bovine b1-4 N-acetylgalactosaminyltransferase Y289L/C342T 

Abbreviation 

b4GalTm

Source

Bovine

Activity

Transfer Gal, GalNAc, GlcNAc to GlcNAc via an b1,4-linkage

Expression

&

Purification

Expression system:    pET23a 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

1) Suspend cells in ice-cold 40 mL Buffer-A + 10% glycerol + 0.2% Trixon X100

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 -20 °C for up to 6 months

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, 50 % Glycerol

Optimal Condition

37 °C

7.5

MgCl2

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:   

UDP-Gal/GalNAc/GlcNAc

GlcNAc or Glc

Donor:

Acceptor:

Kinetics: 

Substrate Specificity

  • Ramakrishnan B. and Qasba P.K. Structure-based Design of b1,4-Galactosyltransferase I (b4Gal-T1) with Equally Efficient N-Acetylgalactosaminyltransferase Activity. J Biol Chem, 2002, 277, 20833.

References