OVEREXPRESSION AND PURIFICATION OF NONGLYCOSYLATED RECOMBINANT ENDO-BETA-N-ACETYLGLUCOSAMINIDASE F3

The gene for endo-beta-N-acetylglucosaminidase F-3 was cloned into the high-expression vector PMAL c-2, and expressed in Escherichia coli as a fusion protein, A key step in the purification employed Poros II (H S) chromatography, which greatly facilitated isolation of the enzyme f rom crude intracellular lysates, The unfused enzyme was recovered foll owing digestion with Factor X(a) and was isolated in a homogeneous for m, The enzyme is non-glycosylated and fully active, and is a very usef ul analytical tool for investigating the structure of asparagine-linke d glycans, especially those with core-substituted alpha 1,6 fucosyl re sidues.