WO2014168497A1 - Vecteurs d'intégration, cellule hôte transformée par le vecteur d'intégration et utilisation des vecteurs d'intégration et de la cellule hôte pour la présentation de protéines de fusion sur la surface des spores de bacillus subtilis - Google Patents

Vecteurs d'intégration, cellule hôte transformée par le vecteur d'intégration et utilisation des vecteurs d'intégration et de la cellule hôte pour la présentation de protéines de fusion sur la surface des spores de bacillus subtilis Download PDF

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WO2014168497A1
WO2014168497A1 PCT/PL2014/000036 PL2014000036W WO2014168497A1 WO 2014168497 A1 WO2014168497 A1 WO 2014168497A1 PL 2014000036 W PL2014000036 W PL 2014000036W WO 2014168497 A1 WO2014168497 A1 WO 2014168497A1
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gene
vector according
plasmid
nucleotide sequence
promoter region
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PCT/PL2014/000036
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English (en)
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Krzysztof HINC
Michał OBUCHOWSKI
Anna GRELA
Tomasz ŁĘGA
Adam Iwanicki
Iwona PIĄTEK
Małgorzata STASIŁOJĊ
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Gdański Uniwersytet Medyczny
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Priority claimed from PL403468A external-priority patent/PL240359B1/pl
Priority claimed from PL406271A external-priority patent/PL241078B1/pl
Application filed by Gdański Uniwersytet Medyczny filed Critical Gdański Uniwersytet Medyczny
Publication of WO2014168497A1 publication Critical patent/WO2014168497A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display

Definitions

  • Integration vectors the host cell transformed with the integration vector and the use of integration vectors and the host cell for the display of fusion proteins on the surface of the Bacillus subtilis spores.
  • the invention concerns the integration vectors, the host cell transformed with the integration vector, and the use of the integration vectors and the host cell for the display of fusion proteins on the surface of the Bacillus subtilis spores.
  • a vector must meet a number of basic conditions. It must be able to replicate itself independently in the specific host and must have restriction sites which enable appropriate insertion of a DNA fragment.
  • the vector should also carry a selectable marker so that the host cells which have absorbed the recombinant vector can be distinguished from those which have not absorbed it.
  • the protein and peptide display systems represent an important tool used in biotechnology and molecular biology.
  • the literature of the subject contains many descriptions of display methods which employ bacteriofags or different cells.
  • bacterial spores which are several times more durable than any other carriers used in the heretofore known solutions.
  • a system of 20 vectors was developed enabling easy construction of fusion genes, the products of which would be displayed on spore surface.
  • the vector must meet several key criteria, namely: i) it should be able to replicate itself independently in the host cell of low trophic requirements; ii) it should have unique restriction sites enabling enable easy cloning of the DNA fragment which encodes the protein to be displayed; iii) it should also carry a selectable marker gene allowing for effective selection of the cells which have absorbed the recombinant vector.
  • Bacterial vectors have found an important use consisting in their transferring genes between different organisms, also between unrelated ones.
  • the vectors which enable this way of DNA transfer are called shuttle vectors. They contain replication systems which enable them to replicate in two different organisms. In this way they can be transferred between those organisms without the need to modify the vector in any way.
  • shuttle vectors contain replication systems which enable them to replicate in two different organisms. In this way they can be transferred between those organisms without the need to modify the vector in any way.
  • Bacterial vectors have found an important use consisting in their transferring genes between different organisms, also between unrelated ones.
  • the vectors which enable this way of DNA transfer are called shuttle vectors. They contain replication systems which enable them to replicate in two different organisms. In this way they can be transferred between those organisms without the need to modify the vector in any way.
  • Integration vectors were developed so as to achieve stable gene persistence and expression in a specific organism or tissue. They occur in small number of copies per cell (usually one copy).
  • the vectors contain homologous sequences for sequencing the chromosome of the host, which enable site-specific integration in the chromosome.
  • the system according to the invention is based on the creation of integration vectors and the corresponding recipient strain.
  • the appropriate vector will be inserted into the recipient's chromosome at a predefined site.
  • the target strain will not carry any gene that would condition antibiotic resistance, and as such it will be safe in use. Some of the applications of recombinant spores cause their release into the environment, however the absence of antibiotic resistance genes makes it safe.
  • the described system enables creation of fusion proteins with different coat proteins: CotZ, or CgeA, CotB, CotC, CotG.
  • the proteins are found in the outermost layer of the spore coat called the crust, in effect of which the fusion proteins are displayed on the surface of the endospores. Thanks to the protein "nature" of the coat it is possible to modify it relatively easily. It is only needed to insert a modified coat- protein coding gene (expanded with a fragment encoding the displayed protein) into the bacterial cell.
  • the coat structures develop in the cell spontaneously since spore formation is a process natural for that bacteria.
  • the coat structures are highly flexible thanks to which insertion of a modified coat protein, even if substantially modified, does not affect the coat structure formation process to any significant extent.
  • Proteins CotZ, CgeA, CotB, CotC, CotG have different molecular weights and different numbers of molecules per spore, which enables anticipation and alignment of the 'anchor' protein and the displayed protein.
  • the constructed vectors form groups. Each group is dedicated to one spore protein. Each individual group contains vectors enabling creation of various protein fusions, both C-terminal and N-terminal, with or without a linker, at different sites of integration into the bacterial chromosome. In each of the groups there is one recipient strain prepared to have an appropriate vector integrated in its chromosome. Each group (vectors + strain) has a different system of selecting the appropriate clones.
  • the system according to the invention is composed of 20 plasmids and 3 strains which enable creation of fusions with proteins CotZ, and CgeA, CotB, CotC or CotB in Bacillus subtilis.
  • the system of vectors and bacterial strains according to the invention is designated to display fusion proteins on the surface of the Bacillus subtilis spores.
  • the spore-displayed proteins may serve different functions: they may be used for enzymatic reactions, for binding low molecular weight substances present in the environment, or as antigens stimulating immune response.
  • the developed vectors enable easy bonding of foreign proteins with the natural components of the spore coat and their subsequent expression in recipient strains which are an element of the system.
  • the gist of the invention is the integration vector for the display of of fusion proteins on the spore surface, where the vector contains the DNA sequence encoding protein CgeA or CgeA-linker or CotZ or CotZ-linker or CotB or CotB-linker or linker-CotB or CotC or CotC-linker or linker-CotC or CotG or CotG-linker or linker-CotG.
  • the vector namely plasmid pAGOl containing the nucleotide sequence of gene cgeA together with the promoter region and the multiple cloning sites, as shown on Fig. lA and Fig.lB.
  • the vector namely plasmid pAG02 containing the nucleotide sequence of gene cotZ together with the promoter region and the multiple cloning sites, as shown on Fig. 2A and Fig.2B.
  • the vector namely plasmid pAG03 containing the nucleotide sequence of gene cgeA together with the promoter region, the linker, and the multiple cloning sites, as shown on Fig. 3A and Fig.3B.
  • the vector namely plasmid pAG04 containing the nucleotide sequence of gene cotZ together with the promoter region, the linker, and the multiple cloning sites, as shown on Fig. 4A and Fig.4B.
  • the vector namely plasmid pTLOl containing the nucleotide sequence of gene cgeA together with the promoter region and multiple cloning sites, as shown on Fig. 5A and Fig.5B.
  • the vector namely plasmid pTL02 containing the nucleotide sequence of gene cotZ together with the promoter region, and the multiple cloning sites, as shown on Fig. 6A and Fig.6B.
  • the vector namely plasmid pTL03 containing the nucleotide sequence of gene cgeA together with the promoter region, the linker, and the multiple cloning sites, as shown on Fig. 7A and Fig.7B.
  • the vector namely plasmid pTL04 containing the nucleotide sequence of gene cotZ together with the promoter region, the linker, and the multiple cloning sites, as shown on Fig. 8A and Fig.8B.
  • the vector namely plasmid pCotB-C containing the nucleotide sequence of gene cotB together with the promoter region and the multiple cloning sites, as shown on Fig. 10A and Fig. 10B
  • the vector namely plasmid pCotB-CL containing the nucleotide sequence of gene cotB together with the linker, the promoter region, the and multiple cloning sites, as shown on Fig. 11A and Fig.llB.
  • the vector namely plasmid pCotB-N containing the nucleotide sequence of gene cotB together with the promoter region and the multiple cloning sites, as shown on Fig. 12A and Fig.l2B.
  • the vector namely plasmid pCotB-NL containing the nucleotide sequence of gene cotB together with the linker, the promoter region, and the multiple cloning sites, as shown on Fig. 13A and Fig.l3B.
  • the vector namely plasmid pCotC-C containing the nucleotide sequence of gene cotC together with the promoter region and the multiple cloning sites, as shown on Fig. 14A and Fig.l4B.
  • the vector namely plasmid pCotC-CL containing the nucleotide sequence of gene cotC together with the linker, the promoter region, and the multiple cloning sites, as shown on Fig. 15A and Fig.l5B.
  • the vector namely plasmid pCotC-N containing the nucleotide sequence of gene cotC together with the promoter region and the multiple cloning sites, as shown on Fig. 16A and Fig.l6B.
  • the vector namely plasmid pCotC-NL containing the nucleotide sequence of gene cotC together with the linker, the promoter region, and the multiple cloning sites, as shown on Fig. 17A and Fig.l7B.
  • the vector namely plasmid pCotG-C containing the nucleotide sequence of gene cotG together with the promoter region and the multiple cloning sites, as shown on Fig. 18A and Fig.l8B.
  • the vector namely plasmid pCotG-CL containing the nucleotide sequence of gene cotG together with the linker, the promoter region, and the multiple cloning sites, as shown on Fig. 19A and Fig.l9B.
  • the vector namely plasmid pCotG-N containing the nucleotide sequence of gene cotG together with the promoter region and the multiple cloning sites, as shown on Fig. 20A and Fig.20B.
  • the vector namely plasmid pCotG-NL containing the nucleotide sequence of gene cotG together with the linker, the promoter region, and the multiple cloning sites, as shown on Fig.21A and Fig.21B.
  • the vector which also contains selectable markers selected from the following group: coding gene gltB or trpC.
  • the vector where the markers are selected from among the genes which encode the enzyme of the amino acid synthesis pathway and the genes which condition resistance to chloramphenicol and/or spectinomycin.
  • the vector which also contains selectable markers selected from the following group of genes: trpC, lysA, thrC.
  • the vector where the markers are selected from among the genes which encode the enzyme of the amino acid synthesis pathway.
  • the vector where the gene coding the enzyme of the tryptophan synthesis pathway, trpC, codes for indole-3-glycerol-phosphate synthetase.
  • the vector where the gene coding the enzyme of the lysine synthesis pathway, lysA, codes for the diaminopimelate decarboxylase .
  • the vector where the gene coding the enzyme of the threonine synthesis pathway, thrC, codes for the threonine synthetase.
  • the vector where the selectable markers enable selection of plasmid-containing cells.
  • the host cell transformed with the integration vector, as defined above, represents the Bacillus subtilis strain 168.
  • Fig.lA presents the map of integration vector pAGOl
  • Fig.lB presents the nucleotide sequence of the promoter region, gene cgeA, and the multiple cloning sites.
  • Fig.2A presents the map of integration vector pAG02
  • Fig.2B presents the nucleotide sequence of the promoter region, gene cotZ, and the multiple cloning sites
  • Fig. 3A presents the map of integration vector pAG03
  • Fig.3B presents the nucleotide sequence of the promoter region, gene cgeA, linker, and the multiple cloning sites
  • Fig. 4A presents the map of integration vector pAG04
  • Fig.4B presents the nucleotide sequence of the promoter region, gene cotZ, linker, and the multiple cloning sites
  • Fig. 5A presents the map of integration vector pTLOl
  • Fig.SB presents the nucleotide sequence of the promoter region, gene cgeA, and the multiple cloning sites
  • Fig. 6A presents the map of integration vector pTL02
  • Fig.6B presents the nucleotide sequence of the promoter region, gene cotZ, and the multiple cloning sites
  • Fig. 7A presents the map of integration vector pTL03
  • Fig.7B presents the nucleotide sequence of the promoter region, gene cgeA, linker, and the multiple cloning sites
  • Fig. 8A presents the map of integration vector pTL04
  • Fig.8B presents the nucleotide sequence of the promoter region, gene cotZ, linker, and the multiple cloning sites.
  • Fig.9 presents the protein linker of the sequence between the appropriate gene cotZ, cgeA and the multiple cloning site.
  • Fig.lOA presents the map of plasmid pCotB-C with the individual restriction sites marked.
  • Fig.lOB presents the sequence of plasmid pCotB-C in the GenBank format.
  • Fig.llA presents the map of plasmid pCotB-CL with the individual restriction sites marked.
  • Fig.llB presents the sequence of plasmid pCotC-CL in the GenBank format.
  • Fig. llC presents the nucleotide and amino acid sequence of the linker.
  • Fig.l2A presents the map of plasmid pCotB-N with the individual restriction sites marked.
  • Fig.l2B presents the sequence of plasmid pCotB-N in the GenBank format.
  • Fig.13 A presents the map of plasmid pCotB-NL with the individual restriction sites marked.
  • Fig. 13B presents the sequence of plasmid pCotB-NL in the GenBank format.
  • Fig.l4A presents the map of plasmid pCotC-C with the individual restriction sites marked.
  • Fig.l4B presents the sequence of plasmid pCotC-C in the GenBank format.
  • Fig.l5A presents the map of plasmid pCotC-CL with the individual restriction sites marked.
  • Fig.l6B presents the sequence of plasmid pCotC-CL in the GenBank format.
  • Fig.l6A presents the map of plasmid pCotC-N with the individual restriction sites marked.
  • Fig. 16B presents the sequence of plasmid pCotC-N in the GenBank format.
  • Fig.l7A presents the map of plasmid pCotC-NL with the individual restriction sites marked.
  • Fig.l7B presents the sequence of plasmid pCotC-NL in the GenBank format.
  • Fig.l8A presents the map of plasmid pCotG-C with the individual restriction sites marked.
  • Fig.l8B presents the sequence of plasmid pCotG-C in the GenBank format.
  • Fig.19 A presents the map of plasmid pCotG-CL with the individual restriction sites marked.
  • Fig.l9B presents the sequence of plasmid pCotG-CL in the GenBank format.
  • Fig.20 A presents the map of plasmid pCotG-N with the individual restriction sites marked.
  • Fig.20B presents the sequence of plasmid pCotG-N in the GenBank format.
  • Fig.21A presents the map of plasmid pCotG-NL with the individual restriction sites marked.
  • Fig.21B presents the sequence of plasmid pCotG-NL in the GenBank format.
  • the invention is illustrated with the following exemplary bot not restricting embodiments.
  • the vectors were constructed under the following procedure.
  • the genes encoding proteins CotZ, CgeA were multiplied under the PCR technology together with the promoter region and then cloned into the integration vectors series pDL and pDG1663, which enabled the insertion in the bacterial chromosome at the amyE and thrC loci.
  • the multiple cloning sites were placed near the 3' ends of genes cotZ and cgeA, which allowed inclusion of foreign genes in the reading frame.
  • vectors which contained protein linker of sequence N-GGGEAAAKGGG-C between the respective gene cotZ, cgeA and the multiple cloning site.
  • protein linker of sequence N-GGGEAAAKGGG-C between the respective gene cotZ, cgeA and the multiple cloning site.
  • four versions of the vectors for each gene were constructed (eg. CotZ-C for integration at the amyE locus, CotZ-C for integration at the thrC locus, CotZ-link-C for integration at the amyE locus, and CotZ-link-C for integration at the thrC locus), 8 vectors in aggregate.
  • the recipient strain constructed based on the wild-type strain 168, B. subtilis will be an important element of the integration vector system. Fragments which encode appropriate fusion proteins will be integrated in the chromosome of the recipient strain.
  • the recipient strain has been equipped with selectable marker enabling their efficient and easy identification. The function of the selectable marker is played by the gene encoding the enzyme of the amino acid synthesis pathway, which is a component absolutely necessary for the life of the cell.
  • the recipient strain will carry the deletion mutation eliminating the activity of its gene. It was resolved to select gene trpC encoding the indole-3-glycerol-phosphate synthetase - an enzym participating in the tryptophan biosynthesis.
  • the bacteria sown on the substrate devoid of the above- named amino acid will be unable to grow until they receive the missing gene through transformation with the vector encoding the fusion protein, and the missing enzyme of the synthesis pathway of this amino acid. This stage will make it possible to select the bacteria which have absorbed the DNA of the respective vector.
  • the second selectable marker will condition antibiotic resistance. It will be located at the site of vector integration with the chromosome of the recipient bacteria. If the integration is correct, the vector will be removed from the chromosome, by which the antibiotic sensitivity phenotype will be restored, and this in turn will enable simple selection of appropriate strains following the preliminary selection. Due to the fact that the strains which have lost the gene conditioning resistance will be the correct ones, the application of the described system will not carry the risk of spreading antibiotic resistance in the environment.
  • the chloramphenicol-encoding gene will act as the selectable marker in the case of integration at the amyE locus, and the spectinomycin-encoding gene will serve the same function in the case of the thrC locus.
  • the B. subtilis recipient strains accompanying the integration vector system will be its major element since fragments encoding appropriate fusion proteins will be integrated in the chromosomes of those strains.
  • the recipient strains carry mutations in the genes encoding proteins necessary for the synthesis of the following amino acids: tryptophan, threonine, and lysine.
  • the selected genes were trpC, thrC and lysA.
  • Gene trpC encodes the indole-3-glycerol-phosphate synthetase - an enzym participating in the tryptophan biosynthesis
  • gene thrC codes for the threonine synthetase
  • gene lysA encodes diaminopimelate decarboxylase, which is an intermediate product in the lysine synthesis pathway.
  • the bacteria sown on the substrate devoid of the appropriate amino acid will be unable to grow until they receive the missing gene through transformation with the appropriate integration vector which carries the fusion protein-encoding gene, and the missing enzyme of the synthesis pathway of the appropriate amino acid.
  • Sowing the transformed cells on minimally composted substrate will enable the growth of only those cells which have absorbed the integration vector and integrated it into their chromosomes. This stage will enable selecting the appropriate transformants. Since the selection is effected through complementation of the mutation which prevents amino acid synthesis, the obtained strains carrying the fusion protein-encoding genes do not carry any drug-resistance markers and as such will not pertain to their spreading.

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Abstract

L'invention concerne des vecteurs d'intégration pour la présentation de protéines de fusion sur la surface de spores, qui contiennent la séquence d'ADN codant pour la protéine CgeA ou CgeA-lieur ou pour CotZ ou CotZ-lieur ou CotB ou CotB-lieur ou lieur-CotB ou CotC ou CotC-lieur ou lieur-CotC ou CotG ou CotG ou lieur-CotG. L'invention concerne également la cellule hôte de la souche Bacillus subtilis 168 contenant le vecteur d'intégration, et l'application des vecteurs d'intégration et de la cellule hôte définie pour la présentation de protéines de fusion sur la surface des spores de Bacillus subtilis.
PCT/PL2014/000036 2013-04-08 2014-04-07 Vecteurs d'intégration, cellule hôte transformée par le vecteur d'intégration et utilisation des vecteurs d'intégration et de la cellule hôte pour la présentation de protéines de fusion sur la surface des spores de bacillus subtilis WO2014168497A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
PLP.403468 2013-04-08
PL403468A PL240359B1 (pl) 2013-04-08 2013-04-08 Wektory integracyjne, komórka gospodarza transformowana wektorem integracyjnym oraz zastosowanie wektorów integracyjnych i komórki gospodarza do prezentowania fuzyjnych białek na powierzchni przetrwalników Bacillus subtilis
PL406271A PL241078B1 (pl) 2013-11-26 2013-11-26 Wektory integracyjne oraz komórka gospodarza transformowana wektorem integracyjnym
PLP.406271 2013-11-26

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WO2020157487A3 (fr) * 2019-01-28 2020-09-24 The University Of Nottingham Constructions géniques et leurs utilisations
WO2022171904A1 (fr) 2021-02-15 2022-08-18 Livingmed Biotech S.R.L. Souches de clostridium génétiquement modifiées exprimant des antigènes recombinants et leurs utilisations

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CN105132450A (zh) * 2015-09-09 2015-12-09 齐鲁工业大学 枯草芽孢杆菌芽孢衣壳蛋白Cot表面展示海藻糖合酶的方法
WO2020157487A3 (fr) * 2019-01-28 2020-09-24 The University Of Nottingham Constructions géniques et leurs utilisations
WO2022171904A1 (fr) 2021-02-15 2022-08-18 Livingmed Biotech S.R.L. Souches de clostridium génétiquement modifiées exprimant des antigènes recombinants et leurs utilisations

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