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

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

1. Cloning the ORF of the Gene of Interest into the Shuttle Vector.
   1. 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.
   2. 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's instructions.
   3. 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'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 DH5α E. coli cells when propagating vectors with cloned ORFs.
   4. Transform DH5α E. coli cells with 1 μl of the reaction product and plate the transformed cells onto a Luria broth (LB) agar plate with 50 mg/ml Kanamycin¹³.
   5. Select the Kanamycin resistant colonies.
   6. 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.
2. Transferring the Gene of Interest from the Shuttle Vector into the LAP-tag Vector.
   1. 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'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.
   2. Transform DH5α E. coli cells with 1 μl of the reaction product and plate the transformed cells onto an LB agar plate with 100 mg/ml Ampicillin¹³.
   3. Select the Ampicillin resistant colonies.
   4. 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.

2. Generation of an Inducible Stable Cell Line that Expresses the LAP-tagged Gene of Interest

1. 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.
2. 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 µg/ml and 800 µg/ml.
   NOTE: HEK293 cells grown in -Tet DMEM/F12 media with 100 µg/ml Hygromycin at 37 °C and 5% CO₂ will die within 1-2 weeks.
3. 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'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.
4. One day post-transfection, replace the -Tet DMEM/F12 media with fresh media.
5. 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 µg/ml Hygromycin.
   NOTE: The FRT containing HEK293 cell line also contains the TetR that is associated with Blasticidin resistance, therefore 5 µg/ml Blasticidin is used during the stable cell line selection process to select for the TetR and to minimize leaky expression.
6. Replace Hygromycin-containing -Tet DMEM/F12 media as needed until distinct cell foci appear that resemble opaque spots against the transparent plate.
7. Add 20 μl of trypsin on top of each cell foci and pipet up and down 2 times with a 200 μl pipet tip. Place cells in a 24 well plate and expand the cells by continual growth in Hygromycin-containing -Tet DMEM/F12 media.
8. 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.

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.

1. Cell Growth and Cell Harvesting.
   1. 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 °C and 5% CO₂.
   2. For Tet/Dox inducible cell lines, induce for 10-15 hr at a concentration of 0.2 µg/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 µg/ml Tet/Dox for 10-15 hr is recommended.
   3. Harvest cells by agitation or trypsinization and pellet cells at 875 x g for 5-10 min.
2. Coupling Anti-GFP Antibody to Protein A Beads
   1. Use 40 µg 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 µg is recommended.
   2. Equilibrate 160 µl 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.
   3. Resuspend beads in 500 µl PBST and add 80 µg of affinity-purified rabbit anti-GFP antibody to each tube of 160 µl beads. Mix for 1 hr at room temperature (RT).
   4. 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 µl of the 0.2 M sodium borate, pH 9 to bring the final volume to 1 ml.
   5. Add 100 µl 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 µl of 0.2 M sodium borate, pH 9 and add immediately to the bead suspension.
   6. 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 µl of 0.2 M ethanolamine, 0.2 M NaCl pH 8.5. Beads are stable for several months at 4 °C.
3. Preparation of Buffers for Cell Lysis and Complex Purification
   1. 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.
   2. Prepare LAPX^N 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.
4. Preparation of Cell Lysates
   1. Resuspend 500 µl of PCV into 2.5 ml of LAP300 with 0.5 mM dithiothreitol (DTT) and protease inhibitors. Add 90 µl of 10% nonyl phenoxypolyethoxylethanol (0.3% final) and mix by inversion.
   2. Place on ice for 10 min. Centrifuge at 21,000 x g for 10 min. Collect this low speed supernatant (LSS). Take a 10 µl sample of the LSS for gel analysis.
   3. Transfer LSS to a TLA100.3 tube and spin at 100,000 x g for 1 hr at 4 °C. Collect this high speed supernatant (HSS), in a tube and place on ice. Take a 10 µl 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.
5. First Affinity Capture: Binding to Anti-GFP Beads
   1. Pre-elute antibody coupled beads (use 160 µl 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.
   2. Wash beads 3 times with 1 ml of LAP200^N. Mix HSS extract with antibody beads for 1 hr at 4 °C. Centrifuge at 21,000 x g for 10 min. Take a 10 µl sample of the supernatant (i.e., the flow through (FT)) for gel analysis.
   3. Wash beads 3 times with 1 ml of LAP200^N with 0.5 mM DTT and protease inhibitors. Wash beads 2 times (5 min each) with 1 ml of LAP200^N with 0.5 mM DTT and protease inhibitors. Wash quickly 2 times with 1 ml of LAP200^N with 0.5 mM DTT and no protease inhibitors before adding the tobacco etch virus (TEV) protease.
6. TEV Cleavage
   1. Dilute 10 µg TEV protease in 1 ml of LAP200^N and rotate tubes at 4 °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.
   2. Pellet beads and transfer supernatant to a fresh tube. Rinse beads twice with 160 µl LAP200^N with 0.5 mM DTT and protease inhibitors (triple concentration) to remove any residual protein. Take a 10 µl sample of the supernatant for gel analysis.
7. Second Affinity Capture: Binding to S Protein Agarose
   1. Wash 1 tube of 80 µl S protein agarose slurry (40 µl packed resin) 3 times with 1 ml of LAP200^N.
      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.
   2. Add TEV eluted supernatant to S protein agarose beads and rock for 3 hr at 4 °C. Pellet beads and wash 3 times with 1 ml of LAP200^N with 0.5 mM DTT and protease inhibitors. Wash beads 2 times with 1 ml of LAP100.
8. Protein Elution
   1. Elute proteins off S protein agarose by adding 50 µl of 4x Laemmli sample buffer and heat at 97 °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).

4. Identify Interacting Proteins by Mass Spectrometry Analysis

1. 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.
2. 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.