title: Purification and characterization of microbially expressed neomycin phosphotransferase II (NPTII) protein and its equivalence to the plant expressed protein.
abstract: The gene encoding neomycin phosphotransferase II (NPTII) has been used routinely as a selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the same coding sequence used for plant transformation was introduced into Escherichia coli to produce gram quantities of this protein. A unique, simple, rapid and efficient purification method was developed to purify thirty grams of NPTII protein. The microbially produced NPTII was shown to be chemically and functionally equivalent to the NPTII protein expressed in and purified from genetically engineered cotton seed, potato tubers and tomato fruit. Microbially produced and plant produced NPTII proteins have comparable molecular weights, immuno-reactivities, epitope structures, amino terminal amino acid sequences, biological activities and both lack glycosylation. Demonstrating the equivalence of NPTII protein from these sources establishes the validity of using the microbially produced NPTII to assess the safety of the NPTII protein produced in genetically engineered crops.
authors.affiliation: Agricultural Group, Unit of Monsanto Company, Chesterfield, MO 63198.
authors.forename: R L
authors.initials: RL
authors.lastname: Fuchs
authors.forename: R A
authors.initials: RA
authors.lastname: Heeren
authors.forename: M E
authors.initials: ME
authors.lastname: Gustafson
authors.forename: G J
authors.initials: GJ
authors.lastname: Rogan
authors.forename: D E
authors.initials: DE
authors.lastname: Bartnicki
authors.forename: R M
authors.initials: RM
authors.lastname: Leimgruber
authors.forename: R F
authors.initials: RF
authors.lastname: Finn
authors.forename: A
authors.initials: A
authors.lastname: Hershman
authors.forename: S A
authors.initials: SA
authors.lastname: Berberich
mesh_terms: D000595:Amino Acid Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering; D006031:Glycosylation; D003368:Gossypium / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010945:Plants, Edible / Q000201:enzymology / Q000235:genetics*; D012449:Safety; D017386:Sequence Homology, Amino Acid; D014675:Vegetables / Q000201:enzymology
chemical_list: D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase