Nickel nitrilotriacetic acid

Cloning of SARS-CoV 3C-like Proteinase —The reverse transcriptional mixture of SARS-CoV RNA from supernatant fluid of the virus-infected Vero cells using random primers was generously supplied by Dr. Y. Lu from Zhejiang Provincial Center for Disease Prevention and Control. For cloning of the cDNA of 3C-like proteinase of the virus, the first stand cDNA mixture was subjected to PCR amplification using a pair of specific primers, comprising F99 (5′-AGT GGT TTT AGG AAA ATG GCA TTC CC-3′) and R108 (5′-TTG GAA GGT AAC ACC AGA GC-3′) to amplify a 917-bp fragment containing full-length 3C-like proteinase coding sequence. The PCR products were purified by agarose gel electrophoresis and then cloned directly into pGEM T Easy vector (Promega, Madison, WI). The resultant sequence confirmed that the amplified fragment was the same as that of SARS-CoV 3C-like proteinase.
Construction of Plasmid pET 3CLP-21x —The gene of the SARS 3C-like proteinase was amplified by PCR from the cloning vector (pGEM T Easy) described above using primers 3CLP-Nhe (5′-CACTGCTAGCGGTTTTAGGAAAATGGCATTCCC-3′) and 3CLP-Xho (5′-CACTCTCGAGTTGGAAGGTAACACCAGAGC-3′). The PCR product was digested with NheI and XhoI and ligated with NdeI/XhoI-digested pET21a DNA. The resulting plasmid pET 3CLP-21x encodes a 35.1 kDa protein containing a C-terminal His₆-Taq.
Expression of 3C-like Proteinase —pET 3CLP-21x was transformed into Escherichia coli BL21(DE3) cells. Cultures were grown at 37 °C in 1 liter of LB medium-containing ampicillin (100 μg/ml) until the A₆₀₀ reached 0.8 and then induced with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside at 30 °C for 3 h. The cells were harvested by centrifugation at 5000 × g for 10 min. The pelleted cells were suspended in buffer A (40 mm Tris-HCl, pH 8.0, 100 mm NaCl, 10 mm imidazole, 7.5 mm 2-mercaptoethanol), at 2% of the original culture volume. After cell lysis by ultrasonic, the cell lysate was separated by centrifugation at 24,000 × g for 20 min. The filtrated supernatant was applied to a nickel-nitrilotriacetic acid column (Qiagen) equilibrated by 50 ml of buffer A. After being washed in 100 ml of buffer A, the 3C-like proteinase was eluted with the gradient of 1–100% buffer B (40 mm Tris-HCl, pH 8.0, 100 mm NaCl, 250 mm imidazole, 7.5 mm 2-mercaptoethanol). The eluted enzyme was concentrated and loaded on a gel filtration column Sephacryl S-200 HR (Amersham Biosciences) equilibrated by 180 ml of buffer C (40 mm Tris-HCl, pH 8.0, 100 mm NaCl, 7.5 mm 2-mercaptoethanol). After elution with another 180 ml buffer C, we received over 95% purified 3C-like proteinase.
Analytic Gel Filtration —The aggregation state of the SARS 3C-like proteinase was analyzed using a Superdex 75 HR column (Amersham Biosciences) on ÄKTA fast protein liquid chromatography. Freshly purified protein was diluted to 4 and 0.2 mg/ml and equilibrated at room temperature for 2 h. 400 μl of 4- and 2-ml 0.2 mg/ml samples were injected into the Superdex 75 HR column and eluted with the buffer (40 mm Tris-HCl, pH 8.0, 100 mm NaCl, 7.5 mm 2-mercaptoethanol) at a flow rate of 0.5 ml/min. The eluted peaks were monitored at 280 nm on fast protein liquid chromatography.
CD Spectra —All of the CD spectra of the proteinase and the substrate peptides were recorded on a Jobin Yvon CD 6 spectrometer at 20 °C. The CD spectra of 3C-like proteinase were recorded in 40 mm Tris-HCl buffer, pH 8.0. For near-UV CD spectrum, a cell with a path length of 1 mm was used and the proteinase concentration is 544 μm, whereas a cell with a path length of 0.1 mm and 54.4 μm of proteinase solution was used for far-UV CD spectrum. The substrate peptides were solved in 20 mm Tris-HCl buffer, pH 7.3, and the final concentration was2mm. A cell with a path length of 0.1 mm was used. Each spectrum was the average of four scans corrected by subtracting a spectrum of the buffer solution in the absence of proteinase/peptide recorded under identical condition. Each scan in the range of 184–260 nm for far-UV CD and of 250–320 nm for near-UV CD spectra was obtained by taking data points every 0.5 nm with integration time of 1 s and a 2-nm bandwidth. Thermal denaturation spectrum was recorded by CD at 218 nm using the same condition for far-UV CD spectrum from 10 to 90 °C with an interval of 0.5 °C. Secondary structure content was calculated using the program VARSLC1 (20 (link))
Synthesis of Substrate Peptides —The substrate peptide S01 was synthesized by solid-phase peptide synthesis using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl)/tert-butyl strategy (21 (link)). The cleavage of the peptide from Rink resin and removal of all of the side-chain protecting groups were achieved in trifluoroacetic acid solution. The crude peptide was purified by reversed-phase high performance liquid chromatography (RP-HPLC, LabPrep System, Gilson) on a Vydac C18 semi-preparative column (218TP510, 10 by 250 mm, Vydac) with gradients of water/acetonitrile containing 0.1% trifluoroacetic acid. Peptide homogeneity and identity were analyzed by analytical HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), respectively. Other substrate peptides of HPLC purity from S02 to S11 were purchased from GL Biochemistry Ltd. (Shanghai, China).
Peptide Cleavage —The proteolysis activity of the SARS 3C-like proteinase was determined by peptide cleavage assay. Peptide S01 (Table I) was used as substrate and was incubated with the enzyme in Tris-HCl buffer, pH 7.3, at room temperature. The cleavage mixture was analyzed by RP-HPLC. To verify the cleavage site on the substrate peptide, the two products were purified by semi-preparative RP-HPLC using a 15-min 0–50% linear gradient of acetonitrile in 0.1% trifluoroacetic acid and lyophilized. The relative molecular weights of the products were identified by MALDI-TOF MS (BIFLEX III time-of-flight mass spectrometer, Bruker).Table I

Catalytic parameters and relative cleavage efficiencies of eleven peptides that represent all of the eleven cleavage sites in SARS coronavirus polyprotein

Peptide	Cleavage site	Sequence	Km	kcat	kcat/Km	(kcat/Km)rel
			mm	min-1	mm-1 min-1
S01	P1/P2	TSAVLQ/SGFRK-NH2	1.15 ± 0.28	12.2 ± 2.9	10.6 ± 0.6a	1.00
S02	P2/P3	SGVTFQ/GKFKK	0.583 ± 0.086	2.55 ± 0.35	4.38 ± 0.26a	0.41
S03	P3/P4	KVATVQ/SKMSD			0.353 ± 0.013b	0.03
S04	P4/P5	NRATLQ/AIASE			0.556 ± 0.126b	0.05
S05	P5/P6	SAVKLQ/NNELS			0.202 ± 0.003b	0.02
S06	P6/P7	ATVRLQ/AGNAT	1.44 ± 0.47	3.29 ± 1.07	2.29 ± 0.09a	0.22
S07	P7/P8	REPLMQ/SADAS			0.0176 ± 0.0022b	0.002
S08	P8/P9	PHTVLQ/AVGAC	1.94 ± 0.66	1.68 ± 0.57	0.865 ± 0.028a	0.08
S09	P9/P10	NVATLQ/AENVT			0.976 ± 0.014b	0.09
S10	P10/P11	TFTRLQ/SLENV	0.286 ± 0.020	0.847 ± 0.051	2.96 ± 0.11a	0.28
S11	P11/P12	FYPKLQ/ASQAW	0.549 ± 0.105	1.57 ± 0.28	2.86 ± 0.21a	0.27

a

Determined by Lineweaver-Burk plot.

b

Determined by fitting reaction kinetic data as in Equation 1 at low substrate concentration.

The relative enzyme activity at different pH values was determined in citric acid/phosphate buffer (pH 5, 6, 7, and 8) or glycine/NaOH buffer (pH 9 or 10) containing 6.8 mm dithiothreitol, 2 mm S01 as substrate, and 2.14 μm SARS 3C-like proteinase with a final volume of 50 μl. The cleavage reaction was stopped after 20 min by the addition of 50 μl of 0.1% trifluoroacetic acid aqueous solution and analyzed by RP-HPLC (LabPrep System, Gilson) on a Zorbax C18 analytic column (4.6 × 250 mm, Agilent). Cleavage products were resolved using a 15-min 0–50% linear gradient of acetonitrile in 0.1% trifluoroacetic acid.
To determine the k_cat/K_m for the substrate, 0.2 mm of substrate peptide was incubated with SARS 3C-like proteinase in 40 mm Tris-HCl buffer, pH 7.3. The concentration of the enzyme varied from 0.90 to 22.5 μm because of the different cleavage activity to different substrates. Reaction aliquots were removed at different times within 7 h and analyzed by RP-HPLC as described above. k_cat/K_m was determined by plotting substrate peak area as Equation 1, $\mathrm{lnPA}=C-k_{\mathrm{cat}}{\mathrm{c}}_{\mathrm{E}}t/K_m$ where PA is the peak area of the substrate peptide, c_E is the total concentration of 3C-like proteinase, and C is an experimental constant.
K_m and k_cat of the proteinase for selected substrates were determined by incubation of the substrate peptide at different concentration varying from 2 to 0.1 mm with SARS 3C-like proteinase in 40 mm Tris-HCl buffer, pH 7.3, for 20 min and analyzed by RP-HPLC as described above. The concentration of the enzyme varied from 1.07 to 17.1 μm because of the different cleavage activity to different substrate. Peak areas were calculated by integration and converted to absolute units by using peptide standards. The reaction rate was calculated for all of the cleavage products of two experiments and averaged. K_m and k_cat were calculated by the Lineweaver-Burk plot.