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

Author(s)

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

• Document type: Journal article
• Source: FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY; v. 11, p. 13-pg., 2024-01-08.

Abstract

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)

• FAPESP's process: 16/50413-8 - Pulmonary delivery of a targeted mucosal nanocarrier vaccine for pneumonia
• Grantee: Viviane Maimoni Gonçalves
• Support Opportunities: Regular Research Grants
• FAPESP's process: 18/13469-0 - Development of the purification process of the genetically detoxified recombinant pneumolysin obtained in Escherichia coli
• Grantee: Manuella Cazelato Pires
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 17/24832-6 - Development of vaccines based on recombinant BCG: Tuberculosis, Pertussis, Pneumococcus and Schistosoma
• Grantee: Luciana Cezar de Cerqueira Leite
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 21/02930-1 - DEVELOPMENT OF A PNEUMOCOCCAL VACCINE BASED ON MULTIEPITOPES
• Grantee: Vítor dos Santos Alves
• Support Opportunities: Scholarships in Brazil - Doctorate