Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens

A risk of introducing into the Russian Federation exotic infections including laboratory-confirmed melioidosis regularly recorded worldwide necessitates development and improvement of express diagnostics tools. Cross reactivity between phylogenetically related species of the genus Burkholderia compl...

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Autores principales:	Yu. A. Kuzyutina, I. B. Zakharova, D. V. Viktorov
Formato:	article
Lenguaje:	RU
Publicado:	Sankt-Peterburg : NIIÈM imeni Pastera 2019
Materias:	melioidosis burkholderia pseudomallei ompa omp38 producing strain recombinant antigen epitope Infectious and parasitic diseases RC109-216
Acceso en línea:	https://doaj.org/article/3c830a4e5c6e40608a3b0e862443b25b
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spelling	oai:doaj.org-article:3c830a4e5c6e40608a3b0e862443b25b2021-11-22T07:09:52ZEngineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens2220-76192313-739810.15789/2220-7619-2019-1-203-208https://doaj.org/article/3c830a4e5c6e40608a3b0e862443b25b2019-05-01T00:00:00Zhttps://www.iimmun.ru/iimm/article/view/717https://doaj.org/toc/2220-7619https://doaj.org/toc/2313-7398A risk of introducing into the Russian Federation exotic infections including laboratory-confirmed melioidosis regularly recorded worldwide necessitates development and improvement of express diagnostics tools. Cross reactivity between phylogenetically related species of the genus Burkholderia complicates melioidosis diagnostics by express test methods based on using monoclonal antibodies against pathogen exopolysaccharide epitopes. Searching for target antigens to create the next generation group- and species-specific immunodiagnostic reagents for identifying Burkholderia pseudomallei is still of high priority. The study was aimed at cloning complete coding sequences for cell surface proteins differentiating Burkholderia pseudomallei and optimizing recombinant antigens purification protocol. In silico comparative study allowed to select highly immunogenic B. pseudomallei outer membrane proteins Omp38 and OmpA/МotB as target biomolecules. For cloning, omp38 and ompA/motB gene-specific amplicons were obtained by PCR and ligated with the linear expression vector RIC-Ready pPAL7. Competent E. coli C-Max5α cells were transformed by a ligation mixture for producing recombinant plasmids, which were further purified to transform E. coli BL21 (DE3) cells for robust recombinant protein expression. Due to a potential multimeric protein structure, a standard protein purification protocol from native cell lysate was inefficient, which was modified to increase recombinant protein yield. However, by adding denaturing conditions at intermediate purification steps caused hydrolysis of peptide bonds in the target proteins, presumably between proline and asparagine residues. As a result, N-terminal fragments connecting recombinant proteins to the stationary phase of chromatographic column were eluted and evaluated for linear epitope detection according to their molecular weights. In silico analysis data identified highly antigenic motifs within the polypeptides studied. Thus, strains of E. coli BL21(DE3) BpsOmp39 and E. coli BL21(DE3) BpsOmpА engineered by us produce cell surface proteins Omp38 and ОmpA/МotB derived from melioidosis pathogen, which can be useful for developing diagnostic test systems.Yu. A. KuzyutinaI. B. ZakharovaD. V. ViktorovSankt-Peterburg : NIIÈM imeni Pasteraarticlemelioidosisburkholderia pseudomalleiompaomp38producing strainrecombinant antigenepitopeInfectious and parasitic diseasesRC109-216RUInfekciâ i Immunitet, Vol 9, Iss 1, Pp 203-208 (2019)
institution	DOAJ
collection	DOAJ
language	RU
topic	melioidosis burkholderia pseudomallei ompa omp38 producing strain recombinant antigen epitope Infectious and parasitic diseases RC109-216
spellingShingle	melioidosis burkholderia pseudomallei ompa omp38 producing strain recombinant antigen epitope Infectious and parasitic diseases RC109-216 Yu. A. Kuzyutina I. B. Zakharova D. V. Viktorov Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens
description	A risk of introducing into the Russian Federation exotic infections including laboratory-confirmed melioidosis regularly recorded worldwide necessitates development and improvement of express diagnostics tools. Cross reactivity between phylogenetically related species of the genus Burkholderia complicates melioidosis diagnostics by express test methods based on using monoclonal antibodies against pathogen exopolysaccharide epitopes. Searching for target antigens to create the next generation group- and species-specific immunodiagnostic reagents for identifying Burkholderia pseudomallei is still of high priority. The study was aimed at cloning complete coding sequences for cell surface proteins differentiating Burkholderia pseudomallei and optimizing recombinant antigens purification protocol. In silico comparative study allowed to select highly immunogenic B. pseudomallei outer membrane proteins Omp38 and OmpA/МotB as target biomolecules. For cloning, omp38 and ompA/motB gene-specific amplicons were obtained by PCR and ligated with the linear expression vector RIC-Ready pPAL7. Competent E. coli C-Max5α cells were transformed by a ligation mixture for producing recombinant plasmids, which were further purified to transform E. coli BL21 (DE3) cells for robust recombinant protein expression. Due to a potential multimeric protein structure, a standard protein purification protocol from native cell lysate was inefficient, which was modified to increase recombinant protein yield. However, by adding denaturing conditions at intermediate purification steps caused hydrolysis of peptide bonds in the target proteins, presumably between proline and asparagine residues. As a result, N-terminal fragments connecting recombinant proteins to the stationary phase of chromatographic column were eluted and evaluated for linear epitope detection according to their molecular weights. In silico analysis data identified highly antigenic motifs within the polypeptides studied. Thus, strains of E. coli BL21(DE3) BpsOmp39 and E. coli BL21(DE3) BpsOmpА engineered by us produce cell surface proteins Omp38 and ОmpA/МotB derived from melioidosis pathogen, which can be useful for developing diagnostic test systems.
format	article
author	Yu. A. Kuzyutina I. B. Zakharova D. V. Viktorov
author_facet	Yu. A. Kuzyutina I. B. Zakharova D. V. Viktorov
author_sort	Yu. A. Kuzyutina
title	Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens
title_short	Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens
title_full	Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens
title_fullStr	Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens
title_full_unstemmed	Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens
title_sort	engineering e. coli recombinant strains for high yield production of burkholderia pseudomallei specific antigens
publisher	Sankt-Peterburg : NIIÈM imeni Pastera
publishDate	2019
url	https://doaj.org/article/3c830a4e5c6e40608a3b0e862443b25b
work_keys_str_mv	AT yuakuzyutina engineeringecolirecombinantstrainsforhighyieldproductionofburkholderiapseudomalleispecificantigens AT ibzakharova engineeringecolirecombinantstrainsforhighyieldproductionofburkholderiapseudomalleispecificantigens AT dvviktorov engineeringecolirecombinantstrainsforhighyieldproductionofburkholderiapseudomalleispecificantigens
_version_	1718417911920459776

Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens

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