Construção de novas moléculas recombinantes hipoalergênicas com potencial aplicação imunoterapêutica utilizando abordagens de biologia sintética.

Abstract

The development of modern biotechnology demands satisfactory amounts of soluble and biologically active recombinant proteins, given the growing use of heterologous proteins in research laboratories and therapeutic applications. Therefore, selecting an ideal expression system, including the genetic elements and the host for expression, is a major issue in developing recombinant proteins. Recently, the significant influence that genetic elements have on the production of a recombinant protein has been unveiled, and it is feasible to optimize these elements thanks to advances in the synthetic biology field. Studies of literature data carried out by our research group allowed the assembly of a new Expression Operational Unit (EOU) comprising standardized and well-characterized biological parts to improve the expression of recombinant proteins in bacterial hosts. We also constructed a novel variant of the T7 promoter with increased transcriptional activity (1.7-fold higher) compared to the wild-type T7 promoter. This new EOU generated an improved production of the superfolderGFP reporter protein (sfGFP) in Escherichia coli BL21(DE3) (relative fluorescence units/RFU = 70.62 ± 1.62 A U.) when compared to an expression vector used as control (plasmid BBa_I746909; RFU = 59.68 ± 1.82 A U.). The functionality of this UOE was also evaluated with the production of the recombinant allergen Der p 21 wt and its hypoallergenic mutant variants. Der p 21 is considered an important allergen, with a high binding capacity for IgE antibodies and high allergenic activity. It defines this protein as a potential candidate for allergen-specific immunotherapy (AIT) against allergy triggered by Dermatophagoides pteronyssinus. Thus, hypoallergenic variants of the Der p 21 allergen were constructed to compose vaccine formulations for TIA, following a structural bioinformatics and immunoinformatics approach. Two of these mutants variants (K110G and E87S) displayed a low binding capacity to IgE compared to the allergen rDer p 21. Hence, these new hypoallergenic proteins are promising candidates for composing next-generation allergen-specific immunotherapy formulations in the near future. Moreover, the use of optimized expression vectors, such as the one assembled in this study, offers excellent advantages in the production of recombinant proteins of biotechnological interest, allowing for efficient production with improved yield and quality.