ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

Abstract

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β- d -thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. Key points • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-11758-9.

Efficient production of human interleukin‐3 from Escherichia coli using protein disulfide isomerase b'a' domain

“…Firstly, the slow growth of the ClearColi strain could potentially be overcome by high cell density bioreactor culture using rich, complex media formulations to boost biomass and productivity. [ 38 ] Secondly, the purification processes for scale‐up can be optimized and the stability of the recombinant IL3 under various storage conditions can be investigated. Thirdly, the biological activity of IL3 in different cell lines or in vivo models could be explored to attain a more comprehensive understanding of its therapeutic potential.…”

Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate

“…In the case of two proteins, both options of order should be tested unless there is a hypothesis for a specific order. In our case, this specific order was chosen because PspA is a well-known soluble protein ( Figueiredo et al, 2017 ; Cardoso et al, 2022 ), which may act as a solubility enhancer for PdT, a protein that frequently presents solubility issues in our lab (data not shown). Moreover, the PspA sequence that we selected presents a part of its proline-rich region in C-terminal, consisting of several repeated residues of proline and alanine that could act as a “bridge” between protein domains, since some described rigid linkers are composed of these residues ( Zhao et al, 2008 ).…”

Maternal immunization with pneumococcal surface protein A provides the immune memories of offspring against pneumococcal infection

“…It is controversial whether any contamination in the purified rPspA may have influenced the results ( Supplementary Figure ). To exclude the possibility that the contamination in the recombinant protein might have enhanced the result of the immunized mother or the offspring derived from and/or fostered by the immunized mother, recent techniques that reduce the endotoxin response in mammalian cells by converting LPS to lipid Iva or a baculovirus expression system should be better strategies for future projects ( Ono et al., 2018 ; Cardoso et al., 2022 ). Comparing the control group that were sham-immunized with the control extract (from E. coli harboring pET20b vector alone) would be another method.…”