Activity

Transfer Fuc from GDP-Fuc to GlcNAc of via an a1,3/4-linkage

Expression

&

Purification

Expression system:    pMAL-c2X with MBP, E. coli BL21(DE3)

Induction condition:  0. 1 mM IPTG, 16 °C, 20 h, 200 rpm, Luria LB broth

Purification: 6 mg/L culture

The cell pellet was resuspended in column buffer (Tris-HCl 20 mM, NaCl 200 mM, ethylenediaminetetraacetic acid (EDTA) 1 mM, Triton X-100 0.1%, pH 7.5) were disrupted by sonication on ice for 45 min in 10-s intervals. The cell debris was removed by centrifugation at 4 °C, 24000 x g for 45 min. The supernatant was applied to amylose resin equilibrated in column buffer (Tris-HCl 20 mM, NaCl 200 mM, EDTA 1 mM, pH 7.5). MBP-fused protein was eluted with 20 mM maltose in column buffer, and the fractions were analyzed by SDS-PAGE. Fractions with significant amounts of protein were pooled and concentrated by centrifugation with a centrifugal filter device, divided into aliquots, and stored at -20 °C.

Optimal Condition