E. coli O55 b1-3 galactosyltransferase
Abbreviation
EcWbgO
Source
E. coli O55:H7
Activity
Transfer Gal from UDP-Gal to GlcNAc via a b1,3-linkage
Expression
&
Purification
Expression system: pGEX-4T-1 with N-terminal GST , E. coli BL21(DE3)
Induction condition: 1 mM IPTG, 30 °C, 6 h, 200 rpm, Luria LB broth
Purification:
1) Suspend cells collected from 1 L medium in ice-cold 40 mL Buffer-A
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 GSTrap resin (pre-equilibrated)
5) Wash with 50 mL Buffer-A, finally elute with 10 mL Buffer-B.
6) Concentrate into 2.5 mL and change buffer to Buffer C 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: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3;
B: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0.
C: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20 % Glycerol
Optimal Condition
37 °C
7.0 - 8.5
MnCl2, MgCl2
50 mM Tris-HCl (pH 8.0), 22 mM donor, 20 mM acceptor, 20 mM MnCl2, incubate at 37 °C for 1 h to overnight.
Temperature:
pH:
Metal ion:
Example condition:
UDP-Gal
GlcNAcbR


Donor:
Acceptor:
Kinetics:
Substrate Specificity
-
McArthur J.B., Yu H., and Chen X. A Bacterial b1–3-Galactosyltransferase Enables Multigram-Scale Synthesis of Human Milk Lacto-N-tetraose (LNT) and Its Fucosides. ACS Cata, 2019, 9, 10721.
-
Liu X., Xia C., et al. Characterization and synthetic application of a novel
b1,3-galactosyltransferase from Escherichia coli O55:H7. Bioorg Med Chem, 2009, 17, 4910.
References