Produção e exportação de proteínas recombinantes em Escherichia coli através da fusão com proteína PE – avaliação preliminar para produção de biofármacos.

Abstract

Biopharmaceuticals constitute a great opportunity for the brazilian market. With expiration of patents for some medicines of biological origin, it presents itself as a faster and cheaper production route for the treatment of rare and recurrent diseases. To obtain the final protein product there are several steps, with the final protein extraction and purification steps being the most expensive and costly. In order to optimize these steps, we seek to develop a production system through fusion with the PE protein. The emulsifying protein of Pseudomonas aeruginosa has the potential to cross protein targets from the cytoplasm to the periplasm and extracellular region. For to produce recombinant proteins, the interest proteins was fused with PE protein. Fusions were made with PE signal peptide and others with the entire PE protein. The tested protein constructions were the biosimilar candidates betainterferon 1-b and anakinra, a bone morphogenetic protein (BMP-2), 2xPE and enterokinase. For enterokinase, a fusion with DsbA was also performed. The fusions performed with the PE protein in 2xPE and with the BMP-2 protein aim to understand and evaluate PE ability to facilitate the export of proteins of interest to the extracellular environment. With the fusions of betainterferon 1-b and anakinra to PE, the objective is to obtain them in the periplasm and culture supernatant, facilitating downstream purification steps. As with enterokinase, they were made for the correct cleavage of the previous constructions, eliminating a fused protein from the final protein product without leaving residues remaining. Constructs containing the PE signal and immature PE protein fused to betainterferon 1-b, expression was not observed. BMP-2 fused with whole PE protein appears to be expressed, however the protein did not go to the culture supernatant. anakinra's constructs were positively expressed. The IL-1Ra construct fused to the PE signal peptide was exported to the periplasm and the IL-1Ra construct fused to an entire PE protein had part of its sequence exported to the culture medium. The enterokinase constructs with PE do not express, however, constructs with DsbA were positively expressed and was secreted into the periplasm. Additionally, the enzymatic activity of enterokinase was observed when incubated with the anakinra construct associated with PE signal peptide. Anakinra expressed and purified from the periplasm presented inhibition activity of interleukin 1 receptors in vivo, with decreased of neutrophil count when compared with not treated animals in gout disease induction model. Ultimately, the potential export tool provided by the PE protein was demonstrated with the ps2xPE, imPE_AK and psPE_AK constructs.