Rev serine-to-glutamate point mutations were introduced using a PCR-based protocol adapted from the QuikChange site-directed mutagenesis method (Strategene). PCR amplification was performed with Phusion High-Fidelity PCR Master Mix (NEB) using the expression plasmid pET28a-His-GB1-TEV-Rev (see below) as a template and specific mutagenic primers. PCR products were digested by DpnI. Final mutations were selected by transformation and verified by DNA sequencing.
Protein expression and purification
N-terminally His-tagged Impβ was expressed from a pETM-11 plasmid in E. coli strain BL21 (DE3). Cells were grown in LB medium containing kanamycin (50 μg/ml) at 37°C until reaching an optical density at 600 nm of 0.5–0.6, induced with 0.5 mM IPTG and further incubated for 5 h at 30°C. Bacteria were collected by centrifugation (6,200 g, 20 min, 4°C) and resuspended in lysis buffer [50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM imidazole, 10 mM MgCl2, 2 mM β-mercaptoethanol (BME)] containing nuclease and protease inhibitors [10 μg/ml DNase I, 10 μg/ml RNase A, 2 mM PMSF and 1 tablet/80 ml of complete EDTA-free protease inhibitor cocktail (Roche)]. Cell lysis was carried out by sonication (15 s pulses separated by 30 s, 5 min total sonication time) and the soluble fraction was recovered by centrifugation (50,000 g, 20 min, 4°C). The soluble fraction was then loaded onto a HisTrap FF NiNTA column (GE Healthcare) pre-equilibrated with Buffer A (50 mM HEPES pH 7.5, 200 mM NaCl, 2 mM BME, 10 mM imidazole). After extensive washing with buffer A, the bound protein was eluted in 2 steps, first with 40 mM and then with 300 mM imidazole. Fractions containing Impβ were further purified using a Superdex 200 16/60 gel filtration column (GE Healthcare) pre-equilibrated with 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP). Fractions containing pure Impβ were pooled, concentrated to >70 mg/ml on an Amicon centrifugal filter (30 kDa cutoff, Millipore) and stored as aliquots at -80°C. The Impβ protein concentration was determined using a molar extinction coefficient of 79,051 M-1cm-1, which was experimentally determined by quantitative amino acid analysis.
Rev proteins were expressed from a pET-28a plasmid as fusion constructs bearing an N-terminal His tag followed by the acidic B1 domain of Streptococcal protein G (GB1 domain) and a tobacco etch virus (TEV) protease site. The use of the GB1 tag has been shown to improve Rev solubility and facilitate purification. Transformed E. coli BL21 (DE3) cells were grown in Auto-Induction Medium containing kanamycin (50 μg/ml) for 12 h at 27°C. Bacteria were collected by centrifugation (6,200 g, 20 min, 4°C) and resuspended in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 100 mM Na2SO4, 10 mM MgCl2, 10 mM imidazole, 0.1% Tween 20) containing nuclease and protease inhibitors (as described above for Impβ). Cells were lysed and the lysate was centrifuged as described above for Impβ. The clarified lysate was incubated with RNAse T1 (20 U/ml, Roche), RNase A (20 μg/ml, Euromedex) in the presence of 2 M NaCl for 1.5 h at room temperature before performing a new centrifugation step (50,000 g, 10 min, 4°C). The soluble fraction was loaded onto a HisTrap FF NiNTA column (GE Healthcare) pre-equilibrated with high-salt buffer B (2 M NaCl, 50 mM Tris pH 8, 0.1% Tween 20, 2 mM BME and 10 mM imidazole). After extensive washing with high-salt buffer B followed by a second wash step with low-salt buffer B (containing 250 mM NaCl and no Tween 20), the bound protein was eluted in 2 steps with low-salt buffer B containing first 40 mM and then 300 mM imidazole. Fractions containing His-GB1-Rev protein were diluted with an equal volume of 50 mM Tris pH 8, 125 mM NaCl before loading onto HiTrap Heparin HP columns (GE Healthcare) pre-equilibrated in 50 mM Tris pH 8, 125 mM NaCl. After extensive washing with 50 mM Tris pH 8, 125 mM NaCl, elution was performed using 50% High Salt Buffer (HSB) (50 mM Tris pH 8, 200 mM NaCl, 400 mM (NH4)2SO4, 100 mM Na2SO4, 2 mM BME) and 50% of 2 M NaCl. The His-GB1 tag was then removed from the Rev protein by overnight incubation with His-tagged TEV protease (1:20 w/w TEV/Rev protein ratio) supplemented with 2 mM BME and 2 mM PMSF. The untagged Rev protein was recovered in the flow-through after passing the sample through a new NiNTA column pre-equilibrated as described above. The untagged Rev protein was then diluted with an equal volume of 50 mM Tris pH 8 and then concentrated by repeating the heparin chromatography step described above. Heparin elution fractions containing Rev were applied onto a Superdex 75 16/60 gel filtration column (GE Healthcare) pre-equilibrated with HSB containing 1 mM TCEP. Fractions containing pure Rev were pooled, concentrated on an Amicon centrifugal filter (10 kDa cutoff, Millipore) and stored at -80°C. Rev protein concentration was determined using a molar extinction coefficient of 9476 M-1cm-1, which was experimentally determined by quantitative amino acid analysis.
N-terminally His-tagged TEV protease bearing the point mutation S219V was expressed from a pRK793 plasmid in E. coli strain BL21 (DE3). Cells were grown in LB medium containing ampicillin (100 μg/ml) at 37°C until reaching an optical density at 600 nm of 0.8, induced with 1 mM IPTG and further incubated for 4 h at 30°C. Bacteria were collected by centrifugation (6,200 g, 20 min, 4°C) and resuspended in lysis buffer [phosphate buffered saline (PBS), 10 % glycerol, 25 mM imidazole]. Cell lysis was carried out by sonication (5 s pulses separated by 15 s, 5 min total sonication time). DNA and RNA were precipitated by the addition of 0.1% (v/v) polyethyleneimine (PEI) and the resulting precipitate removed by centrifugation (50,000 g, 20 min, 4°C). The soluble fraction was then loaded onto a HisTrap FF NiNTA column (GE Healthcare) pre-equilibrated with lysis buffer. After extensive washing with lysis buffer containing first 25, then 50 mM imidazole, the bound protein was eluted with PBS, 10% glycerol, 500 mM imidazole. Fractions containing TEV were further purified using a Superdex 200 16/60 gel filtration column (GE Healthcare) pre-equilibrated with PBS, 10% glycerol, 2 mM EDTA, 10 mM DTT. Fractions containing pure TEV were pooled and the concentration was adjusted to 1 mg/ml as determined in a Biorad Bradford Assay. Aliquots were stored at -80°C.
Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC/ESI-MS)
LC/ESI-MS was performed on a 6210 LC-TOF spectrometer coupled to an HPLC system (Agilent Technologies). All solvents used were HPLC grade (Chromasolv, Sigma-Aldrich). Trifluoroacetic acid (TFA) was from Acros Organics (puriss., p.a.). Solvent A was 0.03% TFA in water; solvent B was 95% acetonitrile–5% water–0.03% TFA. Immediately before analysis, protein samples were diluted to a final concentration of 5 µM with solvent A. Protein samples were then desalted on a reverse phase-C8 cartridge (Zorbax 300SB-C8, 5 µm, 300 µm ID´5 mm, Agilent Technologies) at a flow rate of 50 μl/min for 3 min with 100% solvent A and subsequently eluted with 70% solvent B for MS detection. MS acquisition was carried out in the positive ion mode in the 300–3200 m/z range. MS spectra were acquired and the data processed with MassHunter workstation software (v. B.02.00, Agilent Technologies) and with GPMAW software (v. 7.00b2, Lighthouse Data, Denmark).
The masses of the full-length proteins determined by LC/ESI-MS were in excellent agreement with the expected masses (shown in parentheses) and were as follows : 100,298.91 ± 2 Da for Impβ (100,298.91 Da); 13,174.51 ± 0.67 Da for WT Rev (13,175.85 Da); 13,427.53 ± 0.55 Da for 6xSE (13,428.08 Da); 13,259.81 ± 0.12 Da for S5E/S8E (13,259.93 Da); 13,258.78 ± 0.87 Da for S54E/S56E (13,259.93 Da); 13,259.61 ± 0.32 Da for S92E/S99E (13,259.93 Da); 13,216.74 ± 0.86 Da for S54E (13,217.89 Da) and 13,216.74 ± 0.86 Da for S56E (13,217.89 Da). The spectra showed an absence of any contaminating proteins or degradation products.
Differential scanning fluorimetry (NanoDSF)
Impβ and Rev proteins were first independently diluted to 50 μM in 50 mM HEPES pH 7.5, 100 mM NaCl, and then incubated in a 1:1 Impβ:Rev ratio (5 μM each, 15 μl final volume) for 5 min at 25°C. Tryptophan fluorescence at 330 nm and 350 nm were monitored between 20°C and 90°C with a 1°C/min increasing temperature ramp on a Prometheus NT.48 instrument (Nanotemper) using standard grade capillaries (Nanotemper ref. PR-C002). Tm values were determined at the inflection points of the first derivative of the tryptophan fluorescence ratio (F350 nm/F330 nm) curves.
Native gel shift assay
Impβ and Rev proteins were initially diluted in phosphate buffered saline (PBS) to 25 μM, and incubated in a 1:1 Impβ:Rev ratio (3.7 μM each, 10 μl final volume) for 5 min. After adding 1 μl of native loading buffer (62.8 mM Tris HCl pH 6.8, 40% glycerol, 0.01% bromophenol blue), 2.5 μl of samples were analysed on a 10% TGX (Biorad) gel, run under native conditions (0.5x TAE buffer, 4°C, 110 V, 50 min). Gels were stained with Coomassie blue and scanned on a ChemiDoc MP gel imaging system (Biorad). Protein band intensities were quantified using Adobe Photoshop Version CS5 by integrating pixel intensities in a rectangle encompassing the band and subtracting the background signal determined from an adjacent region of the gel.
Fluorescence polarization binding and inhibition assays
A Rev-NLS-TAMRA peptide, corresponding to the Rev NLS (32-EGTRQARRNRRRRWRERQR-50) N-terminally fused to tetramethylrhodamine (TAMRA) was commercially synthesized (Peptide Specialty Laboratories, Heidelberg). Fluorescence polarization (FP) was measured in black 384-well plates (Greiner ref. 781076, Greiner Bio-One) on a Clariostar plate reader (BMG Labtech) using a 540 ± 20 nm excitation filter, a 590 ± 20 nm emission filter and an LP565 dichroic mirror. The FP values were determined using the equation F = (F// - F⊥)/(F// + F⊥), where F// and F⊥ are the fluorescence intensities parallel and perpendicular to the excitation plane, respectively. The concentration of the Rev-NLS-TAMRA peptide was determined using its specific molar extinction coefficient at 556 nm of 89 000 M-1cm-1.
30 μl of Impβ (6.25 μM) were prepared in Fluorescence Polarization (FP) buffer (20 mM Tris pH 8, 100 mM NaCl, 0.05% Tween 20, 1 mM DTT) and used to perform 14 two-fold serial dilution of Impβ (3.1 μM to 0.2 nM) by mixing 15 μl of sample with 15 μl of FP buffer. A sample containing only FP buffer was used as a reference. 2x Rev-NLS-TAMRA dilution buffer was prepared containing 200 nM of Rev-NLS-TAMRA diluted in FP buffer and dispensed into 16 wells of a black 384-well plate (10 μl/well). To each well 10 μl of Impβ dilutions or reference buffer were added and mixed by pipetting. The plate was incubated for 20 min at room temperature before reading.
The concentration of Rev variants were adjusted to 100 μM in HSB, before preparing a 30 μl aliquot at 70 μM using 2x Rev-NLS-TAMRA dilution buffer. Then 14 2-fold serial dilutions of each Rev variant (35 μM to 4.3 nM) were performed by mixing 15 μl of sample with 15 μl of Rev dilution buffer. A sample containing only Rev dilution buffer was used as a reference. A 2x Impβ solution containing 390 nM Impβ and FP buffer was prepared and dispensed into 16 wells of a black 384-well plate (10 μl/well). In each well, 10 μl of Rev dilutions or reference buffer were added and mixed by pipetting. The plate was incubated for 20 min at room temperature before reading.
Curves obtained with each phosphomimetic mutant were independently fitted and the IC50 values were determined with GraphPad Prism 6 software. The option “Log [Agonist] vs. response (3 parameters)” was used to fit binding data to the equation F=Fmin + (Fmax - Fmin)/(1 + 10LogEC50 - X), where Fmin and Fmax are the minimal and maximal values of fluorescence polarization and X is the common logarithm of Impβ concentration. The option “Log [Inhibitor] vs. response (3 parameters)” was used to fit inhibition data to the equation F=Fmin + (Fmax - Fmin)/(1 + 10X-LogIC50), where X is the common logarithm of Rev concentration.