2.1 Construction of pET32a/ShK expression plasmid
DNA encoding the amino acid sequence of ShK and an N-terminal enterokinase cleavage site was synthesized by PCR using two overlapping primers and was cloned into a pET-32a expression vector (Novagen, USA). For efficient protein expression in bacterial cells, the DNA template was designed based on optimized codons for expression in E. coli. The forward primer (5’-CCAAGAGAATTCGATGATGATGATAAACGCAGCTGCATTGATACCATTCCGAAAAGCCGCTGCACCGCGTTTCAGTGCAAACAT) contained an EcoRI restriction endonuclease cleavage site (underlined) and enterokinase recognition site (double underlined). The reverse primer (5’AAGATCAAGCTTCTAGCAGGTGCCGCAGGTTTTGCGGCAAAAGCTCAGGCGATATTTCATGCTATGTTTGCACTGAAACGCGGT) contained a HindIII restriction endonuclease cleavage site (underlined). The PCR reaction mixture contained 50 pmol of each primer and 2.5 U of Taq DNA polymerase in a final volume of 50 µL. PCR amplification was performed by heating at 94 °C for 5 min followed by 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min and a final extension at 72 °C for 5 min. The amplified PCR product was digested with the restriction endonucleases EcoRI and HindIII and ligated into the thioredoxin-fusion tag-containing pET-32a vector. The sequence of the Trx-ShK fusion protein was confirmed by bi-directional nucleotide sequencing using the T7 promoter and terminator primers.
2.2 Expression of Trx-ShK fusion protein
E. coli BL21(DE3) cells transformed with the pET-32a-ShK expression vector were grown overnight at 37 °C in Luria-Bertani (LB) medium containing 100 µg/mL ampicillin. 1 L LB broth was inoculated with 1% of overnight culture and incubated at 37 °C with agitation at 200 rpm until the optical density at 600 nm (OD600) reached 0.5. The culture was incubated at 18 °C for 1 h before the culture was induced with isopropyl-β-D-thiogalactoside (IPTG, Astral Scientific) at a final concentration of 1 mM. The culture was further grown overnight at 18 °C and the cells were pelleted by centrifugation at 6000 g for 10 min at 4 °C and then stored at – 80 °C until further processing. Expression was analyzed by SDS polyacrylamide gel electrophoresis (12 % Bis-Tris SDS-PAGE gel, Bio-Rad) and the identity of the Trx-ShK fusion protein was confirmed by in-gel digestion with trypsin followed by LC/MS/MS analysis (Joint ProteomicS Laboratory, Parkville, Australia).
2.3 Refolding of Trx-ShK fusion protein
The cell pellet was thawed and lysed at room temperature for 30 min in a solution of 5 mL bugbuster master mix (Novagen) per gram of wet cell pellet containing 1 × protease inhibitor cocktail (lacking EDTA) (Roche, Germany). The lysed cells were centrifuged at 20,000 g for 20 min at 4 °C to pellet the inclusion bodies and the pellet was first washed with ice-cold buffer containing 2 M urea, 20 mM Tris, pH 8.0, 0.5 M NaCl, 2 % Triton X-100 and then briefly sonicated before spinning at 20,000 g for 10 min at 4 °C. The cell pellet was washed repeatedly with the urea-containing buffer solution and then with buffer alone. Subsequently, the inclusion bodies were resuspended in solublization buffer (20 mM Tris, pH 8.0, 0.5 M NaCl, 20 mM imidazole, 6 M guanidine hydrochloride, 1 mM DTT) and stirred for 60 min at room temperature. The sample was centrifuged for 20 min at 20,000 g at 4 °C and the supernatant was filtered through a 0.22 µm filter before being loaded onto a metal-chelating affinity column (5 mL Ni Sepharose, GE healthcare, USA) with a flow-rate of 1 mL/min. The column was washed with 2 column volumes of solublization buffer followed by 10 column volumes of wash buffer (20 mM Tris, pH 8.0, 0.5 M NaCl, 20 mM imidazole) containing 1 mM DTT, and 6 M urea. Immobilized protein was refolded on-column with a linear gradient of 6 to 0 M urea and 1 to 0 mM DTT at a flow-rate of 1 mL/min over 50 min at room temperature. The immobilized refolded protein was eluted with 20 column volumes of elution buffer (20 mM Tris, pH 8.0, 0.5 M NaCl, 0.5 M imidazole).
2.4 Purification of recombinant ShK
The Trx-ShK fusion protein was dialyzed extensively against enterokinase cleavage buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 2 mM CaCl2) at 4 °C for 24 h and then incubated with enterokinase (0.06 % w/w) at room temperature for 48 h. The cleaved protein was loaded onto a C18 reverse-phase high-performance liquid chromatography (RP-HPLC) column (Phenomenex, 100 × 10mm) equilibrated in buffer A (99.9 % water and 0.01 % TFA). Proteins were eluted with an acetonitrile gradient in 0.01 % TFA (0–26 % over 30 min followed by 26–64 % over 5 min). Eluted fractions containing ShK were lyophilized for storage.
2.5 Expression of 15N-labelled ShK
15N-labelled Trx-ShK fusion protein was expressed in E. coli BL21(DE3) using M9 minimal medium supplemented with 100 µg/mL ampicillin. The M9 medium contained 1 g/L 15NH4Cl (Cambridge Isotope Labs) as the sole nitrogen source and 4 g/L glucose as the carbon source. Cultures were grown at 37 °C until mid-log phase (OD600 =0.5) was reached, cooled at 18 °C for 1 h and then induced by adding IPTG to a final concentration of 1 mM. The cultures were incubated overnight with agitation at 18 °C. The cells were pelleted by centrifugation at 6000 g for 10 min at 4 °C, lysed with bugbuster master mix containing protease inhibitors, and protein in the pellet (inclusion bodies) was purified as outlined in sections 2.3 and 2.4 above.
2.6 Determination of recombinant protein concentration, purity and mass
Protein and peptide concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce) with bovine serum albumin as a standard. The BCA assay was calibrated against peptide content by amino acid analysis on several ShK samples (Florey Neuroscience Institutes, Australia). Protein purity was determined by SDS-PAGE on 12 % gels using Coomassie blue staining and analytical RP-HPLC.
Protein mass was determined by electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry using a Waters LCT TOF LC/MS Mass Spectrometer coupled to a 2795 Alliance Separations module and Masslynx software version 4.1 (Waters, Australia). Mass spectra were created by averaging the scans across each total ion current peak and subtracting background. The mass spectrometer conditions were as follows: ionisation mode electrospray ionisation, desolvation gas flow 550 L/h, desolvation temperature 250 °C, source temperature 110 °C, capillary voltage 2400 V, sample cone voltage 60 V, scan range acquired 100–1500 m/z, scan time: 1 sec, internal reference ions positive ion mode = m/z = 556.2771.
2.7 NMR spectroscopy
NMR spectra were recorded on samples of 0.2 mM unlabelled or 0.6 mM 15
N-labelled ShK in 90 % H2
O/10 % 2
O at pH 4.9. The 1D 1
H NMR spectrum was acquired on a Bruker DRX 600 MHz spectrometer while the 2D 1
N HSQC and SOFAST-HMQC spectra were acquired on a Varian INOVA 600 MHz spectrometer. Both spectrometers were equipped with cryogenic probes. The water resonance was suppressed using the WATERGATE pulse sequence (Piotto et al., 1992
). All spectra were collected at 20 °C and referenced to the water resonance. Spectra were acquired over 16384 (1D) or 4096 (2D) data points, with a 1
H spectral width of 14 ppm. The 1D spectrum was acquired with 1024 scans. The HSQC was acquired with 32 scans per increment using 2048 points in the proton dimension and 192 increments in the 15
N dimension. The SOFAST-HMQC spectrum (Schanda and Brutscher, 2005
) was acquired with 32 scans per increment using 2048 points in the proton dimension and 40 increments in the 15
N dimension. The 15
N dimension of the SOFAST-HMQC spectrum was extended to 120 points with linear prediction. The 15
N dimension of both spectra was apodized with a shifted sine-bell apodization function and zero filled to 1024 complex points prior to Fourier transformation. The spectral widths were 14.0 and 40 ppm in the 1
H and 15
N dimensions, respectively. The 3D 15
N-edited NOESY-HSQC spectrum was acquired with a mixing time of 120 ms, 16 scans per increment, 2048 complex points in the acquisition dimension, and 96 and 80 increments in the indirect 1
H and 15
N dimensions, respectively.
2.8 Animals, cells and cell lines
L929 mouse fibroblast cells stably expressing mKv1.1 and mKv1.3 channels (Grissmer et al., 1994
) were gifts from Dr. K. George Chandy (University of California, Irvine). They were maintained in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10 IU/mL penicillin, 0.1 µg/mL streptomycin, 2 mM L-glutamine, 10 % heat-inactivated fetal bovine serum, and 0.5 mg/mL G418 (EMD Chemicals, Gibbstown, NJ).
Female Lewis rats (8–10 weeks old) were purchased from Harlan-Sprague Dawley (Indianapolis, IN, USA) and housed under pathogen-free conditions with food and water ad libitum
. Animals were euthanised under a Baylor College of Medicine Animal Use and Care Committee approved protocol for blood, spleen, and thymus collection, as described (Beeton and Chandy, 2007
Freshly prepared buffy coats were purchased from the Gulf Coast Regional Blood Center (Houston, TX) under a protocol approved by the Baylor College of Medicine Institutional Review Board. Mononuclear cells were isolated from the buffy coats using Histopaque-1077 gradients (Sigma, St Louis, MI) and used immediately.
cells expressing high levels of Kv1.3 channels upon activation were a gift from Dr. Alexander Flügel (University of Göttingen, Germany) and were maintained in culture as described previously (Kawakami et al., 2005
; Matheu et al., 2008
2.9 Electrophysiological analysis
Experiments were conducted at room temperature in the whole-cell configuration of the patch-clamp technique. The patch pipettes had a resistance of 2–4 MΩ when filled with a solution containing (in mM): 145 KF, 10 HEPES, 10 EGTA, and 2 MgCl2, pH 7.2, 290 mOsm. The bath solution contained (in mM): 160 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, pH 7.2, 300 mOsm. Kv currents were elicited every 30 s by 200-ms depolarizing pulses from a holding potential of −80 mV to 40 mV. Kd values and Hill coefficients were determined by fitting the Hill equation to the reduction of peak current measured at 40 mV.
2.10 T lymphocyte proliferation assays
Proliferation assays were conducted as described previously (Beeton et al., 2001a
; Beeton et al., 2005
; Matheu et al., 2008
). Briefly, mononuclear cells (105
/well) or rat ovalbumin-specific TEM
/well) were plated into 96-well plates and pre-incubated with the peptide blockers for 45 min at 37 °C in RPMI medium (Invitrogen) supplemented with 10 IU/mL penicillin, 0.1 µg/mL streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, 1% RPMI vitamins, 50 µM β-mercaptoethanol, and 1% heat-inactivated fetal bovine serum (human cells) or 1% Lewis rat serum (rat cells). Human T lymphocyte activation and proliferation was induced by the addition of 60 ng/mL anti-CD3 antibodies (Clone OKT3, eBioscience, San Diego, CA). Rat splenocytes were stimulated with 1 µg/mL concanavalin A (Sigma). Rat TEM
cells were stimulated with 10 µg/mL ovalbumin in the presence of irradiated thymocytes as antigen-presenting cells. Cells were cultured for 72 h at 37 °C, 5% CO2
, and [3
H] thymidine was added during the last 16–18 h of culture. Cells were lysed by freezing at −20 °C. After thawing, DNA was harvested onto fibreglass filters using a cell harvester (Inotech Biosystems International, Rockville, MD). [3
H] thymidine incorporation into the DNA of proliferating cells was measured using a β-scintillation counter (Beckman Coulter, Brea, CA).