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

PAMELA KIKOT; DANIEL WILLE; HILDA CORREA; MARIANO GRASSELLI

Otro; REUNIÓN CONJUNTA SAIB-SAMIGE; 2020

SAIB SAMIGE

High cell density cultivation is a highly desirable feature in industrial processes. The advantages of high cell densitycultivation are the improvement of productivity, such as high volumetric productivity, reduced culture volume, whichmakes downstream processing easier, facilitated cell separation, improved yield in product recovery, reduced amountof wastes and costs for production, and reduced investment for equipment. The currently used protein productiontechnology is based on genetically modified microorganisms such as Escherichia coli (E. coli). It is one of the mostwidely 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, wellcharacterized and can easily be manipulated.In this study, we tried to construct a high productivity process for recombinant proteins for its use in ELISA and otherimmunochemical methods. We studied the effects of medium composition, strategies for nutrient feeding, temperaturemodifications, and supply of pure oxygen gas on the growth of the recombinant E. coli strain BL21(DE3). We focusedon high-level fed-batch fermentative expression of an engineered Staphylococcal aureus protein A (SpA) domain,called AviPure. Due to high selectivity and good physiochemical stability protein A is a preferred generic ligand foraffinity purification of antibodies and molecules tagged with an antibody Fc region. For this reason the molecule hasbeen used for several immunological, and purification applications, therefore there is need for high level production ofthe protein.The cultures were conducted in a 5-liter bioreactor (BIOSTAT Aplus, Sartorius) in Riesenberg mineral medium.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 levelmaintained at required saturation (pO2) by using filtered air and with stirring speed in cascade mode in order to achieveand keep pO2 level constant. Calibrated peristaltic pumps were used to control the feed rate for feed media (300 g/lglucose) which was determined by the metabolic rate of the culture. The 600 nm optical density obtained for bothcultures was up to 170 uA. The amount of protein purified by IMAC chromatography were 1,6 g for AviPure.