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

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 onedimensional ¹H NMR spectroscopy and circular dichroism. This protocol is applicable for the purification of other zinc finger containing proteins.

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

Figure 1

High-pressure homogenizer (Emulsiflex C5), cooled with the Fisher Scientific Isotherm Model 900 cooler

Figure 2. Coomassie-stained SDS-Page of purified p300-CH1 domain expressed from E. coli

1. Bacterial lysate upon expression. 14 µl of bacterial lysate before capture from 50 ml of total lysate sample from 4 L bacterial growth medium. 2. Clarified bacterial lysate. 14 µl lysate after capture from 50 ml of total lysate sample from 4 L bacterial growth medium. 3. GST-tagged p300-CH1 immobilized to GSH-Agarose beads. 10 µl beads after capture from 3 ml bed volume of GSH-Agarose beads. 4. Bead-immobilized protease cleavage product: Uncut GST-tagged p300-CH1 (37 kDa) and GST (25 kDa). 10 µl beads after cleavage from 3 ml bed volume of GSH-Agarose beads. 5. Supernatant protease cleavage product: p300-CH1 and free GST impurity. 14 µl supernatant after cleavage from 20 ml total supernatant. 6. Free GST impurities captured with GSH-Agarose beads. 10 µl GSH-Agarose bead capture (GST + GST-p300 + Prescission) from 2 ml bed volume of GSH-Agarose beads. 7. Purified recombinant p300-CH1. 14 µl sample from 20 ml of total recombinant p300-CH1 sample.

Figure 3. CD spectra of purified p300-CH1 in the absence (red line) or presence (black line) of 3 equivalents of Zn²⁺

40 µl of p300-CH1 (700 µM in alkaline resuspension buffer supplemented with reducing agent) were loaded in a 0.1 mm quartz cuvette. EDTA treated sample contained 700 µM p300-CH1 and 2.1 mM EDTA (3 stoichiometric equivalents). Collected 3 scans at normal sensitivity in Jasco J-810 spectropolarimeter.

Figure 4. Expansion of 1D ¹H NMR spectrum, highlighting amide–NH’s, aromatic and NH side chain resonances of purified natural abundance p300-CH1 in the presence of 3 equivalents of Zn²⁺

300 µl of purified recombinant p300-CH1 (µM) was filter centrifuged into deuterated buffer (10% Deuterium Oxide (v/v) with 50 mM Tris-d₁₁, 150 mM NaCl, and 5 mM DTT-d₁₀, see Recipes) and injected into Varian INOVA 600MHz NMR spectrometer.