Produção de uma mini pró-insulina Lys-Pro Glargina Canina

Abstract

If there is a hormone that represents the boundary between health and disease, that hormone is insulin. In canine diabetes mellitus, its deficiency necessitates continuous administration of the hormone and, despite advances in the development of analogues for human use, formulations developed specifically for dogs remain limited. In this context, protein engineering and recombinant production in Escherichia coli represent promising strategies for obtaining molecules adapted to the particularities of the canine species. However, the expression of low-molecular-weight proteins in bacterial systems may be limited by low intracellular stability, aggregation, and possible proteolytic degradation. This study aimed to produce a recombinant canine Lys-Pro Glargine mini-proinsulin in E. coli and evaluate the influence of induction conditions on its expression profile. The synthetic insK gene, comprising 252 base pairs, was designed in silico from the native canine insulin sequence (GenBank: V00179.1), incorporating the structural modifications of interest, reduction of the C-peptide, and codon optimization for bacterial expression. The gene was commercially synthesized as a GeneBlock and subcloned into the pGSM-tGH expression vector using the restriction enzymes NdeI and BamHI. The recombinant construct, designated pGSMK, showed a restriction digestion profile compatible with the presence of the insert and was subsequently introduced into E. coli BL21(DE3). Expression was initially evaluated under conventional conditions using 0.5 mM IPTG and incubation at 37 °C. Under these conditions, SDS-PAGE analysis did not show an evident increase in the intensity of a band in the region corresponding to the expected molecular mass of the recombinant protein. Based on this result, an alternative strategy consisting of culture pre-cooling, thermal adaptation, induction with 0.1 mM IPTG, and incubation at 18 °C for 17 hours was evaluated. The low-temperature protocol resulted in a more intense band at the electrophoretic migration front, compatible with the low theoretical molecular mass of the canine mini-proinsulin. Expression of canine proinsulin was confirmed by ELISA using an anti-human insulin antibody. The 18 °C protocol was the most promising condition among those evaluated, providing an experimental basis for subsequent purification, identification, and functional characterization of the recombinant protein.