Production optimization of recombinant scorpion antivenom through biotechnological pathway: study of temperature of induction and modeling of biokinetics on E. coli strains

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Scorpionism is a serious public health problem in tropical areas, especially in Africa, southern India, the Middle East, and Latin America. There are about 12 species of scorpions that are dangerous to humans and all belong to the Buthidae family. In particular, venom on scorpion Androctonus australis hector is particularly toxic to humans. Serotherapy uses antibodies or fragments of antibodies to target the neurotoxins of the scorpion venom. Nanobodies, camelid-derived antibodies, are more effective than the conventional fragments of antibodies due to their low molecular weight (15 kDa). The Laboratoire des Venins et Molécules Thérapeutiques of the Pasteur Institute of Tunis (LVMT-IPT, Tunis, Tunisia) has produced the bispecific nanobodies against the neurotoxins of the Androctonus australis hector scorpion. In this doctoral project, the production of the bispecific nanobodies NbF12-10 and CH10-12 as recombinant proteins in Escherichia coli was studied. Two clones of the NbF12-10 strain were used (NN and NO), and strain E. coli WK6 was used as reference. The four strains were characterized in rich medium (TB) and defined minimal medium (MM) in shake-flask cultures. In TB, all strains grow at an average μ=0.4h-1. The strains WK6 and CH10-12 had a μmax=0.6h-1 in MM. The clone NbF12-10 NO could not grow in MM, and the clone NbF12-10 NN had a μmax=0.2h-1 in MM. the yield of biomass on glucose, YX/S, was 0.4 g/g in MM for all strains and the yield of acetate on glucose was between 0.06 and 0.14 g/g. The periplasmic extraction of the nanobodies NbF12-10 and CH10-12 was tested in TB, an improvement of 300% in the release of periplasmic proteins was achieved, compared to conventional methods. The strain CH10-12 produced 20-fold higher nanobody than the strain NbF12-10 NN (1.58 vs 0.08 mg/L). The quantification of the nanobody was made through a densitometry protocol developed during this thesis. The protocol uses a processing image program (ImageJ, NIH, USA) to quantify the optical density profiles of electrophoresis gels. The band of 50 kDa of the molecular weight marker was used as reference for 750 ng of recombinant protein. The production of the nanobodies CH10-12 and NbF12-10 were tested in bioreactor cultures in high cell density cultures in MM ([X]> 25 g cdw/L) through a specific fed-batch strategy. The effect of the temperature (28 – 37°C) during protein expression and the duration of the induction phase (6 – 38 h) were studied. The lower temperatures (28°C, 30°C) produced the highest titer of nanobody CH10-12 (2.3 mg/L) after 10 h and 12 h of induction, respectively. In high temperature cultures (33°C, 37°C) the production of the nanobody was lower (0.4, 0.2 mg/L). In cultures where the induction phase was longer (>30 h), the production of the nanobody CH10-12 was hampered and reached a plateau after 10 h (32°C, 0.7 mg/L) to 25 h (29°C, 1.4 mg/L). The nanobody NbF12-10 also reached a plateau after 10 h of induction (0.7 mg/L). The metabolic burden was lowered by decreasing the temperature of induction, allowing the production of higher titers of recombinant nanobody. The effect of the low specific growth rate (0.02h-1) and the long exposure to the inducer (IPTG, >30 h) could have produced a stringent response in the strain. High maintenance metabolism was found during the induction phase, and the consumption of citrate could hint an inadequate composition of the culture medium for the induction phase, where the microorganism produces internal metabolites to then produce the nanobody. Two non-specific proteins were found in the purified nanobody samples: a low molecular weight (11 kDa), degradation product of the nanobody caused by a high temperature (proteolytic degradation), and a high molecular weight protein (84 kDa), protein aggregate produced as a response to an inadequate medium composition and the low specific growth rate. The generation of unknown proteins in the purified samples of nanobody could be a response of the strain to the low specific growth rate and the long exposure time to the inducer. Finally, A mathematical model has been proposed to validate the hypothesis of the thermo-dependence of the production of recombinant nanobodies with a Luedeking-Piret kinetics modified with Arrhenius-type kinetic parameters. The model showed that the highest production was obtained at the lowest temperature (28°C), and that the Escherichia coli strain NbF12-10 NN showed the same trend as E. coli CH10-12 cultures.

Keywords: recombinant protein, biotechnological pathway, Escherichia coli, industrialization