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Purifying Properly Folded Cysteine-rich, Zinc Finger Containing Recombinant Proteins for Structural Drug Targeting Studies: the CH1 Domain of p300 as a Case Example

YK Yong Joon Kim
SK Stefan Kaluz
AM Anil Mehta
EW Emily Weinert
SR Shannon Rivera
Erwin G. Van Meir Erwin G. Van Meir

Published: Sep 5, 2017 DOI: 10.21769/BioProtoc.2537 Views: 12319

Reviewed by: Alessandro Didonna

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Abstract

The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies. p300-CH1 has 3 zinc fingers and 9 cysteine residues, posing challenges associated with reagent compatibility and protein oxidation. A protocol has been developed to overcome such issues by incorporating zinc during expression and streamlining the purification time, resulting in a high yield of optimally folded protein (120 mg per 4 L expression media) that is suitable for structural NMR studies. The structural integrity of the final recombinant p300-CH1 has been verified to be optimal using one-dimensional 1H NMR spectroscopy and circular dichroism. This protocol is applicable for the purification of other zinc finger containing proteins.

Keywords: p300 search CBP search HIF search CH1 search Cysteine search Zinc finger search Hypoxia search Recombinant protein purification search

Background

The growth of solid tumors is associated with the development of hypoxic areas due to inappropriate vascular irrigation. In response to a hypoxic microenvironment, tumor cells overexpress Hypoxia Inducible Factors (HIF), a family of heterodimeric transcription factors (Semenza, 2002; Brat and Van Meir, 2004; Kaur et al., 2005). HIFs bind to p300, a transcriptional coactivator, to form a complex that induces HIF target genes, thereby activating the hypoxia response pathway and promoting tumor growth (Kasper and Brindle, 2006; Liu, 2008). The binding domains involved with the HIF/p300 protein-protein interface are the cysteine-histidine-rich region 1 (CH1) domain of p300 and the C-terminal activating domain (CAD) of HIF-1α (Dames et al., 2002; Freedman et al., 2002). The hypoxia response pathway facilitates tumor growth under oxygen limiting conditions. Inhibiting this pathway is a goal for targeted anti-cancer therapy (Post et al., 2004; Belozerov and Van Meir, 2006; Mooring, 2011; Tan et al., 2011; Mun et al., 2012; Wilkins et al., 2016). Small molecule compounds have recently been developed to bind to p300-CH1 and inhibit p300/HIF complexation, inhibiting the hypoxia response pathway and reducing tumor growth (Shi et al., 2012; Yin et al., 2012; Burroughs et al., 2013). Furthermore, the p300-CH1 domain has been reported to interact with over 30 additional transcription factors related to cancer and other diseases (Kasper and Brindle, 2006).

This protocol has been developed to purify the p300-CH1 peptide with the purity and structural integrity necessary to conduct structural NMR studies for studying protein-protein and protein-ligand interactions. p300-CH1 poses many challenges for recombinant protein purification, as it contains three zinc fingers and 9 total cysteine residues. p300-CH1 has been reported to be structurally compromised without a 3:1 stoichiometric ratio of zinc (De Guzman et al., 2005). Without zinc, cysteine residues typically found at zinc fingers can form unwanted disulfide linkages that are thermodynamically more stable than cysteine-zinc interactions. Proteins without zinc cations occupying their native zinc fingers are prone to oxidation and readily form disulfide linkages, resulting in unwanted protein conformations and protein aggregation.

Introducing zinc (II) to a protein purification protocol is complicated by the fact that zinc (II) interacts with certain buffer salts, reducing agents, and hydroxide ions to form precipitates. Unwanted zinc (II) precipitation can prevent the formation of crucial zinc fingers and even leach out existing zinc fingers to denature the protein. In addition, the reducing agents and buffer salts that precipitate with zinc may be unavailable to serve their respective purposes for the purification. Zinc (II) cations form white ZnOH precipitates with neutral to alkaline buffer conditions (pH 7.0 and above). Zinc (II) also forms precipitates with phosphate buffer and thiol reducing agents (BME and DTT). The buffers and reagents used for the purification procedure must be carefully selected to be compatible with zinc. As a precaution, zinc stocks and zinc containing solutions must be pH adjusted with zinc present to verify that ZnOH precipitates do not form during purification. This is especially important in purifying high yields of properly folded zinc-finger containing proteins.

The purification protocol has been designed with the following strategies to minimize oxidation and maximize yield:
1. Occupy cysteine residues with zinc (II) throughout the protocol
2. Streamline the duration of expression and cleavage
3. Use reducing agents whenever possible

The protocol successfully overcomes issues of reagent compatibility and oxidation, yielding 120 mg of purified recombinant p300-CH1 from a 4 L bacterial culture. Concentration of recombinant p300-CH1 was measured by UV-Vis absorbance at a wavelength of 280 nm. The extinction coefficient of p300-CH1 is calculated to be 5,690 L mol-1 cm-1 by ExPASy ProtParam tool (Shi et al., 2012). According to circular dichroism and 1H NMR, the purified p300-CH1 recombinant protein product is optimally folded without the need for any further modification post-purification. The protocol and its strategies can be applied to other cysteine containing proteins systems to improve yield and purity.

Materials and Reagents

  1. 14 ml polypropylene round-bottom tube (Corning, Falcon®, catalog number: 352057 )
  2. 1.7 ml polypropylene microcentrifuge tube (Posi-Click Eppendorf) (Denville Scientific, catalog number: C2170 )
  3. Polypropylene micropipette tips
  4. 50 ml polypropylene conical bottom tube (Corning, catalog number: 430291 )
  5. Two-sided disposable plastic cuvettes 10 mm (VWR, catalog number: 97000-586 )
  6. 250 ml wide-mouth plastic bottle (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: N411-0250 )
  7. 15 ml polypropylene conical bottom tube (Corning, catalog number: 430052 )
  8. Chemically competent E. coli (One Shot® BL21 Star (DE3)) (Thermo Fisher Scientific, InvitrogenTM, catalog number: C601003 ); store at -80 °C
  9. pGEX-6P-1 plasmid expression vector (GE Healthcare, catalog number: 28-9546-48 ) containing GST-p300-CH1 domain cDNA
    Note: The p300-CH1 insert was isolated from a Thrombin-site containing expression vector (pGEX-2T vector containing p300-CH1 insert) (http://web.expasy.org/protparam/) using BamHI and EcoRI restriction enzymes. The insert was cloned into the pGEX-6P-1 vector.
  10. Ice
  11. LB Miller broth powdered (Fisher Scientific, catalog number: BP1426-2 )
  12. Ampicillin sodium salt powdered (Sigma-Aldrich, catalog number: A9518 )
    Note: Prepare 100 mM stock (1,000x) in deionized water. Filter sterilize and store at -20 °C.
  13. Plasmid DNA Extraction Miniprep Kit (GeneJET) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K0502 )
  14. BamHI restriction enzyme FastDigest (800 µl, 1 reaction/1 µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0054 ); store at -20 °C
  15. Restriction enzyme buffer FastDigest (10x; 5x 1 ml) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: B64 ); store at -20 °C
  16. EcoRI restriction enzyme FastDigest (800 µl, 1 reaction/1 µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: FD0274 ); store at -20 °C
  17. Quick-Load 1 kb DNA ladder (New England Biolabs)
  18. 5x nucleic acid loading buffer (10 ml, premixed) (Bio-Rad Laboratories, catalog number: 1610767 )
  19. Deionized water
  20. Acrylamide electrophoresis gels (Criterion TGX Precast Gels; 18 well comb, 30 µl loading volume per well, 1.0 mm thickness, 4-20% gel percentage) (Bio-Rad Laboratories, catalog number: 5671094 )
  21. Agar powdered (Fisher Scientific, catalog number: BP24662 )
  22. 1x TAE running buffer
  23. Ethidium bromide (10 mg/ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15585011 )
  24. Glycerol 100% (EMD Millipore, catalog number: GX0185 )
  25. Isopropyl β-D-1-thiogalactopyranoside (IPTG) powdered (Gold Bio, catalog number: I2481C100 )
    Note: Prepare 10 ml of 1 M (10,000x) stock in deionized water and store at -20 °C.
  26. Zinc chloride (ZnCl2), 99.999% trace metals basis powdered (Sigma-Aldrich, catalog number: 229997 )
    Note: Prepare as 100 mM stock in deionized water.
  27. Bleach
  28. Liquid nitrogen
  29. DNase I (2,000 U/ml; 1 ml) (New England Biolabs, catalog number: M0303S ); store at -80 °C
  30. RNase A (1 ml, 1 mg/ml) (Bioo Scientific, catalog number: 344005 )
  31. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (PEFA-BLOC) powdered (Biosynth, catalog number: A-5440 )
  32. Benzamidine HCl, > 99% powdered (RPI, catalog number: B12000-100.0 )
  33. Glutathione agarose beads (10 ml 50% slurry in 0.05% sodium azide solution) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 16100 )
  34. HRV 3C Cysteine Protease PreScission Site (1 mg lyophilized) (AG Scientific, catalog number: H-1192 )
    Note: Prepare as 2 mg/ml in Prescission Protease buffer, and store as 50 µl aliquots; store at -80 °C.
  35. Coomassie Brilliant Blue G-250 (Bio-Rad Laboratories, catalog number: 1610406 )
  36. Ethylenediaminetetraacetate acid disodium salt (EDTA)
  37. Tris base (White Crystals or Crystalline Powder/Molecular Biology) (Fisher Scientific, catalog number: BP152 )
  38. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-10 )
  39. Sodium hydroxide (NaOH) (BioUltra, for molecular biology, 10 M in H2O) (Sigma-Aldrich, catalog number: 72068 )
  40. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (powder, ≥ 98% purity, 10 g) (Sigma-Aldrich, catalog number: C4706 )
  41. Tris-d11
  42. DTT-d10
  43. Acidic resuspension buffer (pH 6.3) (see Recipes)
  44. Alkaline resuspension buffer (pH 8.0) (see Recipes)
  45. HRV3C PreScission Site Protease buffer (pH 8.0) (see Recipes)
  46. 10% (v/v) deuterated Buffer for NMR (pH 8.0) (see Recipes)
  47. LB Miller medium (see Recipes)

Equipment

  1. Variable Micropipets (Edvotek, catalog numbers: 591 , 5911 , 5921 )
  2. Large silicone non-stick kitchen spatula
  3. Orbital shaker (FormaOrbital Shaker, Thermo Electron)
  4. Autoclave (STERIS, model: SV-120 )
  5. Benchtop shaker (Jeio Tech, Lab Companion, model: SK-300 )
  6. Centrifuge (Beckman Coulter, model: Avanti J-20XP )
  7. Centrifuge rotor with 250 ml rotor cavities (16,000 rpm rotor) (Beckman Coulter, model: J-LITE® JLA-16.250 )
  8. 2 L glass flask (Corning, PYREX®, catalog number: 4980-2L )
  9. -80 °C freezer
  10. High pressure homogenizer (Avestin, model: EmulsiFlex-C5 )
  11. Fisher Scientific Isotherm Model 900 cooler (Fisher Scientific, model: Model 900 )
  12. Ultracentrifuge (Beckman Coulter, model: OptimaTM XE-90K )
  13. Ultracentrifuge rotor with 50 ml rotor cavities (Beckman Coulter, model: SW 40 Ti )
  14. Vertical rotating wheel (Labquake Tube Shaker Rotisserie) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: C415110 )
  15. Benchtop centrifuge (Thermo Fisher Scientific, model: SorvallTM ST 16 , catalog number: 75004240)
  16. Swinging bucket rotor with 400 ml rotor cavities (Thermo Fisher Scientific, Thermo ScientificTM, model: TX-400 , catalog number: 75003629)
  17. Protein electrophoresis apparatus (Bio-Rad Laboratories, model: CriterionTM Cell , catalog number: 1656001)
  18. Spectropolarimeter (JASCO, model: J-810 ; 163-900 nm)
  19. Spectrophotometer (Eppendorf, model: BioPhotometer® plus , catalog number: 952000006)
  20. 600 MHz NMR spectrometer (Varian, INOVA)

Procedure

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.

Category

Cancer Biology > Cancer biochemistry > Protein

Biochemistry > Protein > Isolation and purification

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