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. 2023 Apr;174(6):749-753.
doi: 10.1007/s10517-023-05785-3. Epub 2023 May 10.

Preparation a Recombinant Form of Pneumolysin Protein from Streptococcus pneumoniae

D S Vorobyev  1 , A V Sidorov  2 , A A Kaloshin  2 , N A Mikhailova  2 , A V Poddubikov  2 , I M Gruber  2
Affiliations

Preparation a Recombinant Form of Pneumolysin Protein from Streptococcus pneumoniae

D S Vorobyev et al. Bull Exp Biol Med. 2023 Apr.

Abstract

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.

Keywords: Streptococcus pneumoniae; affinity chromatography; nucleotide sequence; recombinant pneumolysin; sequencing.

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