Helicobacter pylori a1-3/4 fucosyltransferase C-terminal 66AA truncate

Abbreviation 

Hpa3FT

Source

Helicobacter pylori NCTC11639

Activity

Transfer Fuc from GDP-Fuc to GlcNAc of LacNAc via an a1,3-linkage

Expression

&

Purification

Expression system:    pET22b with C-terminal His6-tag , E. coli BL21(DE3)

Induction condition:  0. 2 mM IPTG, 16 °C, 20 h, 200 rpm, Luria LB broth

Purification: 20 mg/L culture

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 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, 20 % Glycerol

Optimal Condition

37 °C

7.5

MnCl2

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

LacNAc, Siaa2-3LacNAc

Donor:

Acceptor:

Kinetics:

Substrate Specificity

  • Lin S., Yuan T., Li J., Lin C. Carboxyl Terminus of Helicobacter Pylori alpha1,3- fucosyltransferase Determines the Structure and Stability. Biochemistry, 2006, 45, 8108.

  • Wang S., Zhang Q., et al. Facile Chemoenzymatic synthesis of O-mannosyl glycans. Angew Chem In Ed, 2018, 57, 9268.

References