RESUMO
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.
Assuntos
Escherichia coli/genética , Expressão Gênica , Engenharia Genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plantas Comestíveis/genética , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Glicosilação , Gossypium/enzimologia , Concentração de Íons de Hidrogênio , Canamicina Quinase , Dados de Sequência Molecular , Peso Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plantas Comestíveis/enzimologia , Segurança , Homologia de Sequência de Aminoácidos , Verduras/enzimologiaRESUMO
The synthesis, processing and secretion of insulin-like growth factor-1 (IGF-1 or somatomedin-C) fused to LamB and OmpF secretion leader sequences in Escherichia coli have been investigated. Expression and secretion of IGF-1 was achieved. The major portion of this secreted IGF-1 accumulated in the periplasmic space as insoluble aggregates. A small amount of IGF-1 was found folded in its native conformation in the medium. The lamB and ompF signal sequences were fused to the 5' coding sequence of IGF-1. Fusion of the lamB signal sequence directly to IGF-1 (lamB-IGF-1) resulted in accumulation of 16-20 micrograms/A550/ml of correctly processed IGF-1 in the periplasmic space. The processing efficiency of LamB-IGF-1 and OmpF-IGF-1 was enhanced in an E. coli strain bearing a prlA4 mutation. Amino acid sequence analysis of IGF-1 secreted into the periplasm and exported into the medium confirmed the precise removal of the LamB or OmpF signal sequence. IGF-1 synthesized in E. coli was demonstrated to be active in a cell proliferation bioassay.
Assuntos
Escherichia coli/genética , Genes Sintéticos , Genes , Fator de Crescimento Insulin-Like I/genética , Somatomedinas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura , Vetores Genéticos , Fator de Crescimento Insulin-Like I/biossíntese , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por RestriçãoRESUMO
Partial misincorporation of Lys for Arg has been observed for the Arg residues of IGF-1 when the molecule is expressed in Escherichia coli using a synthetic gene with the low frequency AGA codon encoding all six Arg residues and yeast preferred codons encoding the remaining residues. The Lys for Arg substitution at these residues could not be detected when a gene containing E. coli preferred codons, with the codon CGT coding for all Arg residues, was used for the expression of the protein. Similarly, no misincorporation of Lys for Arg could be detected when a gene containing Escherichia coli preferred codons at all positions, except for an AGA codon at Arg (36), was utilized.
