors.

Watch this Scientific Journal Video about Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks at JoVE.com

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...

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JoVE Journal

Biology

9.8K Views.  University of California, Los Angeles. We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks. 

Video: Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Biochemistry

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Genetics

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

Developmental Biology

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Immunology and Infection

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

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