Campylobacter jejuni α2-3,8 Sialyltransferase

Abbreviation 

CjCstII

Source

Campylobacter jejuni

Activity

Transfer Sia via an α2-3 and α2-8 linkage

Expression

&

Purification

Expression system:    pET15b 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

6.0

MgCl2

50 mM Tris-HCl (pH 6.0), 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

Sia

Donor:

Acceptor:

Kinetic:

cstii-1.png

Substrate Specificity

  • Cheng, J.;  Yu, H.;  Lau, K.;  Huang, S.;  Chokhawala, H. A.;  Li, Y.;  Tiwari, V. K.; Chen, X., Multifunctionality of Campylobacter jejuni sialyltransferase CstII: characterization of GD3/GT3 oligosaccharide synthase, GD3 oligosaccharide sialidase, and trans-sialidase activities. Glycobiology 2008, 18 (9), 686-97.

References