E. coli O86 a1-3galactosyltransferase

Abbreviation 

EcWbnI

Source

E. coli O86:H2

Activity

Transfer Gal from UDP-Gal to H-antigen via an a1,3-linkage

Expression

&

Purification

Expression system:    pET15b 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: >3 mg/L culture

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 1 mL Ni-NTA resin (pre-equilibrated)

5) Wash with 50 mL Buffer-A+10 glyc, 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 3 months

Buffers:A: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 10% glycerol;

            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 - 8.5

MnCl2

50 mM Tris-HCl (pH 8.0), 22 mM donor, 20 mM acceptor, 20 mM MnCl2, incubate at 30 °C for 1 h to overnight.

Temperature

pH

Metal ion:

Example condition:   

UDP-Gal

H-antigen, only tolerate type I, III, and IV H-antigen

Donor:

Acceptor:

Substrate Specificity

  • Ye F., Liu X., et al. Diversity-Oriented Enzymatic Modular Assembly of ABO Histo-blood Group Antigens. ACS Cata, 2016, 6, 8140.

  • Yi W., Shao J., et al. Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide. JACS, 2005, 127, 2040.

References