A new vector for heterologous gene expression in Escherichia coli with increased stability in the absence of antibiotic.

Expression vectors for industrial production should be stable and allow tight control of protein synthesis. This is necessary to ensure plasmid transmission to daughter cells in order to achieve a stable population capable of synthesizing high amounts of the target protein. A high-copy-number plasmid, pAE, was previously used for laboratory-scale production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and the Schistosoma mansoni fatty acid binding protein (rSm14), but it was unstable for large-scale production. Therefore, here we evaluated a new expression vector derived from pAE, pAR-KanI, which combines two plasmid replication strategies: a high-copy plasmid pUC origin of replication as pAE, and a par locus sequence derived from pSC101, which is typical of low copy plasmids, for rhG-CSF and rSm14 production in Escherichia coli. Clones bearing these constructs were cultivated in two complex media (2YT and auto-induction) and both yielded higher-than-95% resistant colonies, before and after induction, either with or without antibiotics. In 2YT medium, we obtained 244 µg/mL of rSm14, 181 µg/mL and 392 µg/mL for rhG-CSF, with and without glucose, respectively. In auto-induction medium without antibiotics, 147 µg/mL of rSm14 and 162 µg/mL of rhG-CSF were obtained. The new vector presented high stability for the production of both recombinant proteins in complex media in Escherichia coli, even in the absence of antibiotics, making the pAR-KanI a promising vector for industrial production of recombinant proteins.

Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content.

Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 °C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 ± 2.5% of PspA4Pro with 97.8 ± 0.36% purity and reduced endotoxin concentration by >99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.

Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate.

Streptococcus pneumoniae is a bacterial pathogen exclusive to humans, responsible for respiratory and systemic diseases. Pneumococcal protein vaccines have been proposed as serotype-independent alternatives to currently used conjugated polysaccharide vaccines, which have presented limitations regarding their coverage. Previously in our group, pneumococcal surface protein A (PspA) and detoxified pneumolysin (PdT) were genetically fused and the hybrid protein protected mice against pneumococcal challenge, offered higher cross-protection against different strains and showed greater opsonophagocytosis rate than co-administered proteins. As juxtaposed fusion was unstable to upscale production of the protein, flexible (PspA-FL-PdT) and rigid (PspA-RL-PdT) molecular linkers were inserted between the antigens to increase stability. This work aimed to produce recombinant fusion proteins, evaluate their stability after linker insertion, both in silico and experimentally, and enable the production of two antigens in a single process. The two constructs with linkers were cloned into Escherichia coli and hybrid proteins were purified using chromatography; purity was evaluated by SDS-PAGE and stability by Western blot and high performance size exclusion chromatography. PspA-FL-PdT showed higher stability at -20°C and 4°C, without additional preservatives. In silico analyses also showed differences regarding stability of the fusion proteins, with molecule without linker presenting disallowed amino acid positions in Ramachandran plot and PspA-FL-PdT showing the best scores, in agreement with experimental results. Mice were immunized with three doses and different amounts of each protein. Both fusion proteins protected all groups of mice against intranasal lethal challenge. The results show the importance of hybrid protein structure on the stability of the products, which is essential for a successful bioprocess development.