Development of biotechnological processes for the production of recombinant therapeutic proteins with high socio-economic impact

AMARANTO M.; GODINO A.; RODRIGUEZ WALKER M.; DANIOTTI J.L.; BARRA J.L.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Reunión Conjunta de Sociedades de Biociencias

The aim of this work is to develop the biotechnological processesfor the production of three recombinant proteins for therapeuticuse. The α-galactosidase A (α-GAL) and the α-Glucosidase(α-GAA) are used for the treatment of Fabry disease and Pompedisease, respectively. These diseases are ultra-orphan pathologieswhose treatments are amongst the most expensive on a cost-per patientbasis (U$S 150.000-3.000.000/year). The DNaseI is usedfor the treatment of cystic fibrosis, one of the most common lethalinherited genetic diseases in Caucasian population (approximately1/2500 births). Initially, we analyzed the transient expression ofthese proteins in Chinese hamster ovary (CHO-K1) cells. PlasmidpCI-neo (Promega) was used as the expression vector, which promoteconstitutive expression of cloned DNA inserts in mammaliancells. The coding sequence of each protein followed by HA-tag andpreceded by the signal peptide were synthesized (GenScript, USA)and cloned into pCI-neo downstream of CMV promoter region. Forthe three proteins, the sequences of coding regions and signal peptidesused in the constructions were identical to the correspondinghuman sequences. An additional plasmid was constructed for theα-GAL, in which the signal peptide sequence was identical to thecorresponding CHO-K1 cell sequences and the coding sequencewas optimized for the codon usage in this cell line. CHO-K1 cellswere transfected with the plasmids and the expression of the proteinswas analyzed by Western blot and by immunostaining usinganti-HA antibodies, followed by confocal microscopy observation. Allthree proteins were successfully expressed. However, the optimizedsequence seems to work worse than the non-optimized sequence.Treatment of transfected CHO-K1 cells with tunicamycin (N-glycosylationinhibitor) modified the relative migration of the three proteinsindicating that the expressed proteins are glycosylated, requirementfor the functionality of these proteins.