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CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii
可用于刚地弓形虫中邻位标记的CRISPR介导的BirA标记   

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PLOS Pathogens
May 2017

Abstract

Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this method provides a robust approach to defining protein networks. This approach detects interaction within intact cells, it is applicable to both soluble and insoluble components, including large proteins complexes that interact with the cytoskeleton and unique microtubule organizing center that comprises the apical complex in apicomplexan parasites.

Keywords: Mass spectrometry (质谱法), Protein interaction network (蛋白质相互作用网络), Proximity labeling (邻位标记), CRISPR (CRISPR), BioID (BioID ), Proximity ligation (邻位连接)

Background

Analysis of protein-protein interactions is a key endeavor in addressing how proteins assemble and function as macromolecular complexes. Traditionally, protein complexes have been identified through co-immunoprecipitation (co-IP) with subsequent mass spectrometry analysis. However, some protein complex substituents can be artificially lost or gained during the lysis, pull-down, and washing steps of co-IP, which is especially problematic for insoluble membrane or structural proteins that require aggressive solubilization. As an alternative to co-IP, proximity-dependent biotin identification (BioID) provides a ‘snapshot’ of proteins in close proximity to a target protein of interest during normal cellular homeostasis (Roux et al., 2012). BioID utilizes a promiscuous Escherichia coli biotin protein ligase (BirA) fused to a target protein of interest. Biotin supplementation licenses the BirA fusion to biotinylate near-neighbors within 30 nm (Roux et al., 2012; Van Itallie et al., 2013), with a static labeling radius of ≤ 10 nm (Kim et al., 2014). Biotinylated proteins may be captured by affinity chromatography and identified by mass spectrometry (Roux et al., 2012).

Toxoplasma gondii belongs to the phylum Apicomplexa composed of thousands of obligate parasites. Due to ease of in vitro cultivation and genetic manipulation, T. gondii is considered a model organism for studying the biology of apicomplexans. Recently, Chen et al. (2015) adapted BioID for use in T. gondii, identifying several novel protein components of the inner membrane complex (IMC). BioID has since been employed in T. gondii research to identify interactors of kinases (Gaji et al., 2015), calmodulins (Long et al., 2017a), and to define the protein repertoire of other cellular compartments including the parasitophorous vacuole (Nadipuram et al., 2016), sutures of the IMC (Chen et al., 2017), and the apical complex (Long et al., 2017b). Here we will describe the protocol for generating a BirA gene fusions using CRISPR/Cas9 tagging (Shen et al., 2014; Shen et al., 2017), in vivo BirA biotin labeling and purification of biotinylated proteins from parasites, and identification of captured biotinylated proteins by mass-spectrometry. Since analysis of mass spectrometry datasets can be complicated by non-specific hits, we provide a method to filter out false-positive interactions and rank true-positives using Straightforward Filtering IndeX program (SFINX) (http://sfinx.ugent.be/) (Titeca et al., 2016). Candidate interactors that emerge from BioID/SFINX analysis should also be validated by secondary analyses. Therefore we also provide instructions for demonstrating co-localization by a complementary proximity ligation assay.

Materials and Reagents

  1. Pipette tips (Corning, catalog numbers: 4713 , 4712 )
  2. 1.7 ml Eppendorf tube (Corning, Costar®, catalog number: 3620 )
  3. T25, T175 flasks (Corning, catalog numbers: 430639 , 431080 )
  4. Syringes 10cc, 20cc (BD, catalog numbers: 302995 , 302830 )
  5. 22 G blunt needle (CML Supply, catalog number: 901-22-100M )
  6. D3 polycarbonate membrane (GE Healthcare, catalog number: 110612 )
  7. 4 mm electroporation cuvette (Harvard Apparatus, catalog number: 450126 )
  8. 24- and 96-well plates (MIDSCI, catalog numbers: TP92024 , TP92696 )
  9. Coverslips (Fisher Scientific, catalog number: 12-545-80 )
  10. 2 ml Eppendorf tubes (Fisher Scientific, catalog number: 054-08-138 )
  11. 1 L Stericup Filter Units (Merck, Millipore Sigma, catalog number: SCVPU11RE )
  12. Cryovials (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5000-0050 )
  13. 5 ml, 10 ml, 25 ml pipettes (Fisher Scientific, catalog numbers: 13-676-10H , 13-676-10J , 13-676-10K )
  14. C18 CSH column (WATERS, catalog number: 186005295 )
  15. T. gondii RH∆ku80hxgprt strain (a gift from Dr. Vernon Carruthers, University of Michigan Medical School, Ann Arbor)
  16. Q5 Site-mutagenesis Kit with E. coli competent cells (New England Biolabs, catalog number: E0554S )
  17. Plasmids available at https://www.addgene.org/
    1. pSAG1::CAS9-U6::sgUPRT (Addgene, catalog number: 54467 )
    2. pLinker-BirA-3HA-HXGPRT-LoxP (Addgene, catalog number: 86668 )
    3. pLinker-6HA-HXGPRT-LoxP (Addgene, catalog number: 86552 )
    4. pLinker-2Ty-HXGPRT-LoxP (Addgene, catalog number: 86664 )
  18. LB broth (BD, catalog number: 244610 )
  19. Ampicillin (Sigma-Aldrich, catalog number: A0166 )
  20. Plasmid Extraction Kit (Macherey-Nagel, catalog number: 740588.250 )
  21. M13 reverse universal primer
  22. Q5 DNA polymerase (New England Biolabs, catalog number: M0491S )
  23. PCR Cleanup Kit (Macherey-Nagel, catalog number: 740609.250 )
  24. Trypsin for tissue culture (Sigma-Aldrich, catalog number: T3924 )
  25. Mycophenolic acid (Sigma-Aldrich, catalog number: M3536 )
  26. Xanthine (Sigma-Aldrich, catalog number: X4002 )
  27. Formaldehyde 10% ultrapure EM grade (Polysciences, catalog number: 04018-1 )
  28. Mouse anti-HA antibodies (BioLegend, catalog number: 901501 )
  29. Rabbit anti-GAP45 (a gift from Dr. Dominique Soldati-Favre, University of Geneva Medical School, Geneva, Switzerland)
  30. Goat anti-Mouse IgG (H+L) Secondary Antibody Conjugated with Alexa Fluor-488 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11001 )
  31. IRDye 680CW Goat anti-Mouse IgG (H+L) (LI-COR, catalog number: 926-68070 )
  32. Streptavidin Alexa Fluor-488 conjugate (Thermo Fisher Scientific, catalog number: S32354 )
  33. IRDye 800CW streptavidin (LI-COR, catalog number: 925-32230 )
  34. IRDye 680CW Goat anti-rabbit IgG (H+L) (LI-COR, catalog number: 926-68071 )
  35. Goat anti-Rabbit IgG (H+L) Secondary Antibody Conjugated with Alexa Fluor-594 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11037 )
  36. Liquid nitrogen
  37. FBS (GE Healthcare, catalog number: SH30071.03HI )
  38. 20% DMSO
  39. Streptavidin magnetic beads (Thermo Fisher Scientific, PierceTM, catalog number: 88816 )
  40. Ammonium bicarbonate (Sigma-Aldrich, catalog number: 11213 )
  41. DTT
  42. Iodoacetamide (IAM) (Sigma-Aldrich, catalog number: I1149 )
  43. DUOlink In Situ Red Starter Mouse/Rabbit (Sigma-Aldrich, catalog number: DUO92101 )
  44. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 12800017 )
  45. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
  46. HEPES (Sigma-Aldrich, catalog number: H3375 )
  47. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
  48. Gentamicin (Sigma-Aldrich, catalog number: G1272 )
  49. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: 1551128 )
  50. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 1551139 )
  51. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  52. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 793639 )
  53. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  54. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  55. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  56. D-biotin (Sigma-Aldrich, catalog number: 47868 )
    Note: This product has been discontinued.
  57. Tris (Sigma-Aldrich, catalog number: T1503 )
  58. EGTA (Sigma-Aldrich, catalog number: E3889 )
  59. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  60. NP-40 (Sigma-Aldrich, catalog number: I8896 )
  61. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  62. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  63. Deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  64. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  65. DOC, deoxycholate (Sigma-Aldrich, catalog number: 30970 )
  66. Lithium chloride (LiCl) (Sigma-Aldrich, catalog number: 62476 )
  67. D10 medium (see Recipes)
  68. Cytomix buffer (see Recipes)
  69. Phosphate-buffered saline (PBS) (see Recipes)
  70. D-biotin stock (see Recipes)
  71. Cytoskeleton buffer (see Recipes)
  72. 5x SDS sample buffer (see Recipes)
  73. Buffer 1 (see Recipes)
  74. Buffer 2 (see Recipes)
  75. Buffer 3 (see Recipes)
  76. Buffer 4 (see Recipes)

Equipment

  1. Eppendorf centrifuge (Eppendorf, model: 5810 R )
  2. Incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: Model 370 )
  3. Class II biological safety hood (Baker Co., model: SterilGARD® II, ClassII type A/B3 )
  4. BTX ECM-830 electroporator (Harvard Apparatus, model: ECM 830 )
  5. Hemocytometer (Hausser Scientific, catalog number: 3120 )
  6. Inverted phase contrast microscope (Nikon Instruments, model: Eclipse TS100 )
  7. 200 µl pipette
  8. Sonic Dismembrator 550 (Fisher Scientific, model: Model 550 )
  9. Magnetic stand (Thermo Fisher Scientific, catalog number: 12321D )
  10. Odyssey imaging system (LI-COR, model: Odyssey® CLx )
  11. Liquid nitrogen tank (Airgas, model: NI230LT22 )
  12. Q-exactive HF mass spectrometer (Thermo Fisher Scientific, Thermo ScientificTM, model: Q ExactiveTM HF )

Software

  1. sgRNA selection website: http://grna.ctegd.uga.edu/, Mascot, version 2.5.1 (Matrix Science)
  2. SFINX analysis website: http://sfinx.ugent.be/, Scaffold version 4.6.1 (Proteome Software Inc.)

Procedure

  1. Designing a Cas9/sgRNA plasmid targeting a specific gene for C-terminal tagging with BirA
    Note: The protocol described here is adapted from Shen et al., 2017. We recommend reading this review for a background of CRISPR genome editing in T. gondii before attempting this protocol.
    1. sgRNA selection
      1. In http://toxodb.org/toxo/, navigate to your gene of interest (GOI) webpage. This may be accomplished using a gene search based on expression or functional properties or simply by entering a known gene ID or keyword. Be careful to choose the correct T. gondii strain for your work. Download the genomic sequence from 500 bp upstream of the start codon to 500 bp downstream of the stop codon.
      2. Copy the first 200 bp following the stop codon and paste at http://grna.ctegd.uga.edu/. Enter a job name, select the T. gondii lineage database, and keep all other default settings.
      3. From the generated list of sgRNA sequences, select a 20 nt sgRNA sequence that has a 3’ NGG protospacer adjacent motif (PAM) motif. The sgRNA sequence can be either on the forward or reverse DNA strand.
    2. Generation of a Cas9/sgRNA plasmid containing the gene specific sgRNA
      1. Order DNA oligos for PCR mutagenesis of pSAG1::CAS9-U6::sgUPRT (Addgene #54467). The reverse primer (5’-AACTTGACATCCCCATTTAC-3’) is universal for all mutagenesis reactions with this Cas9/sgRNA plasmid. The forward primer (5’-[20 nt sgRNA sequence in 5’ to 3’ direction]–GTTTTAGAGCTAGAAATAGC-3’) will be unique for every sgRNA. For example, if the desired protospacer + PAM site is 5’-GCTGGTCTATCGCTAGCTCGAGG, the forward primer would be 5’-GCTGGTCTATCGCTAGCTCGGTTTTAGAGCTAGAAATAGC. Specific examples for individual genes can be found in these recent papers (Brown et al., 2017; Long et al., 2017a and 2017b).
      2. Use Q5 site-directed mutagenesis Kit (New England Biolabs) to mutate the sgRNA sequence of pSAG1::CAS9-U6::sgUPRT (Addgene #54467). We recommend reactions consisting of 6.25 µl 2x Q5 Hot-start enzyme mix (included in kit), 0.625 µl of each primer (working stock 10 µM), 1 µl (1 ng) pSAG1::CAS9-U6::sgUPRT, and 4 µl ddH2O. For thermal cycling, we recommend 25 cycles of 95 °C for 30 sec, 60 °C for 20 sec, and 72 °C for 5 min.
      3. Resolve 1 µl of the PCR reaction on a DNA gel to confirm the successful reaction with a DNA band at about 10 kb. Ligate 1 µl of the Q5 PCR reaction with the KLD mix (included in kit) according to the manufacturer’s instructions.
      4. Transform 2.5 µl of the KLD reaction into the supplied E. coli competent cells in the kit and grow on LB agar plates containing 100 µg/ml ampicillin 37 °C overnight.
      5. Grow three single colonies of E. coli overnight in 3 ml LB broth containing 100 µg/ml ampicillin at 37 °C. Pellet the bacterial cultures using centrifugation 3,000 x g for 10 min. Extract the plasmids from the bacterial pellets using a Plasmid Extraction Kit (Macherey-Nagel). Send the three plasmids for Sanger sequencing to confirm the successful insertion of sgRNA in the Cas9/sgRNA plasmid using M13 reverse universal primer. Select sequence-confirmed plasmid for further editing.

  2. Generation of a BirA tagging line using CRISPR/Cas9 tagging
    1. Generation of a gene-specific BirA tagging cassette
      1. Design forward and reverse primers for amplification of the BirA tagging cassette. The forward primer contains a 40 nt 5’ homology sitting right upstream of the stop codon (note that the stop codon was excluded), and follows with GCTAGCAAGGGCTCGGGC for anchoring at the linker in a tagging plasmid pLinker-BirA-HXGPRT-LoxP (Addgene #86668). The linker sits in front of the tag BirA and serves as a spacer between the tag and endogenous protein. It also works as an anchoring site for the forward primer. The reverse primer contains a 40 nt 3’ homology arm adjacent to the Cas9 cleavage site at the sgRNA sequence (3 bp upstream of the PAM site), and follows with ATAGGGCGAATTGGAGCTCC for anchoring at the end of the HXGPRT selection cassette in the tagging plasmid. Recommended scale of synthesis is 25 nmole DNA and oligos should be processed using a standard desalting procedure.
      2. Use the manufacturer’s protocol of Q5 DNA polymerase (New England Biolabs) to amplify a BirA cassette with the forward and reverse primers in a 90 µl PCR reaction (Figure 1). The tagging plasmid (1 ng in 90 µl of reaction) is used as a DNA template, and the annealing temperature is set at 60 °C for 2 min.
      3. Resolve 2 µl of the PCR reaction on a DNA gel to confirm the successful reaction to see if a PCR product of 3.5 kb is present.


        Figure 1. Diagram of CRISPR/Cas9 tagging strategy. The efficiency of Cas9 site-specific cleavage combined with integration using short regions of homology allows for rapid tagging of genes of interest. Gene-specific homology regions consisting of 42 bp flanking regions (denoted as HR1 (purple) and HR2 (blue)) are combined with conserved linker regions (L (red) and T (black)) to generate amplicons. The conserved linkers flank the central tagging construct containing a BirA tag (green), a stop codon (s), and a generic 3’ UTR (yellow). Primers HR1 and HR2 are used to amplify the gene-specific BirA amplicon from the tagging plasmid. The BirA tagging cassette is then cloned into a gene-specific pCas9/sgRNA plasmid and transfected into the RH∆ku80 line, followed by drug selection. Diagram was adapted from (Long et al., 2017a).

    2. Transfection into a T. gondii recipient line and screening clonal lines
      Note: Throughout this protocol, all manipulation of live host cells or parasites should be performed with aseptic technique in a BSL2 certified biosafety cabinet or hood.
      1. Purify the BirA cassette with a Gel Extraction and PCR Cleanup Kit (Macherey-Nagel), according to the manufacturer’s protocol.
      2. Elute the DNA cassette by adding 10-15 µl of the Cas9/sgRNA plasmid (10-20 µg of plasmid), and elute the column again with 5 µl ddH2O, to make a cassette-Cas9/sgRNA mixture.
      3. Heat the cassette-Cas9/sgRNA mixture at 75 °C for 10 min in a 1.5 ml Eppendorf tube, and centrifuge to collect liquid on the wall.
      4. Culture human foreskin fibroblasts (HFF) in T25 flasks with D10 medium (Recipe 1) in a 37 °C, 5% CO2 incubator until they reach confluency. Trypsinize HFF cells using standard cell culture techniques to passage the cells by splitting 1:4.
      5. Infect completely confluent HFF monolayer in T25 flasks with the T. gondii line to be tagged at the density of 2.5 x 106 parasites per T25 flask. Here, T. gondii RH∆ku80hxgprt is used. Grow the T. gondii line for ~2 days until natural egress from HFF monolayer has occurred. Fresh HFF flasks will be needed for following transfection.
      6. To harvest the parasites, scrape the monolayer to remove from the flask and collect 5 ml of culture medium containing parasites. Release any remaining intracellular T. gondii by passing through a 22 G needle with a 10 ml syringe, carefully remove the needle and safely discard into a sharps container, and filter the parasites by passing through 3.0 μm polycarbonate membranes to remove host cell debris. Centrifuge the parasites in the flow-through at 800 x g, 18 °C for 10 min, and wash the parasite pellet with 10 ml cytomix buffer (Soldati and Boothroyd, 1993; Recipe 2) once and centrifuge the resuspension again. The typical yield from a single T25 flask of HFF is ~5-8 x 107 parasites in total.
      7. Re-suspend the parasite harvested from one T25 in 2 ml cytomix buffer for transfection.
      8. Combine 200 µl of the parasite suspension (5-8 x 106 parasites), and the cassette-Cas9/sgRNA mixture (Figure 1) in a 4 mm electroporation cuvette (BTX), and perform the electroporation with the following protocol: 1,700 V, 176 μsec of pulse length, two pulses with 100 msec interval with a BTX ECM-830 electroporator.
      9. Immediate after the electroporation, transfer the parasite suspension into fresh T25 flasks with confluent HFF and incubate them at 37 °C, 5% CO2.
      10. Twenty-four hours after electroporation and recovery, begin drug selection with 25 µg/ml mycophenolic acid supplemented with 25 µg/ml xanthine. Passage parasites as needed in drug selection medium until a drug-resistant population emerges (~2-3 passages).
      11. Following natural egress, count parasites with a hemocytometer, and dilute parasites to 3 parasites/150 µl, and add this dilution of parasites in 150 µl D10 medium into 96-well plates with confluent HFF, and grow parasites at 37 °C, 5% CO2 for 6 days without movement.
      12. Visually looking for wells containing only one plaque in the 96-well plates under an inverted-phase contrast microscope. For wells containing only one T. gondii plaque, mix the parasites and medium in wells with 200 µl pipette. Keep growing the parasites for another 3 days until parasite natural egress. Protocols for plaque formation and expansion of T. gondii in microtiter well plates have also been thoroughly described previously (Roos et al., 1994).
      13. Pick 10 clones from wells and inoculate half of the culture in wells with HFF in a 96-well plate and another half in 24-well plates with coverslips and HFF monolayer. Let the parasites on coverslips grow for 24 h, and fixed with 4% formaldehyde in PBS (Recipe 3), and proceed with membrane permeabilization using 0.25% Triton X-100 in PBS containing 10% FBS, and followed with indirect fluorescence microscopy using primary antibodies against HA (mouse) and GAP45 (rabbit), and then secondary antibodies against mouse and rabbit conjugated with Alexa Fluor 488 and 594 respectively. Parasites are then imaged under a fluorescent microscope to look clones that are positive for HA staining (green channel).
      14. Diagnostic PCR for testing the insertion of BirA at the targeted locus is used to confirm the purity of clones using one primer sitting 200 bp upstream of the translational stop codon, and another primer sitting 300 bp downstream of the stop codon. Correctly tagged T. gondii clones do not produce the endogenous PCR product, which is only detected from the parental line RH∆ku80hxgprt.
      15. Western blot can also be performed to confirm the expression of endogenous BirA-3HA tagging line using commercial anti-HA antibodies and secondary antibodies used for detection. For Western blotting, we typically load 106 cell equivalents per lane and resolve by separation on 10% PAGE gels prior to transfer to nitrocellulose. Our preferred method of detection with LI-COR IR dye secondary antibodies; however, optimal loading conditions may vary depending on detection method. Protocols for detection of epitope tagged proteins in T. gondii have been detailed previously (Brown et al., 2017; Long et al., 2017a and 2017b).
      16. Once positive clones are confirmed, they should be transferred to T25 flasks and preserved in cryo-vials in liquid nitrogen storage. Five million parasites should be inoculated in a T25 flask and grown for 1 day. The infected HFF monolayers should be washed twice with warm PBS and trypsinized with 1 ml trypsin solution to detach the monolayers (diluted in an equal volume of PBS). The trypsinization should be stopped by adding 1 ml of 50% FBS in D10 medium followed by addition of 1 ml 20% DMSO in the D10 medium for preservation. Transfer the mixture into cryovials and put in an isopropanol container for freezing at -80 °C. Transfer the cryovials into a box in liquid nitrogen on the next day.

  3. Purification of biotinylated proteins for Mass-spectrometry
    1. Confirmation of BirA activity in vivo
      1. Grow RH∆ku80hxgprt line and the BirA tagging line in T25 flasks containing HFF monolayer (containing 5 ml D10 medium) for 24 h at 37 °C, 5% CO2 in incubator and add 50 µl of 16 mM D-biotin solution (Recipe 4) in DMEM to make a final concentration at 160 µM. Grow the parasites for another 20 h.
      2. Harvest the parasites by passing through 22 G needles and D3 polycarbonate membrane to remove HFF debris, and collect the flow-through and centrifuge at 800 x g, 18 °C for 10 min to pellet the parasites. Wash parasites with cold PBS to remove excess biotin, and centrifuge and resuspend the parasite pellets in 80 µl PBS, and add 20 µl SDS sample buffer. Boil the samples at 100 °C for 5-10 min.
      3. Resolve the parasite lysate using SDS-PAGE, and perform Western blot using Streptavidin-800CW (LI-COR) to detect biotinylated proteins in the parasite lines. The BirA lane should exhibit additional bands not found in the parental RH∆ku80hxgprt line, as shown in Figure 2A with CaM1-BirA and CaM2-BirA.
      4. RH∆ku80hxgprt line and the BirA line can also be grown on coverslips with HFF monolayer in 24-well plates with addition of 160 µM D-biotin for 24 h, and used for testing by indirect immune-fluorescence microscopy using streptavidin Alexa Fluor 488, to detect biotinylated proteins at the localization of BirA protein (Figure 2B).
    2. Purification of biotinylated proteins (Figure 2C)
      1. Inoculate the parental line RH∆ku80hxgprt line and the BirA tagging line in T175 flasks containing confluent HFF monolayer at the dosage of 2 x 107 parasites and grow for 24 h, and add D-biotin to the final concentration of 160 µM. Keep growing parasites in D-biotin for another 20 h until natural egress.
      2. Harvest parasites by passing through a 22 G needle and D3 polycarbonate membrane to remove debris, centrifuge and wash the parasite pellet with 50 ml cold PBS three times to remove excess D-biotin.
      3. Resuspend parasites in 2 ml cytoskeleton buffer (Recipe 5), and sonicate the resuspension with a microtip in Sonic Dismembrator 550 (Fisher Scientific) using a setting: 15 sec pulse, 15 sec interval time, and 2 min total pulse time.
      4. Incubate the parasite lysis on ice for 10 min, and transfer to 2 ml Eppendorf tubes, and centrifuge at 20,000 x g, 4 °C for 20 min. Transfer the supernatant to fresh tubes, and repeat the centrifugation to obtain clearer supernatant.
      5. Transfer the supernatant to 2 ml Eppendorf tubes containing 50 µl streptavidin magnetic beads (Pierce) that have been washed and balanced with cytoskeleton buffer for three times. Resuspend the supernatant and beads well and incubate at 4 °C overnight.
      6. Wash beads twice with 1.5 ml cytoskeleton buffer, and then wash once with 1.5 ml buffer 1 (Recipe 6), wash twice with 1.5 ml buffer 2 (Recipe 7), once with 1.5 ml buffer 3 (Recipe 8), twice with 1.5 ml buffer 4 (Recipe 9) and twice with 1.5 ml PBS. Each wash step needs to incubate for 5 min and the last two wash steps with buffer 4 and PBS should be performed to remove remaining SDS. The beads are pulled down by a magnetic stand, and supernatants are discarded.
      7. Transfer 10% of beads from the last wash step, and the beads are resuspended in 20 µl PBS and 5 µl 5x SDS sample buffer (Recipe 6) for SDS-PAGE and Western blot detection with streptavidin LI-COR C800. The remaining fraction of the beads (90%) can be stored temporarily at -80 °C.


        Figure 2. Strategy and examples of BirA tagging in T. gondii. A. Western blot detection of biotinylated proteins in the RH∆ku80hxgprt lines expressing CaM1-BirA or CaM2-BirA. IRDye 800CW streptavidin was used to detect biotinylated proteins. α-HA antibodies were used to detect BirA-3HA fusions, and α-aldolase was used as a loading control, and separately visualized by IRDye 680CW Goat anti-Mouse IgG and IRDye 680CW Goat anti-rabbit IgG. B. IFA detection of biotinylated proteins in the CaM1-BirA line. Streptavidin Alexa Fluor-488 was used to detect biotinylated proteins, and CaM1-BirA-3xHA detected by α-HA antibodies and visualized with anti-rabbit secondary antibodies conjugated with Alexa Fluor 599. Scale bar = 2 µm. C. Diagram of purification of biotinylated proteins from BirA-expressing T. gondii lines. Parasites were solubilized and biotinylated proteins were captured by streptavidin beads, as described in the Methods. This diagram is based on the original description in (Roux et al., 2012).

  4. Mass-spectrometry analysis of biotinylated proteins (performed by Dr. Michael Naldrett at the Proteomics & Metabolomics Facility, Center for Biotechnology, University of Nebraska, Lincoln (https://biotech.unl.edu/)
    1. Sample preparation
      1. Dissolve sample beads in 200 µl of 100 mM ammonium bicarbonate and reduce by adding 4 µl of 2 mM DTT for 1 h at 37 °C.
      2. Alkylate by adding 22 µl of 100 mM Iodoacetamide (IAM) for 20 min at 22 °C in the dark.
      3. Trypsin (5 µl of 0.1 mg/ml) is added per sample, and digestion is carried out overnight at 37 °C.
    2. LC-MS/MS
      Run 5 µl tryptic digest sample on a NanoLC-MS/MS using a 2 h gradient on a 0.075 x 250 mm C18 Waters CSH column feeding into a Q-exactive HF mass spectrometer.
    3. Spectral analysis
      1. Use Mascot to analyze results from the mass-spectrometry by searching the T. gondii ME49_20150114 (8322 sequences), cRAP_20150130 (117 sequences) and custom (containing a streptavidin sequence) databases assuming digestion by trypsin. Mascot is searched with a fragment ion mass tolerance of 0.060 Da and a parent ion tolerance of 10.0 PPM.
      2. Deamidated of asparagine and glutamine, oxidation of methionine, carbamidomethyl of cysteine and biotin of lysine and the N-terminus should be specified in Mascot as variable modifications.
      3. Use Scaffold version 4.6.1 to validate MS/MS based peptide and protein identifications. Peptide identifications are considered valid if they are supported by greater than 95.0% probability based on the Scaffold delta-mass correction (Keller et al., 2002). We generally accept protein identifications if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Lower cutoffs can also be used for exploratory work, but may lead to more false positive identifications.

  5. Validation of putative interactor proteins
    Note: This step is suggested as a follow-up to BioID experiments but will likely take several months to complete. Some BioID experiments may only require a survey of bait-BirA ‘neighbors’ without validating specific interactors.
    1. Localization of putative interactor proteins
      1. Interactors identified in the protein interactome may not have been previously localized. To verify the possible interactions at the right location, verifying their localization would be the first step.
      2. The candidate protein coding genes can be endogenously tagged by HA or Ty epitope tags using a CRISPR tagging strategy, as described above for the BirA tagging. The only difference is that the BirA tag is replaced by HA or Ty tags in the PCR template for generation of tagging cassette. Here we recommend the plasmids stored in Addgene pLinker-6HA-HXGPRT-LoxP (#86552) and pLinker-2Ty-HXGPRT-LoxP (#86664).
      3. Once the HA or Ty tagging strain is generated, the strains can be grown on HFF monolayer on coverslips in a 24-well plate for indirect immune-fluorescence microscopy.
      4. The interactors co-localized with the bait proteins will go on for bioinformatic analysis in ToxoDB, such as cellular compartment, molecular function.
      5. The stability and function of the protein complex can be determined by conditional depletion of individual substituents using an auxin inducible degron system (Long et al., 2017a; Brown et al., 2017). A detailed protocol for using the auxin-inducible degron system is also available at Bio-protocol (Brown et al., 2018).
    2. Interaction confirmation by another proximity approach–proximity ligation assay (PLA)
      1. The protein interaction can be further confirmed by tagging one gene with HA and another gene with Ty in the same line using the CRISPR/Cas9 tagging strategy described above. In this situation, the Ty tag plasmid is recommended to harbor another resistance marker DHFR not HXGPRT.
      2. The HA tag and Ty tag in one T. gondii line can be recognized by rabbit HA and mouse Ty antibodies, and followed with corresponding secondary antibodies conjugated with PLA probes. The PLA procedures can follow the manufacturer’s protocol with DUOlink In Situ Red Start Kit (Sigma-Aldrich). The PLA approach detects protein interactions within 30 nm distance, which is similar to the distance of BirA labeling (≤ 30 nm) (Soderberg et al., 2006).

Data analysis

Analysis of protein interactome
Straightforward filtering index (SFINX) (http://sfinx.ugent.be/) is a program that provides a statistically robust method to filter false positives and identify bona fide interactions using peptide counts from proteomic studies (Titeca et al., 2016). Here we combine the proximity labeling approach with this program to build a proximity based protein-protein interaction network.
SFINX analysis:

  1. Datasets from two or more independent experiments should be combined into one Scaffold file. Set the program at ‘total unique peptide count’ with protein threshold 95% and a minimal number of peptides 2.
  2. Export the datasets with ‘current view’, and edit the excel file to remove unnecessary columns and rows, leave the gene accession number, the independent mass-spectrometry experiments (projects)’s name and peptide counts. Type in row names in the first row of the first column, as the SFINX website suggests in the info column. Save this basic file as Comma Separated Values file (.csv), or other file formats as suggested by the software.
  3. Create another .csv file (bait file) with the accession number of the gene of interest for BirA tagging in the experiments.
  4. Upload the basic file and bait file to the website and pick the correct file format. A protein-protein network will immediately be created with a strictness bar shown on the left. The strictness bar is based on the SFINX scores (derived from P scores), which is used to rank true positives. In our case, strictness 1 was taken, corresponding to P < 0.00004.
  5. The filtered data can be downloaded as an excel .tsc file by clicking ‘complete output’ or ‘cutoff output’. The downloaded file lists the interactors (preys), and scores and P scores for the interaction between interactors and BirA proteins (baits).
  6. To produce high quality protein-protein interaction networks with SFINX3, multiple bait proteins (i.e., BirA fusions) involved in the same pathway or location should be analyzed by mass spectrometry.

Notes

  1. We have also successfully used the statistical features provided within Scaffold to define meaningful interactions among replicates datasets. When running comparisons from replicate experiments, we analyze data using Student’s t-test either with or without correction for multiple comparisons. Corrected tests are much more stringent and hence less likely to identify false positives. However, they also greatly limit the number of potential interacting proteins. Where discovery is the main objective, it may be a better strategy to accept a higher false positive rate and validate potential interactions using some secondary analysis.
  2. Extra caution should be used when working with the DHFR resistance cassette as this confers resistance to pyrimethamine, the most commonly used drug to treat toxoplasmosis. The plasmids have been designed with loxP sites flanking the DHFR resistance cassette, making it possible to remove this region by transient transfection of Cre, flowed by cloning and screening by PCR.
  3. For optimal performance of SFINX, it is important to have two or more biological replicates for each BirA-labeled protein. The method is quite robust to differences in labeling efficiency between replicates, however, it is helpful to have similar overall peptide coverage between experimental runs. In our experience, SFINX is quite conservative in predicting interactions that exceed a statistical threshold for being unlikely to be due to chance. The output file gives posterior probabilities that can be used to set a threshold for what the user is comfortable accepting. In any cases, these outputs are only predictive of proximity interactions and follow up analysis using some other methods is necessary to confirm interactions.

Recipes

  1. D10 medium
    1 packet of DMEM powder
    3.7 g sodium bicarbonate
    2.38 g HEPES
    10 ml of 200 mM L-glutamine
    1 ml of 10 mg/ml gentamicin
    Q.S. to 1 L with deionized H2O
    Filter sterilize with 1 L Stericup Filter and store at 4 °C
  2. Cytomix buffer
    8.66 mM K2HPO4
    1.36 KH2PO4
    120 mM KCl
    0.15 mM CaCl2
    5 mM MgCl2
    25 mM HEPES
    2 mM EDTA
    Adjust pH to 7.6
    Filter sterilize with Stericup Filter and store at 4 °C
  3. Phosphate-buffered saline (PBS)
    137 mM NaCl
    10 mM phosphate
    2.7 mM KCl
    Adjust pH to 7.4
    Autoclave and store at 4 °C
  4. D-biotin stock
    160 mM D-biotin in DMEM
  5. Cytoskeleton buffer
    10 mM Tris, pH 7.4
    100 mM NaCl
    1 mM EDTA
    1 mM EGTA
    1% Triton X-100
    1% NP-40
    10% glycerol
    0.2% SDS
    0.5% deoxycholate
  6. 5x SDS sample buffer
    Bromophenol blue 0.25%
    DTT 0.5 M
    Glycerol 50%
    SDS 10%
  7. Buffer 1
    20 mM Tris pH 7.5
    2% SDS
  8. Buffer 2
    0.1% DOC
    1% Triton X-100
    500 mM NaCl
    1 mM EDTA
    50 mM HEPES
    Adjust pH to pH 7.5
  9. Buffer 3
    250 mM LiCl
    0.5% NP-40
    0.5% DOC
    1 mM EDTA
    10 mM Tris pH 8.1
  10. Buffer 4
    50 mM Tris, pH 7.4
    50 mM NaCl

Acknowledgments

This work was supported by a grant from the NIH (AI034036) to L.D.S. and adapted from protocols described in Long et al. (2017a and 2017b). The authors declare that there are no conflicts or competing interests.

References


  1. Brown, K. M., Long, S. and Sibley, L. D. (2017). Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii. MBio 8(3).
  2. Brown, K. M., Long, S. and Sibley, L. D. (2018). Conditional knockdown of proteins using auxin-inducible degron (AID) fusions in Toxoplasma gondii. Bio-protocol 8(4): e2728.
  3. Chen, A. L., Kim, E. W., Toh, J. Y., Vashisht, A. A., Rashoff, A. Q., Van, C., Huang, A. S., Moon, A. S., Bell, H. N., Bentolila, L. A., Wohlschlegel, J. A. and Bradley, P. J. (2015). Novel components of the Toxoplasma inner membrane complex revealed by BioID. MBio 6(1): e02357-02314.
  4. Chen, A. L., Moon, A. S., Bell, H. N., Huang, A. S., Vashisht, A. A., Toh, J. Y., Lin, A. H., Nadipuram, S. M., Kim, E. W., Choi, C. P., Wohlschlegel, J. A. and Bradley, P. J. (2017). Novel insights into the composition and function of the Toxoplasma IMC sutures. Cell Microbiol 19(4).
  5. Gaji, R. Y., Johnson, D. E., Treeck, M., Wang, M., Hudmon, A. and Arrizabalaga, G. (2015). Phosphorylation of a myosin motor by TgCDPK3 facilitates rapid initiation of motility during Toxoplasma gondii egress. PLoS Pathog 11(11): e1005268.
  6. Keller, A., Nesvizhskii, A. I., Kolker, E. and Aebersold, R. (2002). Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74(20): 5383-5392.
  7. Kim, D. I., Birendra, K. C., Zhu, W., Motamedchaboki, K., Doye, V. and Roux, K. J. (2014). Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc Natl Acad Sci U S A 111(24): E2453-2461.
  8. Long, S., Brown, K. M., Drewry, L. L., Anthony, B., Phan, I. Q. H. and Sibley, L. D. (2017a). Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii. PLoS Pathog 13(5): e1006379.
  9. Long, S., Anthony, B., Drewry, L. L. and Sibley, L. D. (2017b). A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii. Nat Commun 8(1): 2236.
  10. Nadipuram, S. M., Kim, E. W., Vashisht, A. A., Lin, A. H., Bell, H. N., Coppens, I., Wohlschlegel, J. A. and Bradley, P. J. (2016). In vivo biotinylation of the Toxoplasma parasitophorous vacuole reveals novel dense granule proteins important for parasite growth and pathogenesis. MBio 7(4).
  11. Roos, D. S., Donald, R. G. K., Morrissette, N. S. and Moulton, A. L. (1994). Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell Biol 45: 28-61.
  12. Roux, K. J., Kim, D. I., Raida, M. and Burke, B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196(6): 801-810.
  13. Shen, B., Brown, K., Long, S. and Sibley, L. D. (2017). Development of CRISPR/Cas9 for efficient genome editing in Toxoplasma gondii. Methods Mol Biol 1498: 79-103.
  14. Shen, B., Brown, K. M., Lee, T. D. and Sibley, L. D. (2014). Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/Cas9. MBio 5(3): e01114-01114.
  15. Soderberg, O., Gullberg, M., Jarvius, M., Ridderstrale, K., Leuchowius, K. J., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L. G. and Landegren, U. (2006). Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3(12): 995-1000.
  16. Soldati, D. and Boothroyd, J. C. (1993). Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science 260(5106): 349-352.
  17. Titeca, K., Meysman, P., Gevaert, K., Tavernier, J., Laukens, K., Martens, L. and Eyckerman, S. (2016). SFINX: Straightforward filtering index for affinity purification-mass spectrometry data analysis. J Proteome Res 15(1): 332-338.
  18. Van Itallie, C. M., Aponte, A., Tietgens, A. J., Gucek, M., Fredriksson, K. and Anderson, J. M. (2013). The N and C termini of ZO-1 are surrounded by distinct proteins and functional protein networks. J Biol Chem 288(19): 13775-13788.

简介

定义蛋白质相互作用网络可以为蛋白质复合物如何控制复杂的生物学问题提供关键信息 在这里我们定义了一种基于接近度的标记方法,使用宽容的生物素连接酶来定义细胞内寄生虫弓形虫的蛋白质网络。 当与基于CRISPR / Cas9的标记结合使用时,这种方法提供了一种可靠的方法来定义蛋白质网络。 这种方法检测完整细胞内的相互作用,它适用于可溶性和不可溶性成分,包括与细胞骨架相互作用的大型蛋白质复合物和独特的微管组织中心,其中包括顶尖复合体在顶尖复合寄生虫中。

【背景】分析蛋白质 - 蛋白质相互作用是解决蛋白质如何组装和作为大分子复合物的关键努力。传统上,通过免疫共沉淀(共-IP)和随后的质谱分析已经鉴定出蛋白质复合物。然而,一些蛋白质复合物取代基可能在co-IP的裂解,下拉和洗涤步骤期间人为失去或获得,这对于不溶性膜或需要侵蚀性溶解的结构蛋白质尤其成问题。作为co-IP的替代物,邻近依赖性生物素鉴定(BioID)提供了在正常细胞稳态期间紧邻目标靶蛋白的蛋白质“快照”(Roux等人,2012年)。 BioID利用融合到感兴趣的靶蛋白的混杂的大肠杆菌生物素蛋白连接酶(BirA)。生物素补充使得BirA融合物在30纳米内允许生物素化的近邻生物体(Roux et al。,2012; Van Itallie et al。,2013) ≤10nm(Kim等人,2014)。生物素化的蛋白质可以通过亲和层析来捕获并通过质谱鉴定(Roux等人,2012)。

弓形虫属于由数以千计的专性寄生虫组成的门Apicomplexa。由于易于进行体外培养和遗传操作, gondii 被认为是研究apicomplexans生物学的模式生物。最近,Chen等人(2015年)调整了BioID以用于 T。 gondii ,鉴定了内膜复合物(IMC)的几种新型蛋白质组分。 BioID已被聘用于 T。鉴定激酶相互作用蛋白(Gaji等人,2015),钙调蛋白(Long等人,2017a),并定义蛋白质库的其他细胞区室,包括寄生泡(Nadipuram等人,2016),IMC缝合(Chen等,2017)和顶端复合物(Long et al。,2017b)。这里我们将描述使用CRISPR / Cas9标签产生BirA基因融合体的方案(Shen等人,2014; Shen等人,2017), 体内BirA生物素标记和从寄生虫中纯化生物素化蛋白质,以及通过质谱鉴定捕获的生物素化蛋白质。由于质谱数据集的分析可能因非特定匹配而变得复杂,因此我们提供了一种方法来过滤出假阳性相互作用并使用直接过滤IndeX程序(SFINX)对真阳性进行排名( http://sfinx.ugent.be/ )(Titeca等人,2016年)。从BioID / SFINX分析中出现的候选互动子也应该通过二级分析进行验证。因此,我们还提供了通过互补接近连接测定来证明共定位的说明。

关键字:质谱法, 蛋白质相互作用网络, 邻位标记, CRISPR, BioID , 邻位连接

材料和试剂

  1. 移液器吸头(康宁,产品目录号:4713,4712)
  2. 1.7ml Eppendorf管(Corning,Costar ,目录号:3620)
  3. T25,T175烧瓶(Corning,目录号:430639,431080)
  4. 注射器10cc,20cc(BD,目录号码:302995,302830)
  5. 22 G钝针(CML Supply,目录号:901-22-100M)
  6. D3聚碳酸酯膜(GE Healthcare,目录号:110612)
  7. 4毫米电穿孔比色杯(哈佛设备,目录号:450126)
  8. 24孔板和96孔板(MIDSCI,目录号:TP92024,TP92696)
  9. Coverslips(Fisher Scientific,目录号:12-545-80)
  10. 2ml Eppendorf管(Fisher Scientific,目录号:054-08-138)
  11. 1升Stericup过滤器(Merck,Millipore Sigma,目录号:SCVPU11RE)
  12. 冷冻管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:5000-0050)
  13. 5ml,10ml,25ml移液管(Fisher Scientific,目录号:13-676-10H,13-676-10J,13-676-10K)
  14. C18 CSH柱(WATERS,目录号:186005295)
  15. 吨。 gondii RHΔ ku80 Δ hxgprt 株(安娜堡密歇根大学医学院Dr. Vernon Carruthers赠送)
  16. Q5用大肠杆菌感受态细胞(New England Biolabs,目录号:E0554S)定点诱变试剂盒
  17. 质粒可在 https://www.addgene.org/ 获得
    1. pSAG1 :: CAS9-U6 :: sgUPRT(Addgene,目录号:54467)
    2. pLinker-BirA-3HA-HXGPRT-LoxP(Addgene,目录号:86668)
    3. pLinker-6HA-HXGPRT-LoxP(Addgene,目录号:86552)
    4. pLinker-2Ty-HXGPRT-LoxP(Addgene,目录号:86664)
  18. LB肉汤(BD,目录号:244610)
  19. 氨苄青霉素(Sigma-Aldrich,目录号:A0166)
  20. 质粒提取试剂盒(Macherey-Nagel,目录号:740588.250)
  21. M13反向通用底漆
  22. Q5 DNA聚合酶(New England Biolabs,目录号:M0491S)
  23. PCR Cleanup Kit(Macherey-Nagel,产品目录号:740609.250)
  24. 用于组织培养的胰蛋白酶(Sigma-Aldrich,目录号:T3924)
  25. 霉酚酸(Sigma-Aldrich,目录号:M3536)
  26. 黄嘌呤(Sigma-Aldrich,目录号:X4002)
  27. 甲醛10%超纯EM级(Polysciences,目录号:04018-1)
  28. 小鼠抗HA抗体(BioLegend,目录号:901501)
  29. 兔抗GAP45(瑞士日内瓦日内瓦大学医学院Dominique Soldati-Favre博士赠送)
  30. 与Alexa Fluor-488(Thermo Fisher Scientific,Invitrogen TM,目录号:A-11001)共轭的山羊抗小鼠IgG(H + L)二抗
  31. IRDye 680CW山羊抗小鼠IgG(H + L)(LI-COR,目录号:926-68070)
  32. 链霉亲和素Alexa Fluor-488偶联物(Thermo Fisher Scientific,目录号:S32354)
  33. IRDye 800CW抗生蛋白链菌素(LI-COR,目录号:925-32230)
  34. IRDye 680CW山羊抗兔IgG(H + L)(LI-COR,目录号:926-68071)
  35. 与Alexa Fluor-594(Thermo Fisher Scientific,Invitrogen TM,目录号:A-11037)共轭的山羊抗兔IgG(H + L)二抗
  36. 液氮
  37. FBS(GE Healthcare,目录号:SH30071.03HI)
  38. 20%DMSO
  39. 链霉亲和素磁珠(Thermo Fisher Scientific,Pierce TM,目录号:88816)
  40. 碳酸氢铵(Sigma-Aldrich,目录号:11213)
  41. DTT
  42. 碘乙酰胺(IAM)(Sigma-Aldrich,目录号:I1149)
  43. DUOlink原位Red Starter小鼠/兔(Sigma-Aldrich,目录号:DUO92101)
  44. DMEM(Thermo Fisher Scientific,Gibco TM,目录号:12800017)
  45. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
  46. HEPES(Sigma-Aldrich,目录号:H3375)
  47. L-谷氨酰胺(Sigma-Aldrich,目录号:G7513)
  48. 庆大霉素(Sigma-Aldrich,目录号:G1272)
  49. 磷酸二氢钾(K 2 HPO 4)(Sigma-Aldrich,目录号:1551128)
  50. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:1551139)
  51. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  52. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:793639)
  53. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  54. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  55. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  56. D-生物素(Sigma-Aldrich,目录号:47868)
    注意:此产品已停产。
  57. Tris(Sigma-Aldrich,目录号:T1503)
  58. EGTA(Sigma-Aldrich,目录号:E3889)
  59. Triton X-100(Sigma-Aldrich,目录号:T8787)
  60. NP-40(Sigma-Aldrich,目录号:I8896)
  61. 甘油(Sigma-Aldrich,目录号:G5516)
  62. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  63. 脱氧胆酸盐(Sigma-Aldrich,目录号:D6750)
  64. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  65. DOC,脱氧胆酸盐(Sigma-Aldrich,目录号:30970)
  66. 氯化锂(LiCl)(Sigma-Aldrich,目录号:62476)
  67. D10中(见食谱)
  68. Cytomix缓冲液(见食谱)
  69. 磷酸盐缓冲盐水(PBS)(见食谱)
  70. D-生物素库存(见食谱)
  71. 细胞骨架缓冲液(见食谱)
  72. 5倍SDS样品缓冲液(见食谱)
  73. 缓冲区1(请参阅食谱)
  74. 缓冲区2(请参阅食谱)
  75. 缓冲区3(见食谱)
  76. 缓冲区4(见食谱)

设备

  1. Eppendorf离心机(Eppendorf,型号:5810 R)
  2. 培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:370型)
  3. II类生物安全罩(Baker Co.,型号:SterilGARD II,Class II A / B3型)
  4. BTX ECM-830电穿孔仪(哈佛仪器,型号:ECM 830)
  5. 血细胞计数器(Hausser Scientific,目录号:3120)
  6. 倒置相差显微镜(尼康仪器,型号:Eclipse TS100)
  7. 200μl移液器
  8. Sonic Dismembrator 550(Fisher Scientific,型号:Model 550)
  9. 磁力支架(赛默飞世尔科技,产品目录号:12321D)
  10. 奥德赛成像系统(LI-COR,型号:Odyssey®CLx)
  11. 液氮罐(Airgas,型号:NI230LT22)
  12. Q-exactive HF质谱仪(Thermo Fisher Scientific,Thermo Scientific TM,型号:Q Exactive TM HF)

软件

  1. sgRNA选择网站: http://grna.ctegd.uga.edu/ ,Mascot 2.5版。 1(Matrix Science)
  2. SFINX分析网站: http://sfinx.ugent.be/ ,脚手架版本4.6.1(Proteome Software Inc 。)

程序

  1. 设计针对特定基因的Cas9 / sgRNA质粒用于BirA的C端标记
    注:此处描述的方案来自Shen等人,2017。我们建议您在尝试此协议之前阅读此评论,以了解弓形虫中CRISPR基因组编辑的背景。
    1. sgRNA选择
      1. http://toxodb.org/toxo/ 中,导航到您感兴趣的基因(GOI)网页。这可以使用基于表达或功能特性的基因搜索或仅通过输入已知的基因ID或关键字来完成。小心选择正确的 T。 gondii 拉紧你的工作。将起始密码子上游500bp下游的基因组序列下载到终止密码子下游500bp下游。
      2. 复制终止密码子后的前200 bp,并在 http://grna.ctegd.uga.edu/。输入工作名称,选择 T. gondii 沿袭数据库,并保留所有其他默认设置。
      3. 从生成的sgRNA序列列表中选择具有3'NGG原型间隔区相邻基序(PAM)基序的20nt sgRNA序列。 sgRNA序列可以在正向或反向DNA链上。
    2. 产生含有基因特异性sgRNA的Cas9 / sgRNA质粒
      1. 订购用于PCR诱变pSAG1 :: CAS9-U6 :: sgUPRT(Addgene#54467)的DNA寡核苷酸。反向引物(5'-AACTTGACATCCCCATTTAC-3')对于用该Cas9 / sgRNA质粒进行的所有诱变反应是通用的。正向引物(5' - [5'至3'方向上的20nt sgRNA序列] -GTTTTAGAGCTAGAAATAGC-3')对于每个sgRNA将是唯一的。例如,如果所需的原型间隔子 + PAM 位点是5'-GCTGGTCTATCGCTAGCTCG AGG ,则正向引物将是5' - GCTGGTCTATCGCTAGCTCG GTTTTAGAGCTAGAAATAGC。个别基因的具体例子可以在这些最近的论文中找到(Brown等人,2017; Long等人,2017a和2017b)。
      2. 使用Q5定点诱变试剂盒(New England Biolabs)突变pSAG1 :: CAS9-U6 :: sgUPRT(Addgene#54467)的sgRNA序列。我们推荐由6.25μl2x Q5热启动酶混合物(包含在试剂盒中),0.625μl每种引物(工作原液10μM),1μl(1 ng)pSAG1 :: CAS9-U6 :: sgUPRT和4 μlddH 2 O。对于热循环,我们建议25个循环的95℃30秒,60℃20秒和72℃5分钟。
      3. 将1μl的PCR反应溶解在DNA凝胶上以确认与约10kb的DNA带成功反应。根据制造商的说明,用KLD混合液(包含在试剂盒中)提取1μlQ5 PCR反应。
      4. 将2.5μl的KLD反应转化为所提供的 E。大肠杆菌感受态细胞,并在含有100μg/ ml 37℃氨苄青霉素的LB琼脂平板上生长过夜。
      5. 增长三个单一的殖民地的E。在含有100μg/ ml氨苄青霉素的3ml LB培养基中于37℃过夜。使用3000 x g离心10分钟沉淀细菌培养物。使用质粒提取试剂盒(Macherey-Nagel)从细菌沉淀中提取质粒。发送三个质粒用于Sanger测序以确认使用M13反向通用引物成功将sgRNA插入Cas9 / sgRNA质粒中。选择序列确认的质粒进行进一步编辑。

  2. 使用CRISPR / Cas9标签生成BirA标签生产线
    1. 生成基因特异性BirA标签盒
      1. 设计用于扩增BirA标签盒的正向和反向引物。正向引物含有位于终止密码子正上游的40nt 5'同源性(注意终止密码子被排除),并且跟随GCTAGCAAGGGCTCGGGC用于锚定在标记质粒中的接头pLinker-BirA-HXGPRT-LoxP(Addgene# 86668)。接头位于标签BirA的前面,用作标签和内源蛋白质之间的间隔区。它也可以作为正向引物的锚定位点。反向引物含有与sgRNA序列(PAM位点上游3bp)处的Cas9切割位点相邻的40nt 3'同源臂,并且随后与ATAGGGCGAATTGGAGCTCC一起用于锚定在标记质粒中的HXGPRT选择盒的末端。推荐的合成规模为25 nmole DNA和寡核苷酸应使用标准脱盐程序进行处理。
      2. 使用制造商的Q5 DNA聚合酶(New England Biolabs)方案在90μlPCR反应中用正向和反向引物扩增BirA盒(图1)。使用标记质粒(1ng,反应90μl)作为DNA模板,退火温度设定为60℃2分钟。
      3. 在DNA凝胶上解决2μlPCR反应,以确认是否存在3.5 kb PCR产物的成功反应。


        图1.CRISPR / Cas9标记策略的示意图使用短同源性区域将Cas9位点特异性切割与整合结合的效率允许对感兴趣的基因进行快速标记。将由42bp侧翼区(表示为HR1(紫色)和HR2(蓝色))组成的基因特异性同源区与保守的接头区(L(红色)和T(黑色))组合以产生扩增子。保守连接子位于包含BirA标签(绿色),终止密码子和通用3'UTR(黄色)的中央标签构建体的侧翼。引物HR1和HR2用于从标签质粒中扩增基因特异性BirA扩增子。然后将BirA标签盒克隆到基因特异性pCas9 / sgRNA质粒中并转染到RHΔku80系中,然后进行药物选择。图改编自(Long等人,2017a)。

    2. 转染入T。 gondii 收件人行和筛选克隆线
      注意:在本协议中,所有对活宿主细胞或寄生虫的处理都应使用BSL2认证的生物安全柜或通风橱中的无菌技术进行。
      1. 根据制造商的协议,用Gel Extraction和PCR Cleanup Kit(Macherey-Nagel)纯化BirA盒。
      2. 通过加入10-15μlCas9 / sgRNA质粒(10-20μg质粒)洗脱DNA盒,并再次用5μlddH 2 O洗脱柱以制备盒-Cas9 / sgRNA混合物。
      3. 在1.5ml Eppendorf管中于75℃加热盒-CS9 / sgRNA混合物10分钟,并离心以收集壁上的液体。
      4. 在含有D10培养基(配方1)的T25烧瓶中,在37℃,5%CO 2培养箱中培养人类包皮成纤维细胞(HFF),直到它们达到融合。使用标准细胞培养技术将HFF细胞胰蛋白酶消化,以1:4分开传代细胞。
      5. 在T25瓶中感染完全融合的HFF单层,其中T.gondii线以每个T25烧瓶2.5×10 6个寄生虫的密度标记。在这里, T。 gondii 使用RHΔ ku80 Δ hxgprt 。发展 T。 gondii 行约2天,直到从HFF单层发生自然出口。转染后需要新鲜的HFF培养瓶。
      6. 为了收获寄生虫,刮掉单层以从烧瓶中取出并收集5ml含有寄生虫的培养基。释放任何剩余的细胞内T细胞。通过使用10ml注射器穿过22G针头,小心取出针头并将其安全地丢弃到锐器容器中,并且通过穿过3.0μm聚碳酸酯膜过滤寄生虫以去除宿主细胞碎片。将流出物中的寄生虫在800×g,18℃下离心10分钟,并用10ml细胞混合缓冲液(Soldati和Boothroyd,1993;方案2)洗涤寄生物沉淀一次,然后离心再次重新悬浮。来自单个TFF的HFF的典型产量总共约5-8×10 7个寄生虫。
      7. 将从T25中收获的寄生虫重悬于2ml细胞混合缓冲液中用于转染。
      8. 在4mm电穿孔比色杯(BTX)中将200μl寄生虫悬液(5-8×10 6寄生虫)和盒-CSa9 / sgRNA混合物(图1)合并,并进行电穿孔使用以下协议:使用BTX ECM-830电穿孔仪获得1,700 V,176μsec的脉冲长度,两个100 ms间隔的脉冲。
      9. 在电穿孔后立即将寄生虫悬浮液转移到具有融合HFF的新鲜T25烧瓶中,并在37℃,5%CO 2下孵育它们。
      10. 电穿孔和恢复后24小时,开始用25μg/ ml麦考酚酸补充25μg/ ml黄嘌呤进行药物选择。在药物选择培养基中根据需要传代寄生虫直至出现耐药性人群(〜2-3代)。
      11. 在自然出口后,用血细胞计数器计数寄生虫,并将寄生虫稀释至3个寄生虫/150μl,并将该稀释的寄生虫加入150μlD10培养基中至具有融合HFF的96孔板中,并在37℃,5% CO <2>连续6天不动。
      12. 在倒置相差显微镜下观察在96孔板中仅含有一个噬菌斑的孔。对于只含有一个弓形虫噬菌斑的孔,用200μl移液管在孔中混合寄生虫和培养基。继续生长寄生虫3天,直到寄生虫天然出口。斑块形成和扩张的协议。在微滴定孔板中的弓形虫也已经在之前被充分描述过(Roos等,1994)。
      13. 从孔中挑选10个克隆并在96孔板中用HFF接种一半培养物,另一半接种在具有盖玻片和HFF单层的24孔板中。让盖玻片上的寄生虫生长24小时,并用含4%甲醛的PBS(配方3)固定,并用含有10%FBS的PBS中的0.25%Triton X-100进行膜透化,然后用间接荧光显微术使用初级针对HA(小鼠)和GAP45(兔)的抗体,然后分别与Alexa Fluor 488和594缀合的针对小鼠和兔的二抗。然后在荧光显微镜下观察寄生虫,看看HA染色阳性的克隆(绿色通道)。
      14. 用于测试在靶向基因座处插入BirA的诊断性PCR用于确认使用位于翻译终止密码子上游200bp处的一个引物和位于终止密码子下游300bp处的另一个引物的克隆纯度。正确地标记了 T。 gondii克隆不产生内源性PCR产物,该产物仅从亲本细胞系RHΔ ku80 Δ hxgprt 检测到。
      15. 还可以使用商业抗HA抗体和用于检测的第二抗体进行Western印迹以确认内源性BirA-3HA标签系的表达。对于蛋白质印迹,我们通常每道加载10 6个细胞当量并在转移到硝酸纤维素之前通过在10%PAGE凝胶上分离来解析。我们用LI-COR IR染料二抗检测的首选方法;然而,最佳负载条件可能会因检测方法而异。先前已经详细描述了用于检测弓形虫中表位标签化蛋白质的方案(Brown等人,2017; Long等人,2017a,2017a和2017b)。
      16. 一旦确认阳性克隆,应将它们转移到T25烧瓶中并保存在液氮储存的冷冻小瓶中。五百万寄生虫应该接种在T25烧瓶并且生长1天。感染的HFF单层应该用温PBS洗涤两次,并用1ml胰蛋白酶溶液进行胰蛋白酶消化以分离单层(用等体积的PBS稀释)。应在D10培养基中加入1ml 50%FBS终止胰蛋白酶消化,然后在D10培养基中加入1ml 20%DMSO进行保存。将混合物转移到冷冻管中并放入异丙醇容器中在-80℃下冷冻。
        第二天将冷冻管转移到液氮箱中。

  3. 用于质谱分析的生物素化蛋白质的纯化
    1. 确认体内BirA活性
      1. 在含有HFF单层(含有5ml D10培养基)的T25烧瓶中,在37℃,5%CO下,将RHΔku80Δhxgprt系和BirA标记系增长24小时在培养箱中加入50μl16mM D-生物素溶液(配方4)于DMEM中以使终浓度为160μM。将寄生虫再生长20小时。
      2. 通过穿过22G针头和D3聚碳酸酯膜来收获寄生虫以去除HFF碎片,并且在800℃×g,18℃下收集流通和离心10分钟以沉淀寄生虫。用冷PBS清洗寄生虫以除去多余的生物素,然后用80μlPBS离心并重新悬浮寄生虫沉淀,并加入20μlSDS样品缓冲液。
        在100°C煮沸样品5-10分钟。
      3. 使用SDS-PAGE分解寄生虫裂解物,并使用链霉亲和素-800CW(LI-COR)进行蛋白质印迹以检测寄生虫系中的生物素化蛋白质。 BirA泳道应该显示在亲本RHΔemuΔ hxgprt 行中未发现的额外条带,如图2A中用CaM1-BirA和CaM2-BirA所示。 >
      4. RHΔ ku80 hxgprt 线和BirA线也可以在具有HFF单层的盖玻片上在24孔板中生长并添加160μMD-生物素24小时,并用于通过使用链霉亲和素Alexa Fluor 488的间接免疫荧光显微镜检测来检测BirA蛋白定位处的生物素化蛋白质(图2B)。
    2. 生物素化蛋白的纯化(图2C)
      1. 在含有汇合的HFF单层的T175烧瓶中以2×10 7 / s的剂量接种亲本系RHΔ hxgprt 线和BirA标签线>寄生虫并生长24小时,并添加D-生物素至最终浓度为160μM。
        将D-biotin的寄生虫继续生长20小时,直到天然出口。
      2. 通过穿过22G针头和D3聚碳酸酯膜来收获寄生虫以去除碎片,离心并用50ml冷PBS洗涤寄生虫沉淀三次以去除多余的D-生物素。
      3. 在2毫升细胞骨架缓冲液中重悬寄生虫(方案5),并使用设置:15秒脉冲,15秒间隔时间和2分钟总脉冲时间,用Sonic Dismembrator 550(Fisher Scientific)中的微尖超声波再悬浮。
      4. 在冰上孵育寄生虫裂解10分钟,并转移至2ml Eppendorf管中,并以20000×g,4℃离心20分钟。将上清液转移至新鲜试管中,重复离心以获得更清澈的上清液。
      5. 将上清液转移至2ml含有50μl链霉抗生物素蛋白磁珠(Pierce)的Eppendorf管中,所述磁珠已经用细胞骨架缓冲液洗涤并平衡三次。
        重悬上清液和珠子,并在4°C孵育过夜。
      6. 用1.5ml细胞骨架缓冲液洗珠两次,然后用1.5ml缓冲液1(配方6)洗涤一次,用1.5ml缓冲液2(配方7)洗涤两次,用1.5ml缓冲液3(配方8)洗涤一次,用1.5ml缓冲液4(配方9)和1.5ml PBS两次。每个清洗步骤需要孵育5分钟,最后两个清洗步骤用缓冲液4和PBS进行以除去剩余的SDS。用磁力架将珠子拉下,弃上清。
      7. 转移10%来自最后一次洗涤步骤的珠子,并将珠子重悬于20μlPBS和5μl5x SDS样品缓冲液(配方6)中,用链霉亲和素LI-COR C800进行SDS-PAGE和蛋白质印迹检测。珠子的剩余部分(90%)可以暂时储存在-80°C。


        图2. T. gondii 中BirA标签的策略和示例。 :一种。蛋白质印迹检测表达CaM1-BirA或CaM2-BirA的RHΔku80Δhxgprt系中的生物素化蛋白质。使用IRDye 800CW链霉抗生物素蛋白来检测生物素化蛋白质。使用α-HA抗体来检测BirA-3HA融合,并且使用α-醛缩酶作为上样对照,并且通过IRDye 680CW山羊抗小鼠IgG和IRDye 680CW山羊抗兔IgG分别可视化。 B.IFA检测CaM1-BirA系中的生物素化蛋白质。链霉抗生物素蛋白Alexa Fluor-488用于检测生物素化蛋白质,并且通过α-HA抗体检测到CaM1-BirA-3xHA,并用与Alexa Fluor 599缀合的抗兔二抗显色。比例尺=2μm。 C.从表达BirA的表达纯化生物素化蛋白质的图。 gondii 行。如方法中所述,将寄生虫溶解并通过链霉抗生物素蛋白珠捕获生物素化蛋白质。此图基于(Roux et。,2012)中的原始描述。

  4. 生物素化蛋白的质谱分析(由Michael Naldrett博士在Lincoln内布拉斯加大学生物技术中心蛋白质组学和代谢组学设施完成( https://biotech.unl.edu/
    1. 样品制备
      1. 将样品珠溶解在200μl100 mM碳酸氢铵中,并在37°C下加入4μl2 mM DTT 1小时以减少。
      2. 通过在22℃在黑暗中加入22μl100mM碘乙酰胺(IAM)20分钟来烷基化。
      3. 每个样品加入胰蛋白酶(5μl,0.1mg / ml),并在37℃过夜消化。
    2. LC-MS / MS
      在0.075 x 250 mm C18 Waters CSH柱上使用2 h梯度,在NanoLC-MS / MS上运行5μl胰蛋白酶消化样品,进样至Q-exactive HF质谱仪。
    3. 光谱分析
      1. 使用Mascot通过搜索质谱来分析质谱的结果。假设通过胰蛋白酶消化的弓形虫ME49_20150114(8322序列),cRAP_20150130(117序列)和习惯(含有链霉抗生物素蛋白序列)数据库。
        吉祥物是通过0.060 Da的碎片离子质量容差和10.0 PPM的母离子容差进行检索的
      2. 在天冬酰胺和谷氨酰胺脱酰胺的过程中,甲硫氨酸,半胱氨酸的氨基甲酰甲基以及赖氨酸的生物素和N-末端的氧化应在Mascot中规定为可变修饰。
      3. 使用Scaffold 4.6.1版来验证基于MS / MS的肽和蛋白质鉴定。基于Scaffold delta质量校正(Keller等人,2002),肽识别被认为是有效的,如果它们支持超过95.0%的概率。我们通常接受蛋白质鉴定,如果它们的建立概率大于99.0%并且包含至少2个鉴定的肽。较低的临界值也可以用于探索性工作,但可能会导致更多的误报识别。

  5. 推定的相互作用蛋白的确认
    注意:建议将此步骤作为BioID实验的后续步骤,但可能需要几个月才能完成。一些BioID实验可能只需要对诱饵-BirA'邻居'进行调查而不验证特定的交互者。
    1. 推定的相互作用蛋白的定位
      1. 在蛋白质相互作用组中鉴定出的相互作用蛋白可能以前没有定位过。为了验证在正确的位置可能的交互,验证其本地化将是第一步。
      2. 候选蛋白质编码基因可以使用CRISPR标记策略通过HA或Ty表位标签内源标记,如上面关于BirA标签所述。唯一的区别是,PCR模板中的BirA标签被替换为HA或Ty标签以生成标签盒。这里我们推荐保存在Addgene pLinker-6HA-HXGPRT-LoxP(#86552)和pLinker-2Ty-HXGPRT-LoxP(#86664)中的质粒。
      3. 一旦产生HA或Ty标签菌株,就可以在24孔板的盖玻片上用HFF单层培养菌株进行间接免疫荧光显微镜检查。
      4. 与诱饵蛋白共定位的相互作用蛋白将继续用于ToxoDB的生物信息学分析,如细胞区室,分子功能。
      5. 蛋白质复合物的稳定性和功能可以通过使用生长素诱导性决定子系统(Long等人,2017a; Brown等人, 2017年)。使用生长素诱导性决定子系统的详细方案也可在Bio-protocol(Brown等人,2018年)上获得。
    2. 通过另一种接近途径 - 接近结扎测定(PLA)进行相互作用确认
      1. 使用上述CRISPR / Cas9标记策略,通过用HA标记一个基因和用Ty在同一行中标记另一个基因可进一步证实蛋白质相互作用。在这种情况下,推荐Ty标签质粒携带另一个抗性标记DHFR而不是HXGPRT。
      2. 在一个弓形虫线中的HA标签和Ty标签可以被兔HA和小鼠Ty抗体识别,然后是相应的与PLA探针缀合的第二抗体。 PLA程序可以遵循制造商的协议,使用DUOlink原位红色启动试剂盒(Sigma-Aldrich)。 PLA方法在30 nm距离内检测蛋白质相互作用,与BirA标记的距离相似(≤30 nm)(Soderberg et al。2006年)。

数据分析

蛋白质相互作用分析
直接过滤索引(SFINX)( http://sfinx.ugent.be/ < )是一个程序,它提供了一个统计学上可靠的方法来过滤假阳性,并使用来自蛋白质组学研究的肽计数来确定真正的相互作用(Titeca等,2016)。在这里,我们将邻近标记方法与此程序相结合,以建立基于接近度的蛋白质 - 蛋白质相互作用网络。
SFINX分析:

  1. 来自两个或多个独立实验的数据集应该合并成一个Scaffold文件。将程序设置为“总独特肽数”,蛋白质阈值为95%,肽2的数量最少。
  2. 使用'当前视图'导出数据集,并编辑excel文件以删除不必要的列和行,留下基因登录号,独立质谱实验(项目)的名称和肽数。按照SFINX网站在信息栏中的建议,在第一列的第一行中输入行名称。将此基本文件保存为逗号分隔值文件(.csv)或软件建议的其他文件格式。
  3. 在实验中用BirA标记感兴趣基因的登录号创建另一个.csv文件(诱饵文件)。
  4. 将基本文件和诱饵文件上传到网站并选择正确的文件格式。蛋白质 - 蛋白质网络将立即生成,并在左边显示一个严格条。严格栏是基于SFINX评分(来源于 P 评分),该评分用于对真正的评分进行排名。在我们的例子中,严格性1被采用,对应于 P &lt; 0.00004。
  5. 通过点击'complete output'或'cutoff output'可以将过滤的数据作为excel .tsc文件下载。下载的文件列出了交互者(preys),以及评分和交互者与BirA蛋白质(诱饵)之间相互作用的得分和 P 得分。
  6. 为了用SFINX 3产生高质量的蛋白质 - 蛋白质相互作用网络,应当通过质谱法分析涉及相同途径或位置的多个诱饵蛋白质(即,emA,em,BirA融合蛋白)。

笔记

  1. 我们还成功地使用了Scaffold中提供的统计特征来定义重复数据集之间的有意义的交互。当从重复实验进行比较时,我们使用Student's t - 测试来分析数据,无论是否进行多重比较校正。校正后的测试要严格得多,因此不太可能发现误报。然而,它们也极大地限制了潜在的相互作用蛋白质的数量。如果发现是主要目标,那么接受更高的误报率并使用一些次级分析验证潜在的相互作用可能是更好的策略。
  2. 使用DHFR耐药盒时应特别小心,因为这会赋予对最常用于治疗弓形体病的药物乙胺嘧啶的抗性。这些质粒的设计是在DHFR耐药盒侧翼的loxP位点上进行的,可以通过瞬时转染Cre来消除该区域,通过PCR进行克隆和筛选。
  3. 为了获得SFINX的最佳性能,重要的是对于每种BirA标记的蛋白质具有两个或更多个生物学重复。该方法对重复样品之间标记效率的差异非常稳健,但是,在实验运行之间具有相似的整体肽覆盖度是有帮助的。根据我们的经验,SFINX在预测超过统计阈值的交互作用方面相当保守,因为这不太可能是偶然的。输出文件给出后验概率,可用于设置用户可以接受的阈值。在任何情况下,这些输出只能预测邻近相互作用,并且需要使用其他方法进行后续分析以确认相互作用。

食谱

  1. D10中等
    1包DMEM粉末
    3.7克碳酸氢钠
    2.38克HEPES
    10毫升的200毫米L-谷氨酰胺
    1毫升10毫克/毫升庆大霉素
    适量用去离子H 2 O至1L加入 用1升Stericup过滤器过滤灭菌并在4°C储存。
  2. Cytomix缓冲液
    8.66mM K 2 HPO 4 4 1.36 KH 2 PO 4 4
    120 mM KCl
    0.15 mM CaCl 2 2/2 5mM MgCl 2·/ 2 25 mM HEPES
    2 mM EDTA
    调整pH值至7.6
    用Stericup过滤器过滤灭菌并在4°C储存。
  3. 磷酸盐缓冲盐水(PBS)
    137mM NaCl
    10 mM磷酸盐
    2.7 mM KCl
    调整pH值到7.4

    高压灭菌器在4°C储存
  4. D生物素库存
    DMEM中160 mM D-生物素
  5. 细胞骨架缓冲区
    10 mM Tris,pH 7.4
    100 mM NaCl
    1 mM EDTA
    1 mM EGTA
    1%Triton X-100
    1%NP-40
    10%甘油
    0.2%SDS
    0.5%脱氧胆酸
  6. 5倍SDS样本缓冲区
    溴酚蓝0.25%
    DTT 0.5 M
    甘油50%
    SDS 10%
  7. 缓冲区1
    20 mM Tris pH 7.5
    2%SDS
  8. 缓冲区2
    0.1%DOC
    1%Triton X-100
    500 mM NaCl
    1 mM EDTA
    50毫米HEPES
    调整pH值至pH 7.5
  9. 缓冲区3
    250 mM LiCl
    0.5%NP-40
    0.5%DOC
    1 mM EDTA
    10 mM Tris pH 8.1
  10. 缓冲区4
    50 mM Tris,pH 7.4
    50 mM NaCl

致谢

这项工作得到了NIH(AI034036)对L.D.S.的资助。该协议根据Long et al。(2017a和2017b)中描述的协议进行改编。作者声明没有冲突或利益冲突。

参考


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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Long, S., Brown, K. M. and Sibley, L. D. (2018). CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii. Bio-protocol 8(6): e2768. DOI: 10.21769/BioProtoc.2768.
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