Pasteurella multocida  α2-3 Sialyltransferase M144D

Abbreviation 

PmST1-M144D

Source

Pasteurella multocida

Activity

Transfer Sia via an α2-3 linkage

Expression

&

Purification

Expression system:    pET23a with C-terminal His6-tag , E. coli BL21(DE3)

Induction condition:  0.1 mM IPTG, 37 °C, 3 h, 250 rpm, Luria LB broth

Purification

1) Suspend cells in ice-cold 40 mL Buffer-A + 0.1% 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 -9.0

MgCl2

50 mM Tris-HCl (pH 8.5), 22 mM donor, 20 mM acceptor, 20 mM MgCl2, incubate at 37 °C for 30 min.

Temperature

pH

Metal ion:

Example condition:   

CMP-Neu5Ac/Neu5Gc/Kdn

Gal

Donor:

Acceptor:

Kinetics: 

PmST1 M144D kenetic-3.png
PmST1 M144D kenetic.png
PmST1 M144D 4.png
PmST1 M144D kenetic-2.png

Substrate Specificity

  • Sugiarto, G.;  Lau, K.;  Qu, J.;  Li, Y.;  Lim, S.;  Mu, S.;  Ames, J. B.;  Fisher, A. J.; Chen, X., A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating LewisX. ACS Chem Biol 2012, 7 (7), 1232-40.

References