HIGH-LEVEL EXPRESSION OF THE ENDO-BETA-N-ACETYLGLUCOSAMINIDASE F-2 GENE IN ESCHERICHIA-COLI - ONE-STEP PURIFICATION TO HOMOGENEITY

The Endo F-2 gene was overexpressed in E.coli as a fusion protein join ed to the maltose-binding protein. MBP-Endo F-2 was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialy sis rendered the fusion protein active and soluble. MBP-Endo F-2 was d igested with Factor X-a and purified on Q-Sepharose. The enzyme was ho mogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HP LC. Analysis of the amino-terminus demonstrated conclusively that reco mbinant Endo F-2 was homogeneous and identical to the native enzyme.