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1 Precisely regulated galactosylation of nucleoside analogues in aqueous hydrophilic solvents
2 catalyzed by solvent-stable β-galactosidase
3 Youzhi Zhou a, Jianlin Chu a, Jinsong Zhang a, Ke Liu a, Bingfang He b,*
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5 a College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, China.
6 b School of Pharmaceutical Sciences, Nanjing Tech University, China.
7 Fax: + 86 25 5813 9935
8 Tel: + 86 25 5813 9902
9 E-mail: [email protected]
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Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2016
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25 Cloning, heterologous expression and purification of BMG
26 The genomic DNA of Bacillus megaterium YZ08 was extracted using the Genomic DNA extraction
27 kit (HAOJIA, Shanghai, China). The gene sequence of BMG was then obtained using primers of
28 BMG-F (5'-GCGGGATCCGACGACGACGACAAGATGTTAAAAACCGGCAAGAAATT-3') and
29 BMG-R (5'-GCGCTCGAGTTATAGAGGTTTTAGCGTAAACG-3'). The PCR product was ligated
30 into pET28a (Novagen, Germany) vector using ClonExpress II One Step Cloning Kit (Vazyme,
31 China). The verified recombinant plasmid was introduced into E. coli JM109 (DE3) for expression.
32 Transformants were cultivated in LB medium containing kanamycin (50 μg/ml) at 37 ℃, 180 rpm till
33 OD600 ~0.6-1.0, and then induced by the addition of 0.5 mM IPTG at 20 ℃ for 6 h. The cells were
34 harvested by contrifugation and used for protein purification.
35 The recombinant BMG was purified as His-tagged protein in a Ni2+-affinity chromatography. All
36 purification procedures were followed according to the manufacturer's instructions (GE Healthcare).
37 The eluate containing recombinant BMG was incubated for 12 h at 4 ℃ in the presence of
38 recombinant enterokinase (rEK, Duly, Nanjing), which effectively cleaved the BMG after Asp4-Lys.
39 After the cleavage reaction, the reaction mixture was eluted with the binding buffer through a Ni2+-
40 affinity column to remove the His-tagged rEK. The collected eluate without rEK was dialyzed in 20
41 mM Tris-HCl buffer (pH 7.5).
42 The molecular mass of native BMG was analyzed by gel filtration chromatography on a Superdex
43 200 pg gel filtration column (GE Healthcare). Purified BMG and protein standard (Gel Filtration
44 Calibration Kits, GE Healthcare) were both eluted with 50 mM phosphate buffer (pH 7.2) containing
45 150 mM NaCl at a rate of 1 mL/min. The molecular mass of BMG was determined from the standard
46 curve based on elution volumes. Protein concentrations were determined according to Bradford
47 method with bovine serum albumin as the standard.
48 HPLC analysis
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49 The mixture of enzymatic galactosylation was analyzed by RP-HPLC on an Agilent TC-C18 column
50 (4.6 mm × 250 mm, 5 μm) with a flow rate of 1 ml/min. An elution [water/methanol: 95/5 (v/v)] was
51 used for the analysis of the mixture of enzymatic galactosylation of uridine, β-D-
52 arabinofuranosyluracil, 2'-deoxyuridine, 5-methyl-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine and
53 inosine. The retention times of monogalactoside and substrate were 7.7 and 8.2 min (uridine, Fig S2);
54 7.7 and 10.1 min (β-D-arabinofuranosyluracil, Fig S3). The retention times of digalactoside,
55 monogalactoside and substrate were 5.9, 7.9 and 11.4 min (2'-deoxyuridine, Fig S4); 12.3, 16.7 and
56 18.6 min (5-methyl-2'-deoxyuridine, Fig S5); 8.6, 11.2 and 16.7 min (5-fluoro-2'-deoxyuridine, Fig
57 S6); 8.6, 12.3 and 16.7 min (inosine, Fig S7). An elution [water/methanol: 90/10 (v/v)] was used for the
58 analysis of the mixture of enzymatic galactosylation of 2', 3'-dideoxythymidine. The retention times of
59 digalactoside, monogalactoside and 2', 3'-dideoxythymidine were 10.4, 16.8 and 31.9 min (Fig S8).
60 An elution [water/methanol: 80/20 (v/v)] was used for the analysis of the mixture of enzymatic
61 galactosylation of AZT. The retention times of digalactoside, monogalactoside and AZT were 8.1,
62 12.6 and 21.5 min (Fig S9).
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64 Fig S1 SDS-PAGE analysis of the recombinant BMG. Lane 1, marker; Lane 2, total cell protein after
65 IPTG induction; Line 3, BMG purified by Ni2+ column; Lane 4, purified BMG removed of His-tag.
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67 Fig S2 HPLC analysis of the mixture of enzymatic galactosylation of uridine in buffer (balck) and 10 %
68 DMSO (blue).
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70 Fig S3 HPLC analysis of the mixture of enzymatic galactosylation of β-D-arabinofuranosyluracil in
71 buffer (balck) and 10 % DMSO (blue).
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73 Fig S4 HPLC analysis of the mixture of enzymatic galactosylation of 2'-deoxyuridine in buffer (balck)
74 and 10 % DMSO (blue).
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76 Fig S5 HPLC analysis of the mixture of enzymatic galactosylation of 5-methyl-2'-deoxyuridine in
77 buffer (balck) and 10 % DMSO (blue).
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79 Fig S6 HPLC analysis of the mixture of enzymatic galactosylation of 5-fluoro-2'-deoxyuridine in
80 buffer (balck) and 10 % DMSO (blue).
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82 Fig S7 HPLC analysis of the mixture of enzymatic galactosylation of inosine in buffer (balck) and 10 %
83 DMSO (blue).
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85 Fig S8 HPLC analysis of the mixture of enzymatic galactosylation of 2',3'-dideoxythymidine in buffer
86 (balck) and 10 % DMSO (blue).
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88 Fig S9 HPLC analysis of the mixture of enzymatic galactosylation of AZT in buffer (balck) and 10 %
89 DMSO (blue).
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91 Fig S10 Mass spectrometry for AZT monogalactoside.
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93 Fig S11 Mass spectrometry for AZT digalactoside.
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95 Fig S12 13C for the 5'-O-β-galactosyl-3'-azido-3'-deoxythymidine.
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97 Fig S13 1H for the 5'-O-β-galactosyl-3'-azido-3'-deoxythymidin.
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99 Fig S14 Mass spectrometry for 2'-deoxyuridine monogalacroside.
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101 Fig S15 Mass spectrometry for 2'-deoxyuridine digalacroside.
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103 Fig S16 13C for the 5'-O-β-galactosyl-3'-azido-2'-deoxyuridine
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105 Fig S17 1H for the 5'-O-β-galactosyl-3'-azido-2'-deoxyuridine