Pasteurella multocida a1-3 N-acetyl-galactosaminyltransferase
Abbreviation
Pm1138
Source
Pasteurella multocida Pm70
Activity
Transfer GalNAc from UDP-GalNAc to GalNAc via an a1,3-linkage
Expression
&
Purification
Expression system: pET28a 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 + 20 glycerol + 0.1% Triton-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
MnCl2
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-GalNAc and derivative
GalNAc, to form Forssman antigen
Donor:
Acceptor:
Substrate Specificity
-
Houliston R.S., Bernatchez S., et al. Complete chemoenzymatic synthesis of the Forssman antigen using novel glycosyltransferases identified in Campylobacter jejuni and Pasteurella multocida. Glycobiology, 2009, 19, 153.
References