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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

SL Shaojun Long
KB Kevin M. Brown
LS L. David Sibley

发布: 2018年03月20日第8卷第6期 DOI: 10.21769/BioProtoc.2768 浏览次数: 11348

评审: David CisnerosAmit DeyAnonymous reviewer(s)

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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) search Protein interaction network (蛋白质相互作用网络) search Proximity labeling (邻位标记) search CRISPR (CRISPR) search BioID (BioID) search Proximity ligation (邻位连接) search

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∆ku80∆hxgprt 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

English
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文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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微生物学 > 微生物蛋白质组学 > 全生物体

分子生物学 > 蛋白质 > 蛋白质-蛋白质相互作用

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