RESUMO
The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l(-1) at the 200 1 scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.
Assuntos
Reatores Biológicos/microbiologia , Técnicas de Cultura de Células/métodos , Lectinas de Ligação a Manose/biossíntese , Lectinas de Ligação a Manose/química , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Lectinas de Plantas/biossíntese , Lectinas de Plantas/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Galanthus/genética , Galanthus/metabolismo , Lectinas de Ligação a Manose/genética , Dados de Sequência Molecular , Lectinas de Plantas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The kidney bean lectin Phaseolus vulgaris phytohemagglutinin E-form (PHA-E) was expressed and secreted by the methylotrophic yeast Pichia pastoris. To optimise yields of PHA-E, transformants of P. pastoris were selected for high-level production of the recombinant protein. A scaleable process for the production and purification of gram quantities of recombinant PHA-E is reported. PHA-E was secreted at approximately 100 mg/L at the 2- and 200-L scale and was purified to 95% homogeneity in a single step using cation-exchange chromatography. The purified recombinant PHA-E consists of four forms with molecular masses between 28.5 and 31.5 kDa, as assessed by MALDI-TOF, whereas its native counterpart has a molecular mass of approximately 30.5 kDa. Endoglycosidase treatment revealed that the range in size of the recombinant protein was attributed to differences in the nature of the N-linked oligosaccharides bound to the protein. The primary amino acid sequence of the recombinant PHA-E was found to be identical to the native protein and to have an agglutination activity similar to that of native PHA-E. The data presented here suggest that, using P. pastoris, gram quantities of a recombinant phytohemagglutinin E-form can be produced and that the recombinant protein is similar to the protein synthesised in plants with respect to structure and biological activity.
Assuntos
Phaseolus , Fito-Hemaglutininas/isolamento & purificação , Fito-Hemaglutininas/metabolismo , Pichia/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , DNA Recombinante/genética , Engenharia Genética , Glicosilação , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Phaseolus/química , Phaseolus/genética , Fito-Hemaglutininas/biossíntese , Fito-Hemaglutininas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
A publication reporting the harmful effects on the monarch butterfly of maize genetically modified to express insecticidal delta-endotoxins from the soil bacterium Bacillus thuringiensis (Bt) caused much public interest. A series of ecologically based studies were subsequently carried out to evaluate rigorously the impact of pollen from such crops and to quantify the risks. The results demonstrated that the commercial large-scale cultivation of current Bt-maize hybrids did not pose a significant risk to the monarch population. Further studies also demonstrated that Bt-expressing crops posed little risk to other nontarget insects, including beneficial insects such as pollinators and natural enemies.