Control of Proteolysis of Recombinant Proteins in Escherichia coli

摘要: Proteolysis was shown to have a significant impact on the accumulation and final yield of recombinant proteins. Much smaller intracellular concentrations proteins SpA ZZT2 compared their stable versions SpA-βgalactosidase ZZT0 were explained by high proteolysis rate. The reduction rate in Clp protease-deficient strain enabled increase concentration two-fold, but impaired cell growth. A method calculate hypothetical product assuming complete stabilization described, which reveals potential productivity eliminating proteolysis. effects scale-up production protein investigated reactors different configuration scale: well-mixed lab-scale bioreactor, an industrial scale 12-m two-compartment scale-down bioreactor. highest cultivation (50 mg/g), however large-scale declined 8 hours after induction from its value 45 35 mg/g. cultivations even lower than large-scale. higher declining synthesis are thought be reasons for yields cultivations. heterogeneities appeared prevent decline viable count death, usually observed at end fed-batch reactors. Two mixtures amino acids fed together with glucose cultures aiming improve biomass protein. One mixture contained all twenty quantities, would sufficient half proteins, including other only five socalled “protein acids”, reported primarily incorporated into biomass: alanine, arginine, methionine, histidine phenylalanine. Although both cases feeding improved slightly reduced rate, less control. is reason this. acids” shake flask culture inhibited cellular growth, seen as indicator disturbed metabolism caused added acids. novel technique, temperature-limited culture, applied Low temperature surplus limit endotoxin release decrease degradation ZZT2. More work needed maintain desired range avoid acetate during this type cultivation. absence oxygen transition period downstream processing resulted ATP pool 0.5 mM almost stabilisation A. Neither interruption under aerobic conditions nor anaerobic presence could stabilise completely did not 0.75-1 treatment. Therefore, correlated experiments vivo. ADP AMP increased starvation also play role may limiting factor further steps processing. Its decreased 80-90% harvesting centrifugation disruption cells.