Biomedical Chemistry: Research and Methods 2024, 7(3), e00237

Recombinant Proteins Combining the Inter-Domain Region of Pneumococcal Surface Antigen "A" and Adaptive W-Type Polypeptide of Thermotoga Bacteria as a Potential Component Base for the Development of New Diagnostics and Genetically Engineered Subunit Vaccines against Pneumococcal Infection

О.K. Parfenova1*, N.G. Sidorov1,2, E.Y. Kasap1, R.V. Kurkin1, D.V. Grishin1

1Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia; *e-mail: molbiol_ibm@inbox.ru
2I. Mechnikov Research Institute of Vaccines and Sera, 5А Malyi Kazennyi lane, Moscow, 105064 Russia

Keywords: fusion proteins; pneumococcal surface protein; thermostable protein; vaccine-valuable protein; Escherichia coli; heterologous expression

DOI:10.18097/BMCRM00237

The whole version of this paper is available in Russian.

The problems related to the development of pneumococcal vaccines require combination of traditional solutions and alternative systems optimizing this procedure. Recombinant subunit vaccines have undeniable advantages over inactivated and live-attenuated vaccines: they induce cell-mediated and humoral immunologic responses effectively and with high specificity, but without the risks associated with authentic pathogen processing. However, subunit vaccines require specific adjuvants to enhance the immune response or special fusion partners to improve solubility, expression and optimize subsequent fine purification of the protein of interest. In the framework of this work, a structurally conserved region of the most immunogenic region of the vaccine-valuable surface antigen PspA of Streptococcus pneumoniae was chosen as a model protein, and the adaptive polypeptide CheW from the hyperthermophilic microorganism Thermotoga petrophila was used as a promising fusion protein. Appropriate expression plasmid vectors were designed in silico and constructed in vitro. Efficient E. coli producer strains were obtained and appropriate conditions for heterologous production of chimeric proteins were selected. The fusion partner from T. petrophila positively influenced the properties of the resulting constructs such as thermostability, solubility, and homogeneity. During this work, the optimal pH and temperature ranges of the created proteins were determined, and the principles of low-stage purification were elaborated. We obtained and characterized new proteins, which were not previously found in nature in a similar bioconfiguration. The results indicate that the biotechnologically valuable characteristics of the fusion protein were more expressed when the adaptive CheW protein was combined with the N-terminus of the PspA antigen.
Figure 1. Scheme of the design of genetically engineered structures A) PspA-sp-CheW and B) CheW-sp-PspA. Promoter T7 – strong promoter of bacteriophage T7; SD – ribosome binding site or Shine-Dalgarno box; PspA – а nucleotide sequence encoding the inter-domain region of S. pneumoniae surface antigen; CheW – а nucleotide sequence encoding a thermally stable W-type adaptive polypeptide from bacteria of the genus Thermotoga; sp – a nucleotide sequence encoding a separating glycine-serine linker; 6×His – a nucleotide sequence encoding six histidine residues; XbaI, NdeI и XhoI – recognition sites for restriction endonucleases; Terminator – terminator of transcription.

Figure 2. Control of expression of genetically engineered cassettes PspA-sp-CheW and CheW-sp-PspA in 12%SDS-PAGE in the presence of 2-Mercaptoethanol and Coomassie Brilliant Blue R-250. (the volume of samples applied to one track without thermolysis was 9 µl, with thermolysis - 16 µl). A) 1 – Molecular weight marker LRPL; 2 – E. coli BL21(DE3) pLysS [pET- CheW-sp-PspA] (induction+heating 75°С); 3 – E. coli BL21(DE3) pLysS [pET-CheW-sp-PspA] (induction, without heating); 4 – E. coli BL21(DE3) pLysS [pET-CheW-sp-PspA] (before induction+heating 75°С); 5 – E. coli BL21(DE3) pLysS [pET-CheW-sp-PspA] (before induction, without heating); 6 –E. coli BL21(DE3) pLysS (induction+heating 75°С); 7 – E. coli BL21(DE3) pLysS (induction, without heating). В) 1 – Molecular weight marker LRPL; 2 – E. coli BL21(DE3) pLysS [pET-PspA-sp-CheW] (induction+heating 75°С); 3 – E. coli BL21(DE3) pLysS [pET- PspA-sp-CheW] (induction, without heating); 4 – E. coli BL21(DE3) pLysS [pET- PspA-sp-CheW] (before induction+heating 75°С); 5 – E. coli BL21(DE3) pLysS [pET- PspA-sp-CheW] (before induction, without heating); 6 – E. coli BL21(DE3) pLysS [pET-TpeCheW] (induction+heating 75°С); 7 – E. coli BL21(DE3) pLysS [pET-TpeCheW] (induction, without heating); 8 – E. coli BL21(DE3) pLysS [pET-TpeCheW] (before induction, without heating).

Figure 3. Comparison of growth dynamics of E. coli BL21(DE3) pLysS cells prosucing PspA-sp-CheW and CheW-sp-PspA chimeric proteins

Figure 44. Analysis of the soluble fraction of purified recombinant protein CheW-sp-PspA at different temperatures and pH 7.5 in 12% SDS-PAGE. 1 – Molecular weight marker LRPL; 2 – E. coli BL21(DE3) pLysS [pET-CheW-sp-PspA] (induction+heating 75°C); 3 – E. coli BL21(DE3) pLysS [pET- CheW-sp-PspA] (induction+heating 80°C); 4 – E. coli BL21(DE3) pLysS [pET-CheW-sp-PspA] (induction+heating 85°C); 5 – E. coli BL21(DE3) pLysS [pET-CheW-sp-PspA] (induction+heating 90°C); 6 – E. coli BL21(DE3) pLysS [pET-CheW-sp-PspA] (induction+heating 99°C).

FUNDING

The research was supported by the Russian Science Foundation grant No. 23-15-00149.

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