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

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Abstract
The Endo F2 gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2 was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2 was digested with Factor Xa and purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2 was homogeneous and identical to the native enzyme.