E. coli O128 a1-2 fucosyltransferase

Abbreviation 

EcWbsJ

Source

E. coli O128:B12

Activity

Transfer Fuc from GDP-Fuc to Gal via an a1,2-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

5.5-7.5

MgCl2, MnCl2

50 mM Tris-HCl (pH 7.0), 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

Galb1-3GalNAc

Donor:

Acceptor:

Kinetics:

Substrate Specificity

  • Li M., Liu X., et al. Characterization of a Novel a1,2-Fucosyltransferase of Escherichia coli O128:B12 and Functional Investigation of Its Common Motif. Biochemistry, 2008, 47, 378.

  • Su D.M., Eguchi, H., Enzymatic Synthesis of Tumor-Associated Carbohydrate Antigen Globo-H Hexasaccharide. Org Lett, 2008, 10, 1009.

References