Bioprocessing strategies in improving recombinant human interleukin-3 production in Streptomyces lividans fermentation.

摘要

Interleukin-3 is a cytokine, which acts on many target cells within the haemopoietic system, often in synergy with other cytokines. Streptomyces lividans NUMB 11416/IL3 p002 secreting human interleukin-3 was used as the host organism for the study of improving target protein production in a filamentous host. The host loses its plasmids upon sporulation, which dictated the use of mycelial suspension inocula rather than spores. Recombinant human interieukin-3 (rHuIL-3) in culture supernatant suffered proteolysis, compromising its authenticity and reducing overall product titre. Proteases were considered as either general proteases (assayed on casein as substrate) and a sequence-specific protease, namely tripeptidyl peptidase A (tppA) (assayed with an interleukin-3 analog). General proteolytic activity was detected in the culture medium starting from the mid log phase (18 hours post-inoculation). TppA activity was observed to be directly associated with the concentration of rHuIL-3 in the medium. At constant nominal glucose concentration in the medium, low C:N ratios seemed to promote tppA action causing a dramatic increase in compromised rHuIL-3 concentration. An isoelectric-focusing (IEF) gel electrophoresis was developed for the rapid assessment of rHuIL-3 authenticity. The cultures were grown in shake flasks, airlift and stirred bioreactors (STR). Fermentation in STR at high agitation rates (200 rpm) gave rise to more dispersed filamentous growth and higher occurrence of cell damage, contaminating the culture broth with cell lysate. In air-sparged systems, productivity reached a plateau at an aeration rate of 1.5 vvm. Volumetric productivity of rHuIL-3 could be increased via the application of engineering principles to the production process, namely the introduction of the temperature downshift and oxygen-enriched cultures, each producing a maximal rHuIL-3 titre of about 250 mg/L. Fluorescence microscopy of pellets treated with live/dead staining confirmed complete cell viability throughout the production phase of the fermentation.