E. coli O127 b1-3 galactosyltransferase

Abbreviation 

WbiP

Source

E. coli O127

Activity

Transfer Gal from UDP-Gal to GalNAc via a b1,3-linkage

Expression

&

Purification

Expression system:    pET28a with N-terminal GST 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 4 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, MgCl2

50 mM Tris-HCl (pH 7.5), 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

GalNAc, Tn-antigen

Donor:

Acceptor:

Kinetics:

Substrate Specificity

  • Yi W., Perali R.S., et al. Characterization of a Bacterial a-1,3-Galactosyltransferase with Application in the Synthesis of Tumor-Associated T-Antigen Mimics. Biochemistry, 2008, 47, 1241.

References