High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined tothe maltose-binding protein. MBP-Endo F2was found in a highly enrichedstate as insoluble, inactive inclusion bodies. Extraction of the inclusionbodies with 20% acetic acid followed by exhaustive dialysis rendered thefusion protein active and soluble. MBP-Endo F2was digested with FactorXaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, andappeared as a single symmetrical peak on HPLC. Analysis of theamino-terminus demonstrated conclusively that recombinant Endo F2washomogeneous and identical to the native enzyme.