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Supplementary materials
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Flavonoid compound-mediated inhibition of SARS coronavirus 3C-like protease
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expressed in Pichia pastoris
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Thi Thanh Hanh Nguyen · Hye-Jin Woo · Hee-Kyoung Kang · Van Dao Nguyen · Young-Min Kim · Do-Won
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Kim · Sul-Ah Ahn · Yongmei Xia
Doman Kim
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Optimized DNA sequence of 3CLpro of SARS for expression in P. pastoris (Fig S1)
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3CLpro gene incorporated into P. pastoris GS115 genome was tested by colony PCR screening
using 3CLpro primers and α-factor and 3AOX1 primers (Fig S2).
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SDS-PAGE and Western blot analysis of recombinant 3CLpro expressed from P. pastoris
GS115 (Fig S3).
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SDS-PAGE of purified 3CLpro after 80-85% ammonium sulfate fractionation (Fig S4).
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The purification table of SARS-3CLpro expressed in P. pastoris GS115 (Table S1)
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Lineweaver-Burk plot for the determination of the 3CL pro Km value (Fig S5).
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Fig. S1. Nucleotide sequence of the synthetic 3CLpro gene by codon optimization. The synthetic gene is based
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on the amino acid sequence of SARS-3CLpro (GenBank accession no. AY274119).
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Fig. S2. PCR analysis of P. pastoris GS115 clones. 3CLpro gene incorporated into P. pastoris GS115 genome
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was tested by colony PCR screening using 3CLpro primers and α-factor and 3AOX1 primers. Lane C1: P.
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pastoris GS115 control; lane C2: amplicon of P. pastoris clone containing pPICZαA. Lanes 1–7: amplicons of
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pPICZα-3CLpro transformants No. 1–7 with 3CL protease primers, Lanes 8–14: amplicons of pPICZα-3CLpro
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transformants No. 1–7 with α-factor and 3AOX1 primers. N1: set two primer α-factor and 3AOX1, N2: set one
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primer 3CLpro primer, C: control pPICZαA with α-factor and 3AOX1 primers
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Fig. S3. SDS-PAGE and Western blot analysis of recombinant 3CLpro. The recombinant 3CLpro after 4 days
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induction was separated by 12 % SDS-PAGE and visualized by Coomassie blue (A) and Western blotting with
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anti-His antibodies (B). Lane M: molecular weight markers; lane C: P. pastoris GS115 containing pPICZαA
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vector; lane 1: P. pastoris 3CLpro.
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Fig. S4. SDS-PAGE of purified 3CLpro after 80-85% ammonium sulfate fractionation. Lane M: marker;
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lane 1: supernatant before ammonium sulfate fractionation. Lane 2: purified 3CLpro.
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Table S1. Purification table of SARS-3CLpro expressed in P. pastoris GS115.
Steps
Total volume
Protein
Total
Total activity
Specific activity
Purification
Yield 2
(ml)
(mg/ml)
Protein (mg)
(FC)*
(FC/mg)
fold
(%) 3
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Culture supernatant
4000
0.13
520
86.02x104
1654.23
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100
Concentration and buffer exchange
1000
0.30
300
56.86x104
1895.33
1.15
66.10
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(milipore membrane 10 kDa)
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Ammonium sulfate fractionation and
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0.28
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8.49x104
dialysis
6064.29
3.67
9.87 9
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*FC (Fluorescence concentration) (FI= RFU · min-1 x D x V) (D: dilution factor, V: volume)
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1
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Fig. S5:
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various substrate concentrations to obtain Km value of the enzyme. SigmaPlot was used to fit the kinetic data
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using Lineweaver-Burk double reciprocal plots.
Lineweaver-Burk plot for the determination of the 3CL pro Km value. The reaction was done at
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