High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity |
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Authors: | Reddy A; Grimwood BG; Plummer TH; Tarentino AL |
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Institution: | Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, P.O. Box 509, Empire State Plaza, Albany, NY 12201-0509, USA. |
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Abstract: | The Endo F2gene was overexpressed in E.coli as a fusion protein joined to
the maltose-binding protein. MBP-Endo F2was 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 F2was digested with Factor
Xaand 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 F2was
homogeneous and identical to the native enzyme.
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