Inactivation of diphtheria toxin by site-directed mutagenesis
Само за регистроване кориснике
2024
Аутори
Prijić, IvanaPanić, Marko
Simić, Mihajlo
Blagojević, Veljko
Ćuruvija, Ivana
Lukić, Ivana
Dragačević, Luka
Kojić, Milan
Конференцијски прилог (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
Diphtheria toxin is a single polypeptide chain produced by toxigenic strains of Corynebacterium diphtheriae that causes the disease diphtheria in humans by gaining entry into the cytoplasm of cells and inhibiting protein synthesis. Formaldehyde (chemical) detoxification converts diphtheria toxin into toxoid, which is used in diphtheria vaccine production. Recombinant, genetically detoxified diphtheria toxin is superior in terms of safety and purity, but it has still not found its application in recombinant diphtheria vaccine production. Both chemically and genetically inactivated forms of the diphtheria toxin have proven effective as protein carriers in conjugate vaccines. The goal of this study was to create a plasmid construct which can be used to express a genetically inactivated diphtheria toxin. Gene coding for diphtheria toxin was cloned into pMALHisEk expression vector and introduced into DH5α competent Escherichia coli cells. Three site-directed point mutations, which led to th...ree amino acid substitutions (G52E-substitutes glycine with glutamic acid, G79D- substitutes glycine with aspartic acid, E148D- substitutes glutamic acid with aspartic acid) were conducted. A single G52E amino acid substitution is responsible for the loss of the enzymatic activity of the diphtheria toxin. G79D is recognized as a good candidate site for combining with other mutations in vaccine development and E148D may be a good candidate as carrier protein because it could reduce both the stability of NAD binding and catalytic activity of the enzyme. Each individual mutation is sufficient for toxin inactivation, but together they ensure non-toxicity, preventing reversion to the wild-type sequence. All mutations were confirmed by DNA sequencing. Recombinant diphtheria toxoid could serve as a potential vaccine epitope or protein carrier for conjugate vaccines. Further optimization of recombinant protein expression in Escherichia coli should provide sufficient quantities of soluble recombinant protein for further testing of its safety, immunogenicity and protection.
Кључне речи:
diphtheria toxin / genetic detoxification / site-directed mutagenesis / vaccineИзвор:
XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april, 2024, 115-115Издавач:
- Serbian Society for Microbiology
Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200177 (Центар за имунолошка истраживања 'Бранислав Јанковић' Торлак, Београд) (RS-MESTD-inst-2020-200177)
Институција/група
TorlakTY - CONF AU - Prijić, Ivana AU - Panić, Marko AU - Simić, Mihajlo AU - Blagojević, Veljko AU - Ćuruvija, Ivana AU - Lukić, Ivana AU - Dragačević, Luka AU - Kojić, Milan PY - 2024 UR - http://intor.torlakinstitut.com/handle/123456789/877 AB - Diphtheria toxin is a single polypeptide chain produced by toxigenic strains of Corynebacterium diphtheriae that causes the disease diphtheria in humans by gaining entry into the cytoplasm of cells and inhibiting protein synthesis. Formaldehyde (chemical) detoxification converts diphtheria toxin into toxoid, which is used in diphtheria vaccine production. Recombinant, genetically detoxified diphtheria toxin is superior in terms of safety and purity, but it has still not found its application in recombinant diphtheria vaccine production. Both chemically and genetically inactivated forms of the diphtheria toxin have proven effective as protein carriers in conjugate vaccines. The goal of this study was to create a plasmid construct which can be used to express a genetically inactivated diphtheria toxin. Gene coding for diphtheria toxin was cloned into pMALHisEk expression vector and introduced into DH5α competent Escherichia coli cells. Three site-directed point mutations, which led to three amino acid substitutions (G52E-substitutes glycine with glutamic acid, G79D- substitutes glycine with aspartic acid, E148D- substitutes glutamic acid with aspartic acid) were conducted. A single G52E amino acid substitution is responsible for the loss of the enzymatic activity of the diphtheria toxin. G79D is recognized as a good candidate site for combining with other mutations in vaccine development and E148D may be a good candidate as carrier protein because it could reduce both the stability of NAD binding and catalytic activity of the enzyme. Each individual mutation is sufficient for toxin inactivation, but together they ensure non-toxicity, preventing reversion to the wild-type sequence. All mutations were confirmed by DNA sequencing. Recombinant diphtheria toxoid could serve as a potential vaccine epitope or protein carrier for conjugate vaccines. Further optimization of recombinant protein expression in Escherichia coli should provide sufficient quantities of soluble recombinant protein for further testing of its safety, immunogenicity and protection. PB - Serbian Society for Microbiology C3 - XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april T1 - Inactivation of diphtheria toxin by site-directed mutagenesis EP - 115 SP - 115 UR - https://hdl.handle.net/21.15107/rcub_intor_877 ER -
@conference{ author = "Prijić, Ivana and Panić, Marko and Simić, Mihajlo and Blagojević, Veljko and Ćuruvija, Ivana and Lukić, Ivana and Dragačević, Luka and Kojić, Milan", year = "2024", abstract = "Diphtheria toxin is a single polypeptide chain produced by toxigenic strains of Corynebacterium diphtheriae that causes the disease diphtheria in humans by gaining entry into the cytoplasm of cells and inhibiting protein synthesis. Formaldehyde (chemical) detoxification converts diphtheria toxin into toxoid, which is used in diphtheria vaccine production. Recombinant, genetically detoxified diphtheria toxin is superior in terms of safety and purity, but it has still not found its application in recombinant diphtheria vaccine production. Both chemically and genetically inactivated forms of the diphtheria toxin have proven effective as protein carriers in conjugate vaccines. The goal of this study was to create a plasmid construct which can be used to express a genetically inactivated diphtheria toxin. Gene coding for diphtheria toxin was cloned into pMALHisEk expression vector and introduced into DH5α competent Escherichia coli cells. Three site-directed point mutations, which led to three amino acid substitutions (G52E-substitutes glycine with glutamic acid, G79D- substitutes glycine with aspartic acid, E148D- substitutes glutamic acid with aspartic acid) were conducted. A single G52E amino acid substitution is responsible for the loss of the enzymatic activity of the diphtheria toxin. G79D is recognized as a good candidate site for combining with other mutations in vaccine development and E148D may be a good candidate as carrier protein because it could reduce both the stability of NAD binding and catalytic activity of the enzyme. Each individual mutation is sufficient for toxin inactivation, but together they ensure non-toxicity, preventing reversion to the wild-type sequence. All mutations were confirmed by DNA sequencing. Recombinant diphtheria toxoid could serve as a potential vaccine epitope or protein carrier for conjugate vaccines. Further optimization of recombinant protein expression in Escherichia coli should provide sufficient quantities of soluble recombinant protein for further testing of its safety, immunogenicity and protection.", publisher = "Serbian Society for Microbiology", journal = "XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april", title = "Inactivation of diphtheria toxin by site-directed mutagenesis", pages = "115-115", url = "https://hdl.handle.net/21.15107/rcub_intor_877" }
Prijić, I., Panić, M., Simić, M., Blagojević, V., Ćuruvija, I., Lukić, I., Dragačević, L.,& Kojić, M.. (2024). Inactivation of diphtheria toxin by site-directed mutagenesis. in XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april Serbian Society for Microbiology., 115-115. https://hdl.handle.net/21.15107/rcub_intor_877
Prijić I, Panić M, Simić M, Blagojević V, Ćuruvija I, Lukić I, Dragačević L, Kojić M. Inactivation of diphtheria toxin by site-directed mutagenesis. in XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april. 2024;:115-115. https://hdl.handle.net/21.15107/rcub_intor_877 .
Prijić, Ivana, Panić, Marko, Simić, Mihajlo, Blagojević, Veljko, Ćuruvija, Ivana, Lukić, Ivana, Dragačević, Luka, Kojić, Milan, "Inactivation of diphtheria toxin by site-directed mutagenesis" in XIII Congress of microbiologists of Serbia with international participation, Mikromed regio 5, From biotechnology to human and planetary health, 4-6 april (2024):115-115, https://hdl.handle.net/21.15107/rcub_intor_877 .