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Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock

低滴度病毒载体在昆虫细胞中表达和纯化功能性5-HT2A受体

SM Sukanya Mozumder
GM Gopa Mahesh
KS Krishnamoorthi Srinivasan
JS Jayati Sengupta
SM Sujoy Mukherjee

发布: 2020年08月05日第10卷第15期 DOI: 10.21769/BioProtoc.3704 浏览次数: 3967

评审: Anonymous reviewer(s)

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Abstract

The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell cultures. In this protocol, we describe a step-by-step method to express and purify serotonin 5-HT2AR using a baculoviral expression vector system in Sf9 cell cultures, derived from our work with the rat (matching Uniprot ID P14842) and human (matching Uniprot ID P28223) 5-HT2ARs. A unique feature of this method is the utilization of cell culture additives to infect cells at low multiplicity of infection, thereby using several fold less quantity of viral titer compared to prior methods without the additive. This protocol can be tweaked to selectively over-express glycosylated or non-glycosylated forms of the receptor by varying the post-infection harvest times.

Keywords: Serotonin 5-HT2A receptor (5-羟色胺5-HT2A受体) search GPCR production (GPCR生产) search Low MOI expression (低MOI表达) search Glycosylation (糖基化) search baculoviral expression (杆状病毒表达) search Sf9 cell culture (Sf9细胞培养) search

Background

Over the last decade structural investigations on GPCRs have surged due to development of methods for high yield expression of functionally active receptors (Granier and Kobilka, 2012). Among those, the serotonin GPCRs are a small but diverse set of receptors that play important roles in neuromodulation and their dysfunction is linked to a number of mental health disorders (Berger et al., 2009). Of the many methods available employing eukaryotic hosts for protein expression, including yeast, insect and mammalian cells, the use of Sf9 cells to express GPCRs in conjunction with baculoviral expression system has emerged as a prominent one due to its high yield along with reliability (Saarenpaa et al., 2015; Wiseman et al., 2020). However, various methods continue to be developed to overcome the various drawbacks of existing methods. There is no uniform, single method to express all types of GPCRs with sufficiently high yield to enable structural studies. This is partly due to the diversity of these receptors as well as modifications needed in their sequences to enable crystallographic studies. The advent of high-resolution single particle cryo-electron microscopy (CryoEM) circumvents some of the stringent requirements for crystallization and is more amenable towards studies on native receptors. One critical requirement for existing methods using baculoviral expression in insect cells is to obtain a high titer viral stock, which is non-trivial in many cases. Moreover for protein production, infecting cells typically at multiplicities of infection ~1-5 requires the use of significant volumes of viral titer, which gets depleted over time. Using multiple batches of viral titers can increase batch-to-batch variability. Hence, methods that consume much smaller quantities of viral titer are strongly desired. In addition, the native forms of many GPCRs, including serotonin receptors, have extensive glycosylation which is not desirable in all situations. Thus, any method that can provide either predominantly glycosylated or non-glycosylated form of receptor, preferably by a simple change of protocol, is also desirable. Here, we describe a method to express and purify 5-HT2AR using baculoviral expression system in Sf9 cells that requires minimal quantities of viral titers compared to existing methods and can select the extent of glycosylation by varying the post-infection harvest times. This protocol, based on prior work (Wacker et al., 2013; Mahesh et al., 2018; Mozumder et al., 2020), has been standardized to prepare samples for high-resolution CryoEM structure determination and it is applicable to other GPCRs, membrane proteins and other soluble proteins employing a similar expression and purification strategy.

Materials and Reagents

  1. 6-well plate (BD Falcon, catalog number: 353046 )
  2. 24-well plate (BD Falcon, catalog number: 353047 )
  3. Whatman glass microfiber filters, Grade GF/B (Merck Millipore, catalog number: WHA1821025 )
  4. Electroporation cuvettes (Bio-Rad, catalog number: 1652086 )
  5. Sf9 cells, adapted to serum free medium (Life Technologies, catalog number: 11496-015 )
  6. E. coli DH10Bac cells (Life Technologies, catalog number: 10361-012 )
  7. pFastBac HT cloning vector kit, containing control plasmid (Life Technologies, catalog number: 10584-027 ) or pFastBac1 cloning vector kit, containing control plasmid (Life Technologies, catalog number: 10360014 )
  8. Baculoviral Titer kit, containing anti-gp64-PE conjugated antibody and control virus (Expression Systems, catalog number: 97-101 )
  9. Purelink HiPure Plasmid Miniprep Kit (Life Technologies, catalog number: K210002 )
  10. ESF 921 Insect cell culture medium (Expression Systems, catalog number: 96-001-01 )
  11. Grace’s Insect cell culture medium (Life Technologies, catalog number: 11595030 )
  12. Production Boost Additive (Expression Systems, catalog number: 95-006-100 )
  13. Cellfectin II (Life Technologies, catalog number: 10362100 )
  14. Alexa Fluor 488 goat anti-rabbit antibody (Life Technologies, catalog number: A11008 )
  15. Anti-5-HT2AR rabbit antibody (Neuromics, catalog number: RA24288 )
  16. Anti-5-HT2AR rabbit polyclonal antibody (Abcam, catalog number: ab66049 )
  17. Anti-FLAG(R), antibody produced in rabbit (Sigma-Aldrich, catalog number: F7425 )
  18. HRP-conjugated goat anti-rabbit antibody (Merck Millipore, catalog number: AP187P )
  19. Cholesteryl hemisuccinate (Avanti Polar Lipids, catalog number: 850524 )
  20. n-dodecyl-β-D-maltopyranoside (DDM) (Anatrace, catalog number: D310S )
  21. α-iodoacetamide (Merck Millipore, catalog number: 407710 )
  22. [3H]-Ketanserin (Perkin-Elmer, catalog number: NET791250UC )
  23. Ketanserin tartrate (Alfa Aesar, catalog number: J62798 )
  24. Serotonin Hydrochloride (Acros Organics, catalog number: AC215021000 )
  25. SIGMAFAST Protease Inhibitor Cocktail tablet (Sigma-Aldrich, catalog number: S8830 )
  26. Polyethylenimine (Sigma-Aldrich, catalog number: P3143 )
  27. Ni-NTA Agarose (Qiagen, catalog number: 30210 )
  28. Liquid nitrogen
  29. Penicillin Streptomycin (Invitrogen, catalog number: 15070063 )
  30. Kanamycin sulfate (Calbiochem, catalog number: 420411 )
  31. Gentamicin sulphate (Himedia, catalog number: TC026 )
  32. Tetracycline (Himedia, catalog number: TC036 )
  33. Bovine Serum Albumin (Himedia, catalog number: MB083 )
  34. Bluo Gal (Invitrogen, catalog number: 15519028 )
  35. Trypan Blue (Life Technologies, catalog number: 15250 )
  36. Scintillation fluid Cocktail ‘T’ (Spectrochem, catalog number: 0 30399 )
  37. Isopropyl β-d-1-thiogalactopyranoside (IPTG) (Himedia, catalog number: MB072 )
  38. YT HiVeg Broth (Himedia, catalog number: MV1251 )
  39. LB medium (Himedia, catalog number: MV1245 )
  40. Agar Powder (Himedia, catalog number: RM301 )
  41. Na2HPO4 (Himedia, catalog number: MB024 )
  42. KH2PO4 (Himedia, catalog number: TC011 )
  43. HEPES (Calbiochem, catalog number: 5320 )
  44. MgCl2 (SRL, catalog number: 69396 )
  45. KCl (Himedia, catalog number: MB043 )
  46. NaCl (Himedia, catalog number: TC046 )
  47. EDTA (Sigma-Aldrich, catalog number: E5134 )
  48. SDS (SRL, catalog number: 54468 )
  49. Methanol (Finar, catalog number: 67-56-1 )
  50. Glacial Acetic Acid (SRL, catalog number: 90868 )
  51. Glycine (Himedia, catalog number: MB013 )
  52. Tween 20 (Himedia, catalog number: MB067 )
  53. Imidazole (Himedia, catalog number: MB019 )
  54. Glycerol (Himedia, catalog number: MB060 )
  55. BCA Protein Assay Kit (Pierce, catalog number: PI23227 )
  56. LB agar plate for blue-white colony selection (see Recipes)
  57. Sf9 cell culture medium (see Recipes)
  58. Phosphate Buffered Saline (PBS) pH 7.4 (see Recipes)
  59. Hypotonic buffer (see Recipes)
  60. Hypertonic buffer (see Recipes)
  61. Resuspension buffer (see Recipes)
  62. Solubilization buffer (see Recipes)
  63. Equilibration buffer (see Recipes)
  64. Wash buffer I (see Recipes)
  65. Wash buffer II (see Recipes)
  66. Elution buffer (see Recipes)
  67. Radioligand binding buffer (see Recipes)
  68. SDS-PAGE Running buffer (see Recipes)
  69. Coomassie Brilliant Blue staining solution (see Recipes)
  70. SDS-PAGE destaining solution (see Recipes)
  71. Transfer buffer for Western blotting (see Recipes)
  72. TBST buffer (see Recipes)
  73. Blocking buffer (see Recipes)

Equipment

  1. -80 °C freezer (ESCO, model: UUS-439-A-1 )
  2. Bio-safety Cabinet Class II A2 type (ESCO, model: AC2-4S8 )
  3. Electroporator (Bio-Rad, model: Micropulser electroporator , catalog number: 165-2100 )
  4. Flow cytometer (BD Biosciences, BD LSRFortessa, model: LSR Fortessa )
  5. Liquid scintillation counter (Perkin Elmer, model: Tri-Carb 2810 TR )
  6. Ultracentrifuge (Thermo, model: 46901-WX90 ) with T865 rotor for 30 ml tubes
  7. Incubator Shaker (Sartorius, Brand, model: Certomat BS-T ), used for large volume cultures
  8. Incubator Shaker (Jeiotech, Brand, model: ISS-4075R ), used for maintenance and small volume cultures
  9. Bacterial incubator (BIS-1824)
  10. Fluorescence inverted microscope (Leica, model: DMIL LED FluoTrinoculer inverted microscope , serial number: 362094 )
  11. Refrigerated centrifuge (Hermle, model: Z446K ), used for larger volume (> 2 ml) or plate centrifugation
  12. Refrigerated centrifuge (Eppendorf, model: 5417R ), used for smaller volume (≤ 2 ml) centrifugation
  13. Probe Sonicator (Takashi, model: U150 ), equipped with a 6 mm diameter probe
  14. Gel electrophoresis set for SDS PAGE (GE Lifesciences, model: SE250 )
  15. Mini PAGE Blotting System (GE Lifesciences, Brand, catalog number: SBE2010-PB )
  16. Manifold Vacuum Filtration (Millipore, catalog number: M2536-1EA )
  17. Visible Fluorescent Western Blot Imaging System (Azure, model: Azure Biosystems c400 )

Software

  1. FACSDiva v6.2 (BD Biosciences) for flow cytometric analysis
  2. Prism (GraphPad, version 5.01), or free alternatives (e.g., R Bioconductor packages), for fitting of radioligand binding assay data
  3. Leica Microscope Imaging software (Leica), for estimation of cell density, viability and monitoring

Procedure

English
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文章信息

版权信息

© 2020 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Mozumder, S., Mahesh, G., Srinivasan, K., Sengupta, J. and Mukherjee, S. (2020). Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock. Bio-protocol 10(15): e3704. DOI: 10.21769/BioProtoc.3704.

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分类

生物物理学 > 显微技术 > 低温显微镜技术

生物化学 > 蛋白质 > 分离和纯化

分子生物学 > 蛋白质 > 表达

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