DEVELOPMENT OF BIOTECHNOLOGICAL PROCESSES FOR THE PRODUCTION OF RECOMBINANT THERAPEUTIC PROTEINS WITH HIGH SOCIO-ECONOMIC IMPACT

AMARANTO, MARILLA; GODINO, AGUSTINA; RODRIGUEZ-WALKER, MACARENA; DANIOTTI, JOSÉ LUIS; BARRA, JOSÉ LUIS

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

The aim of this work is to develop the biotechnological processes for the production of three recombinant proteins for therapeutic use. The α-galactosidase A (α-GAL) and the α-Glucosidase (α-GAA) are used for the treatment of Fabry disease and Pompe disease, respectively. These diseases are ultra-orphan pathologies whose treatments are amongst the most expensive on a cost-per-patient basis (U$S 150.000-3.000.000/year). The DNaseI is used for the treatment of cystic fibrosis, one of the most common lethal inherited genetic diseases in Caucasian population (approximately 1/2500 births). Initially, we analyzed the transient expression of these proteins in Chinese hamster ovary (CHO-K1) cells. Plasmid pCI-neo (Promega) was used as the expression vector, which promote constitutive expression of cloned DNA inserts in mammalian cells. The coding sequence of each protein followed by HA-tag and preceded by the signal peptide were synthesized (GenScript, USA) and cloned into pCI-neo downstream of CMV promoter region. For the three proteins, the sequences of coding regions and signal peptides used in the constructions were identical to the corresponding human sequences. An additional plasmid was constructed for the α-GAL, in which the signal peptide sequence was identical to the corresponding CHO-K1 cell sequences and the coding sequence was optimized for the codon usage in this cell line. CHO-K1 cells were transfected with the plasmids and the expression of the proteins was analyzed by Western blot and by immunostaining using anti-HA antibodies, followed by confocal microscopy observation. All three proteins were successfully expressed. However, the optimized sequence seems to work worse than the non-optimized sequence. Treatment of transfected CHO-K1 cells with tunicamycin (N-glycosylation inhibitor) modified the relative migration of the three proteins indicating that the expressed proteins are glycosylated, requirement for the functionality of these proteins.