PURIFICATION AND CHARACTERIZATION OF NEOMYCIN PHOSPHOTRANSFERASE II FROM GENETICALLY MODIFIED COTTONSEED (GOSSYPUM HIRSUTUM)

摘要

Expression of the nptII gene [encoding the neomycin phosphotransferase II (NPTII) enzyme] is frequently employed as a selectable marker for plant transformation in a variety of species, including cotton. Therefore, characterization of this enzyme was considered an important part of the regulatory approval process designed to assess the safety of products derived from genetically modified plants. Because of the role of cottonseed in animal feeds and other commercial uses, a purification scheme for NPTII from cottonseed was developed employing solvent extraction, a series of precipitations, DEAE-Sepharose chromatography, and affinity chromatography which yielded a highly enriched preparation that retained enzymatic activity. Further purification was obtained by SDS-PAGE, electrotransfer to PVDF membrane, and direct sequencing of the appropriate band. NPTII derived from cottonseed was characterized by purification behavior, NH2-terminal sequence, SDS-PAGE, immunoblot, ELISA, and enzymatic activity and was found by these criteria to be chemically equivalent to the enzyme derived from an Escherichia coli expression system.