Influence of the mRNA initial region on protein production: a case study using recombinant detoxified pneumolysin as a model

Autor(es)

Fusco, Filipe ; Pires, Manuella Cazelato ; Lopes, Alexandre Paulo Yague ; Alves, Vitor dos Santos ; Goncalves, Viviane Maimoni

• Tipo de documento: Artigo Científico
• Fonte: FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY; v. 11, p. 13-pg., 2024-01-08.

Resumo

Recombinant proteins are of great importance in modern society, mostly as biopharmaceutical products. However, challenging and complex processes with low production yield are major drawbacks. Normally, the optimization to overcome these obstacles is focused on bioreactor and purification processes, and the biomolecular aspects are neglected, seen as less important. In this work, we present how the 5 ' mRNA secondary structure region can be relevant for translation and, therefore, protein production. For this, Escherichia coli BL21(DE3) clones, producing recombinant detoxified pneumolysin (PdT) with and without the N-terminal His-tag, were cultivated in 10-L bioreactors. Another version of the pdt gene (version 2) with synonymous changes in the 5 '-end nucleotide sequence was also obtained. Protein production, plasmid stability, carbon sources, and acetic acid were quantified during the cultures. Furthermore, in silico mRNA analyses were performed using TIsigner and RNAfold. The results showed that the His-tag presence at the N-terminus generated a minimum 1.5-fold increase in target protein synthesis, which was explained by the in silico mRNA analyses that returned an mRNA secondary structure easier to translate and, therefore, higher protein production than without the His-tag. The pdt gene version 2 showed lower 5 ' mRNA opening energy than version 1, allowing higher PdT production even without a tag. This work reveals that simple mRNA analyses during heterologous gene design and production steps can help reach high-recombinant protein titers in a shorter time than using only traditional bioprocess optimization strategies. (AU)

• Processo FAPESP: 16/50413-8 - Administração pulmonar de uma vacina de mucosa para pneumonia
• Beneficiário: Viviane Maimoni Gonçalves
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 18/13469-0 - Desenvolvimento do processo de purificação da pneumolisina recombinante geneticamente destoxificada obtida em Escherichia coli
• Beneficiário: Manuella Cazelato Pires
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 17/24832-6 - Desenvolvimento de vacinas baseadas em BCG recombinante: Tuberculose, Pertussis, Pneumococo e Schistosoma
• Beneficiário: Luciana Cezar de Cerqueira Leite
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 21/02930-1 - Desenvolvimento de vacina pneumocócica baseada em multiepítopos
• Beneficiário: Vítor dos Santos Alves
• Modalidade de apoio: Bolsas no Brasil - Doutorado