Helicobacter mustelae a1-2 fucosyltransferase
Abbreviation
Hma2FucT
Source
Helicobacter mustelae
Activity
Transfer Fuc from GDP-Fuc to Gal via an a1,2-linkage
Expression
&
Purification
Expression system: pET15 with N-terminal His6-tag , E. coli BL21(DE3)
Induction condition: 0. 2 mM IPTG, 16 °C, 20 h, 200 rpm, Luria LB broth
Purification:
1) Suspend cells collected from 1 L medium in ice-cold 40 mL Buffer-A + 10% glycerol
2) Disrupt cells by a CF1 cell disruptor (Constant Systems), 20 kPsi, 4 °C
3) Centrifuge at 12000 x g, 4 °C, 30 min
4) Load supernatant to a 10 mL gravity column filled with 2 mL Ni-NTA resin (pre-equilibrated)
5) Wash with 50 mL Buffer-A, then 50 mM Buffer-B, finally elute with 10 mL Buffer-C.
6) Concentrate into 2.5 mL and change buffer to Buffer D using a PD-10 column.
7) Store at 4 °C for up to 6 months, store at -80 °C for up to 1 year (avoid repeated freeze)
Buffers: A: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole;
B: A + 15 mM imidazole; C: A + 250 mM imidazole.
D: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 50 % Glycerol
Optimal Condition
37 °C
7.5
MnCl2, MgCl2
50 mM Tris-HCl (pH 7.5), 22 mM donor, 20 mM acceptor, 20 mM MgCl2, incubate at 37 °C for 1 h to overnight.
Temperature:
pH:
Metal ion:
Example condition:
GDP-Fuc
Lac/LacNAc/Galb1-3GalNAc
Donor:
Acceptor:
Substrate Specificity
-
Liu Y., Wen L., et al. A general chemoenzymatic strategy for the synthesis of glycosphingolipids. European J Org Chem, 2016, 25, 4315.
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Kamada N., Koizumi S. Alpha-1,2-fucosyltransferase and dna encoding the same. Patent: US20040219553A1. 2004.
References