Optimization of the production process of acra-o157, a recombinant Glycoprotein for the serodiagnosis of hemolytic uremic syndrome

CAILLAVA, ANA J.; GASTÓN E. ORTIZ; CIOCCHINI, ANDRÉS E.; COMERCI, DIEGO J.

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. 2017; 2018

SAIB

Escherichia coli O157:H7 represents the most important etiological agent of Hemolytic Uremic Syndrome associated with Shigatoxin-producing Escherichia coli (STEC) worldwide. Recently, ourgroup have developed a high-performance test for the serodiagnosis of human infection with E. coli O157 employing a new recombinant glycoprotein (AcrA-O157), consisting of the O157 polysaccharide attached to a carrier protein (AcrA), as antigen. Characterizationand optimization of AcrA-O157 production process is important forincreasing productivity and for scaling up. In this work, we evaluatedAcrA-O157 production in different culture media and defined 2xYPas the optimal culture medium. In order to maximize the specificproductivity of AcrA-O157, we analyzed the relationship betweenthe specific product synthesis rate (qp) and the growth rate (m).Exponential fed-batch cultures were designed using a pre-definedexponential feeding strategy based on the desired m and the biomass yield. We analyzed glycosylation rate, product synthesis rateand production kinetics at three different specific growth rates: 0,32h-1 (equal to mmax), 0,21 h-1 and 0,14 h-1 by Western Blot and ELISA. We observed maximum glycosylation degree of AcrA at highest m indicating that the glycosylation rate is affected by the specificgrowth rate. Specific rate of product formation was also influencedby specific growth rate, increasing 3-fold and 7,2-fold at 0,21 h-1 and0,32 h-1, respectively, when compared to 0,14 h-1. Additionally, weobserved a shorter lag phase to reach the maximal specific productivity when working at higher m. Based on these results, we concludethat mmax is the optimal specific growth rate allowing us to achieve thehighest productivity with a shorter lag phase