Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev

Abstract

Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins' application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense.

Figure 1. Comparison between the purification of (His₆)-tagged Rev using either (A) PEI or (B) urea denaturation protocols

Aliquots from individual steps of the purification of (His₆)-tagged Rev by the PEI or the urea protocols were subjected to electrophoresis on a 4–20% acrylamide gel. Lane 1, protein markers; lanes 2 and 6, total E. coli intracellular proteins; lanes 3 and 7, total soluble proteins; lanes 4 and 8, fractions (1 μg) purified by nickel column; lane 5, fraction (1 μg) purified from cationic exchange column. The molecular weight (kDa) of the marker proteins is indicated on the left.

Figure 2. Flowcharts of the purification protocols adopted to remove nucleic acid contaminants from (His₆)-tagged Rev protein

(A) Purification requires a multi-step protocol in order to remove contaminating nucleic acid. Following an initial purification by immobilized-metal affinity chromatography (IMAC), nucleic acids are effectively removed by PEI precipitation and ammonium sulfate fractionation. A final purification by a cationic exchange column yields pure (His₆)-tagged Rev without nucleic acid contamination. (B) Devised one-step purification of (His₆)-tagged Rev based on urea denaturation followed by successive on-column refolding. After urea denaturation, (His₆)-tagged Rev is refolded on IMAC ensuring the removal of contaminants in the final preparation.

Figure 3. Urea-induced unfolding transition of (His₆)-tagged Rev

The change of the dichroic signal at 222 nm (expressed as molar ellipticity per residue, θ) for (His₆)-tagged Rev at 10 μM concentration was monitored at 25 °C as a function of increasing concentrations (from 0 to 8 M) of urea. Data was fitted using Eq. 1–3 (solid line).

Figure 4. CD analysis of purified (His₆)-tagged Rev

CD spectra of purified Rev following nucleic acid removal by (A) PEI precipitation or (B) urea denaturation/on-column refolding. Shown is the molar ellipiticity per residue (θ) corrected for the background buffer. (C) and (D) Multicomponent analysis of the CD spectra of (His₆)-tagged Rev purified by (C) PEI precipitation or (D) urea denaturation/on-column refolding, respectively. The CD spectra A and B were deconvoluted using the algorithms CONTIN/LL (black bars), SELCON 3 (light gray bars), and CDSSTR (dark gray bars). Bars labeled “unordered” include estimated percentage of unordered structures and turns.

Figure 5. Gel-mobility shift assay of HIV-1 RRE stem II with (His₆)-tagged Rev protein

All samples contained 1 × 10⁻⁹ M radiolabeled RNA; samples b-l contained 12.5, 25, 50, 100, 200, 400 800, 1000, 2000, 3000, 4000 × 10⁻⁹ M (His₆)-tagged Rev purified according the (A) PEI or (B) the urea denaturation/on-column refolding protocol, respectively. Samples were resolved on 8% (w/v) polyacrylamide gel cast run at room temperature in a water-cooled electrophoresis apparatus. Observable species are designated F (free RNA) or numbered (1–2) to indicate the (His₆)-tagged Rev:RNA ratio of the corresponding complexes.