RESUMO
We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.
Assuntos
Biotecnologia/métodos , Técnicas Genéticas , Pichia/metabolismo , Proinsulina/genética , Proinsulina/metabolismo , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Clonagem Molecular , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Fermentação , Dosagem de Genes , Vetores Genéticos , Humanos , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Mapeamento de Peptídeos , Peptídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Serina Endopeptidases/metabolismo , Fatores de TempoRESUMO
Using immunosorbents based upon cyanogen bromide-Sepharose CL-4B, we have examined different ligand densities in coupling of monoclonal antibody (MAb) to find the best performance, for recombinant hepatitis B surface antigen (rHBsAg) purification. Three replicates of 5 and 15 cycles of densities ranges: 2.17-2.19, 3.18-3.62, 4.06-4.17, and 5.13-5.40 mg/ml (control); or 1.81-2.47, 3.17-3.41, 4.16-4.28, and 5.16-5.19 mg/ml (control), respectively were evaluated in terms of binding capacity, antigen recovery, ligand leakage and purity of antigen, and compared to the control. Adsorption and antigen recovery of immunosorbents manufactured were not different statistically, eventhough increased 8.08 and 9.90% at a range of 3.17-3.41 mg/ml. At this range, efficiency expressed as productivity and MAb saving was optimal. Ligand leakage and purity of antigen showed similar behaviour among all densities. Aspects related to ligand density in antigen immunoaffinity purification are discussed.
Assuntos
Anticorpos Monoclonais , Cromatografia de Afinidade/métodos , Antígenos de Superfície da Hepatite B/isolamento & purificação , Brometo de Cianogênio , Imunoadsorventes , Ligantes , Pichia/metabolismo , Proteínas Recombinantes/isolamento & purificação , SefaroseRESUMO
Increased expression of recombinant mini-proinsulin in Pichia pastoris in 2.5 l bioreactors was achieved by increasing the cultivation pH from 5.1 to 6.3, by decreasing the temperature from 28 to 22 degrees C, and by periodical addition of ammonium sulfate and EDTA to the culture broth. Using this procedure, mini-proinsulin reached nearly 0.3 g l(-1) in the culture supernatant after 160 h of growth.