RESUMO
Hydroxylamine-induced cleavage at the asparaginyl-glycine dipeptide site inserted between the two moieties of recombinant fusion proteins has been used at both the analytical and the preparative scale to obtain the mature protein. In this study a model protein containing a fusion precursor of insulin-like growth factor I was used to investigate the influence of the operating conditions on the cleavage reaction and the formation of undesired side products such as hydroxamate and deamidated analogs. Moreover, the stability of the cleavage site toward deamidation was examined and a chemometric study performed to define the effect of the reaction conditions on the cleavage yield and on the formation of side products.
Assuntos
Hidroxilamina/farmacologia , Fator de Crescimento Insulin-Like I/biossíntese , Sequência de Aminoácidos , Dipeptídeos/metabolismo , Fermentação , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Ácidos Hidroxâmicos/metabolismo , Fator de Crescimento Insulin-Like I/isolamento & purificação , Ponto Isoelétrico , Metionina/metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massa de Íon Secundário , Tiossulfatos/metabolismoRESUMO
A method is described using fast protein liquid chromatography (FPLC) for the monitoring of protein formation during fermentation. The procedure consists of centrifugation to recover the cells, sonication of the cells, centrifugation to remove cell debris, and analysis of supernatant on a column of Mono Q (a strong anion exchanger). Analysis of peak areas provides quantitative determination of product concentration. Maintenance and life of the Mono Q column is discussed. We find that FPLC is a convenient method for measuring products in cell homogenates because it gives rapid, highly resolved separations.
RESUMO
Proteolytic enzymes are present in many biological raw materials used for the production of proteins and peptides. Such enzymes are active not only in homogenates of bacteria and yeast but also in ascites liquid and cell culture media used for the production of monoclonals. A new protease substrate was used to determine protease activity in homogenates of E. coli and baker's yeast, which are useful for the production of therapeutically valuable polypeptides. Using this substrate we found that cooling and the addition of protease inhibitors had little effect on the proteolytic activity. Most of the proteolytic activity however could be adsorbed to hydrophobic gel media. Data is given on the protease adsorption at different temperatures and ionic strengths.
Assuntos
Bactérias/enzimologia , Peptídeo Hidrolases/análise , Leveduras/enzimologia , Cromatografia de Afinidade , Inibidores de Proteases/farmacologia , Cloreto de Sódio/farmacologiaRESUMO
Preparation of adsorbents with high partition coefficients in polyethylene glycol-dextran and polyethylene glycol-phosphate systems is described. These adsorbents may be used to carry to carry proteins away from the insoluble cell fragments generated during cell disruption. By chromatographic elution, proteins may be selectively desorbed in a reduced volume.