Transfer Sia via an α2-3 linkage; also contains α2-3 sialidase activity.

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

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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