CULTIVATION OF ESCHERICHIA COLI AND RECOMBINANT PROTEIN PRODUCTION FOR ITS USE AS A DIAGNOSTIC REAGENT

KIKOT, PAMELA; WILLE, DANIEL; CORREA, HILDA; GRASSELLI, MARIANO

CABA

Congreso; REUNIÓN CONJUNTA SAIB-SAMIGE; 2020

SAIB

High cell density cultivation is a highly desirable feature in industrial processes. The advantages of high cell density cultivation are the improvement of productivity, such as high volumetric productivity, reduced culture volume, which makes downstream processing easier, facilitated cell separation, improved yield in product recovery, reduced amount of wastes and costs for production, and reduced investment for equipment (1). The currently used protein production technology is based on genetically modifed microorganisms such as Escherichia coli (E. coli). It is one of the most widely used host cells in recombinant protein production and metabolic engineering, due to its short dividing time, ability to use inexpensive substrates and additionally, the genetics of E. coli are comparatively simple, well characterized and can easily be manipulated (2).In this study, we tried to construct a high productivity process for recombinant proteins for its use in ELISA and other immunochemical methods. We studied the effects of medium composition, strategies for nutrient feeding, temperature modifications, and supply of pure oxygen gas on the growth of the recombinant E. coli strain BL21(DE3). Two different proteins were expressed in different E. coli clones. In one hand, we focused on high-level fed-batch fermentative expression of an engineered Staphylococcal aureus protein A (SpA) domain, called AviPure (2). Due to high selectivity and good physiochemical stability protein A is a preferred generic ligand for affinity purification of antibodies and molecules tagged with an antibody Fc region. For this reason the molecule have been used for several immunological, and purification applications, therefore there is need for high level production of the protein (2).The cultures were conducted in a 5 liters bioreactor (BIOSTAT Aplus, Sartorius) in Riesenberg mineral medium [3]. During fermentation process some parameters were observed due to their importance; the change in OD, pH, aeration, antifoam, carbon source, agitation. At first the fermentation was run in batch mode with dissolved oxygen level maintained at required saturation (pO2) by using filtered air and with stirring speed in cascade mode in order to achieve and keep pO2 level constant. Calibrated peristaltic pumps were used to control the feed rate for feed media (300 g/l glucose) which was determined by the metabolic rate of the culture. The 600 nm optical density obtained for both cultures was up to 170 uA. The amount of protein purified by IMAC chromatography were 1,6 g for AviPure. 1.Biosci. Biotech. Biochem., 59 (12), 2263-2267, 1995. Satoshi NAKAGAWA,* Hideki ODA, and Hideharu ANAZAWA2.Kangwa et al. AMB Expr (2015) 5:703.Riesenberg et al (1991). Journal of Biotechnology 20 (1). 17-27.