EP4599068A1 - Vecteur d'expression pour la production de protéines recombinantes dans des cellules hôtes procaryotes - Google Patents

Vecteur d'expression pour la production de protéines recombinantes dans des cellules hôtes procaryotes

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Publication number
EP4599068A1
EP4599068A1 EP22799844.0A EP22799844A EP4599068A1 EP 4599068 A1 EP4599068 A1 EP 4599068A1 EP 22799844 A EP22799844 A EP 22799844A EP 4599068 A1 EP4599068 A1 EP 4599068A1
Authority
EP
European Patent Office
Prior art keywords
plasmid
sequence
protein
seq
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22799844.0A
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German (de)
English (en)
Inventor
Lubomir JANDA
Jan Gebauer
Adam Norek
Sarka KOBZOVA
Michaela STASTNA
Martin FALDYNA
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Veterinary Research Institute
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Veterinary Research Institute
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Publication of EP4599068A1 publication Critical patent/EP4599068A1/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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention provides a novel expression vector particularly suitable for the production of recombinant proteins in prokaryotic host cells by fermentation technology.
  • Plasmids are common extrachromosomal genetic elements that replicate independently of the chromosomes of most prokaryotic but also eukaryotic organisms. Cells often have multiple plasmids of different sizes existing together in varying numbers of copies per cell. They are widely used as carriers of genetic information in biological studies involving the analysis of gene function, protein expression or genome editing.
  • a custom DNA construct is an artificial segment of nucleic acid created by inserting a target DNA fragment(s) into the backbone of a plasmid vector and is a vehicle for transferring the target DNA fragment(s) into a tissue or cell in order to produce recombinant proteins.
  • Recombinant technology plays an irreplaceable role in biotechnology as well as in molecular biology research leading to modem diagnostic and therapeutic approaches.
  • Recombinant proteins find their purpose in many applications - in basic research, they are used for studying cellular processes, protein-protein interactions, immune responses and many others. They are also part of common laboratory techniques like ELISA assays, western blots or immunohistochemical methods. Moreover, they indeed are the cornerstones of recombinant vaccines and also find their role in many therapeutics.
  • prokaryotic cells is the most widely used among others. In general, lower cost, shorter time, high protein yields and reasonable control in large-scale production are the most prominent advantages.
  • the present invention relates generally to an engineered plasmid and selection material for influencing microbial production of useful polypeptides and more particularly to novel plasmid DNA useful in providing exceptionally high levels of exogenous gene expression in E. coli host cells.
  • expression vectors with an optimized list of genes exposed to expression in microbial host cell populations such as E. coli.
  • the plasmid of the invention is a new artificial highly stable vector with a minimal ORI sequence length with a moderate high copy number creating a minimal burden on the host cell.
  • the present invention provides a plasmid vector for protein production using heterologous prokaryotic host cells, such as E. coli.
  • the backbone of the plasmid of the invention was derived from the wildtype Corynebacterium renale ORI sequence of the pCR2 replication family. However, the ORI sequence of replication had to be significantly modified to meet the technological demands of recombinant protein production.
  • the plasmid of the invention is stable for several passages even without the selection pressure; is particularly suitable for use in bioreactors; enables protein expression at room temperature without the need for excessive heating, aeration nor for extensive stirring; enables overexpression of the produced protein molecule in standard medium (e.g. Luria-Bertani broth); allows to express of proteins that are otherwise difficult to produce.
  • standard medium e.g. Luria-Bertani broth
  • expression plasmid refers to a plasmid useful for expression of recombinant proteins.
  • the most preferred plasmid vector of the invention having SEQ ID NO: 1 is also referred to in this text as “pUbExlOO”.
  • the present invention provides an expression vector plasmid of the invention which comprises, in the given order: an origin of replication (ORI sequence); a gene encoding ubiquitin as a leader protein; multiple cloning site (MSC); at least one affinity tag; TEV site; and at least one gene for selection antibiotic resistance; wherein the ORI sequence has a sequence SEQ ID NO: 2:
  • Stabilising element is identical to the terminal sequence of the replication initiation protein (RepE protein) gene.
  • the newly designed complete ORI sequence (SEQ ID NO: 2) of the plasmid including the stabilizing element is only 755 nucleotides long.
  • pBR322 complete ORI sequence of plasmid pET
  • pBR322 complete ORI sequence of plasmid pET
  • Alteration of replication initiation reduces the amount of biomass required to produce protein units. A decrease in the metabolic activity of biomass also reduces oxygen consumption by approximately 10% (depending on the specific protein produced).
  • the plasmid of the invention combines the advantages of stable low-copy and high-copy plasmids. Dozens of templates are able to rapidly produce more protein while maintaining template stability.
  • the plasmid of the invention has the DNA sequence SEQ ID NO: 1 (and may be referred to further in this text as “pUbExlOO”):
  • the plasmid is a double-stranded DNA plasmid.
  • SEQ ID NO: 1 represents one strand, and the other strand is complementary to SEQ ID NO: 1.
  • the plasmid having the sequence SEQ ID NO: 1 was deposited with the German Collection of Microorganisms and Cell Cultures (Leibniz Institute DSMZ, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) by the Depositor: Veterinary Research Institute, Hudcova 296/70, 62100 Bmo, Czech Republic, under the Accession No. DSM 34045, date of deposit: 19.08.2021.
  • the present invention further provides a method of producing a recombinant protein in a heterologous prokaryotic host cell, such as E. coli, said method comprising the steps of: inserting the coding sequence of the protein to be produced into a plasmid according to the invention, transforming the plasmid into a prokaryotic host cell, culturing and harvesting the prokaryotic host cells, disintegrating the prokaryotic host cells to release the produced recombinant protein, optionally purifying the produced recombinant protein and optionally cleaving the leader protein from the produced recombinant protein.
  • a heterologous prokaryotic host cell such as E. coli
  • the present invention provides a sequence SEQ ID NO: 2, and its use as ORI sequence in plasmids. Detailed Description of the Invention
  • the plasmid of the present invention contains, in the listed order: an origin of replication (ORI sequence); a gene encoding ubiquitin as a leader protein; multiple cloning site (MSC); at least one affinity tag; TEV site; and at least one gene for selection antibiotic resistance.
  • ORI sequence origin of replication
  • MSC multiple cloning site
  • TEV site at least one gene for selection antibiotic resistance.
  • recombinantly produced protein refers to the protein whose coding sequence is inserted into the plasmid which is in turn transformed into a prokaryotic host cell which then produces the recombinant protein (recombinantly produced protein).
  • GOI gene of interest
  • Origin of replication (ORI) sequence is a representation of Origin of replication (ORI) sequence:
  • plasmids used in the heterologous E. coli expression systems are based on plasmids ColEl, or on closely related pBR322 and pMBl plasmids. Although most origins of replication have the same ORI basis, the resulting plasmids still produce different copy numbers depending on how they are regulated. Generally, replication control is referred to as "relaxed” or “strict” depending on whether the ORI is upregulated by RNA or proteins. A balance between positive and negative regulation results in plasmid copy number range and can be manipulated by mutations in the sequence. For example, pMBl ORI stores about 20 copies per cell, while pUC (pMBl derivative) - which differs by only two mutations - produces up to 700 copies per cell.
  • RNA II The prototypical ORI sequence of E. coli, ColEl ORI, which is approximately two kbp in length, is replicated via the Theta mechanism.
  • the replication initiation requires the transcription of RNA primer (542 bp), RNA II, from the RNA II promoter (P2).
  • RNA II and the P2 promoter are highly conserved across the whole ColEl plasmid family (e.g. DoriC, pCR2, pET28, pIGAL, pBR322, etc.).
  • RNA II is necessary to initiate replication because the 3'-end serves as a primer for the DNA polymerase I complex and is essential for DNA replication.
  • RNA II activity is mediated by an RNA I molecule (108 bp) that is transcribed from the opposite strand of RNA II from its Pl promoter.
  • the ORI sequence (having the sequence SEQ ID NO: 2) is a newly designed minimal origin devised from the pCR2 origin of replication. Only the core region complemented with replication regulating sequence RepE) necessary for replication in E. coli has been preserved in plasmid of the invention.
  • the ORI sequence of the plasmid of the invention consists of DNA Unwinding Element (DUE), responsible for opening of the double helix for DNA replication, DNA region coding RNA II and RNA I, promoter (Pl) and Stem sequences which form loops participating in the regulation of the replication.
  • DUE DNA Unwinding Element
  • Pl promoter
  • Stem sequences which form loops participating in the regulation of the replication.
  • the essential promoter P2 critical for RNA II primer transcription, was replaced by de-novo designed promoter.
  • the regulation DNA hairpin was replaced by a DNA sequence that cannot fold into a secondary structure element. Moreover, the high AT pair-rich region was utterly redesigned. Removing translation signal hairpin and implementing promotor with low affinity to RNA polymerase results in stable low-speed replication with medium -range copy number of the plasmid of the invention in E. coli host cells.
  • Plasmid of the invention contains in its complete ORI sequence a newly designed P2 promoter and a stabilizing element, which is a sequence identical to the terminal sequence of the replication initiation protein (terminal amino acid sequence of the RepE protein).
  • the RepE protein plays an essential role in initiating replication from the origin, ori2.
  • the RepE protein has two main functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription.
  • Monomers of RepE represent the active form by binding to ori2 to initiate replication, while dimers act as an autogenous repressor by binding to the operator.
  • Increased expression of the RepE gene switches the initiation of replication to autogenous repression, which leads to a reduction in replication and thus to a reduction in the number of plasmid copies.
  • the stabilizing element regulates the number of RepE protein molecules so that the replication of the plasmid is not blocked. This occurs through hybridization of the antisense RNA with the complementary region of the gene for the RepE protein.
  • the plasmid of the invention was tested for stability in two host strains, DH10 ⁇ and BL21 (DE3). In both host strains, it showed a comparable stability to the reference plasmid pET28 (Fig. 1) at a one third of the plasmid size.
  • the ubiquitin (Ub) gene encodes a protein that interacts with most other proteins with a Kdd affinity > 0.3 mM, which obscures the hydrophobic segments of the fusion protein with a direct effect on protein expression and solubility.
  • ubiquitin was an eukaryotic 8.5 kDa protein with many functions. Its sequence is highly conserved from unicellular organisms to humans. Its presence in the sequence facilitates the production, isolation and identification of the protein from the host production system. Ubiquitin also increases the solubility of the fusion protein produced (Rogov VV, Rozenknop A, Rogova NY, Lohr F, Tikole S, Jaravine V, Gimtert P, Dikic I, Dotsch V.
  • the gene encoding ubiquitin is optimized for codon usage in E. colt and preferably has a DNA sequence SEQ ID NO: 3. This gene sequence corresponds to the amino acid sequence SEQ ID NO: 4.
  • MCS Multiple cloning site
  • the DNA sequence for a recombinantly produced protein is inserted into the multiple cloning site (MCS) of the expression vector.
  • MCS multiple cloning site
  • the multiple cloning site or polylinker forms a short DNA segment (SEQ ID NO: 5) that contains a series of 8 restriction enzyme sites (RE) which are sites for Kpnl, Ncol, BamHI, PstI, SacI, Hindlll, Notl, Sall.
  • the plasmid of the invention preferably contains two versions of polyhistidine tags in its sequence, a 14xHisTag and a 6xHisTag.
  • a peptide tag containing 14 histidines (14xHisTag) and additional amino acids (G, S and T) forming loops in its secondary structure is attached on the N-terminus of a recombinantly produced protein. Due to its structure, the 14xHisTag elicits higher affinity to divalent cations and therefore may improve the purification process by metallochelating methods.
  • Proteins expressed from the vector plasmid of the invention have the ability to cleave the 14xHisTag after purification due to the presence of TEV cleavage site in between of a tag sequence and the protein of interest.
  • the 14xHisTag DNA sequence is preferably SEQ ID NO: 6, and the corresponding amino acid sequence is SEQ ID NO: 7.
  • the expression vector of the invention also contains a conventional 6xHisTag sequence behind the multiple cloning site (MCS), which can be used as a C-terminal peptide tag for the metallochelation carrier.
  • MCS multiple cloning site
  • 6xHisTag in a protein sequence may improve detection of a protein by commercially available specific anti-HisTag antibody.
  • the gene of interest GOI, gene encoding the recombinant protein to be produced
  • GOI gene encoding the recombinant protein to be produced
  • sequence encoding 6xHisTag is SEQ ID NO: 8, and the corresponding amino acid sequence is SEQ ID NO: 9.
  • TEV site is a short amino acid sequence enabling the cleavage of ubiquitin sequence and 14xHisTag from the recombinantly produced protein by a TEV protease.
  • TEV cleavage site is in the plasmid of the invention encoded by a DNA sequence SEQ ID NO: 10, and the corresponding amino acid sequence is SEQ ID NO: 11.
  • the preferred selection antibiotic is kanamycin
  • the preferred selection antibiotic resistance gene is a codon optimised kanamycin resistance gene having the sequence SEQ ID NO: 12.
  • the kanamycin resistance gene Km R which is used in a number of E. coli expression plasmids, including pET plasmids, was originally isolated from transposable element Tn903 in E. coli. This gene encodes an aminoglycoside 3 '-phosphotransferase (kanamycin kinase; neomycin-kanamycin phosphotransferase). Aminoglycoside phosphotransferases inactivate aminoglycoside antibiotics through phosphorylation. The original gene had a CAI (Codon adaptation index) of 0.39 and was improved to 0.95 after codon usage optimization.
  • the kanamycin resistance gene sequence is preferably SEQ ID NO: 12, its complementary sequence is SEQ ID NO: 13, and the corresponding amino acid sequence is SEQ ID NO: 14.
  • Figtire 1 The plasmid of the invention stability in E. coli BL21 (DE3) cells - a comparative study of plasmid retention under non-selective conditions for four subsequent passages. Bars indicate optical density after 24 h of cultivation with and without the addition of antibiotics. On the left side, results for cells transformed with the plasmid having SEQ ID NO: 1 are depicted. On the right side, results of control cells transformed with pET28 are depicted.
  • the plasmid map comprises the DNA sequence of SEQ ID NO: 2 encoding ORI gene, the ubiquitin leader protein of SEQ ID NO: 3, 14xHisTag sequence for affinity purification (SEQ ID NO: 6) and optimized gene for kanamycin resistance (SEQ ID NO: 12).
  • the DNA sequence for the gene of interest can be inserted into the multiple cloning site (MCS - (SEQ ID NO: 5) of the expression vector plasmid of the invention.
  • MCS - multiple cloning site or polylinker forms a short segment of DNA that contains a series (generally up to 8 REs).
  • Restriction enzyme (RE) sites - are Kpnl, Ncol, BamHI, PstI, Sad, Hindlll, Notl, Sall. Examples
  • the DNA sequence for a recombinantly produced protein is inserted into the multiple cloning site (MCS) of the expression vector plasmid having the sequence SEQ ID NO: 1 (plasmid pUbExlOO).
  • MCS multiple cloning site
  • the multiple cloning site is formed by a short DNA segment that contains a series of short DNA sequences recognised by restriction enzymes in following order Kpnl, Ncol, BamHI, PstI, Sad, Hindlll, Notl, Sall.
  • a cleavage site may be included between the leader protein Ubq with 14xHis-Tag peptide for IMAC affinity purification (Immobilized Metal Chelate Affinity Chromatography) and the protein of interest to cleave off the leader peptide and purify recombinant protein.
  • the gene of interest may be inserted in any of the cleavage sites in the MCS. All genes were codon optimised and shared common restriction sites at 5’- and 3’- ends of coding sequences. The incorporation procedure, as well as protein expression experiments, were done under the same conditions.
  • Gene for the recombinantly produced protein was processed by restriction enzymes Ncol/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C.
  • the restriction mixture was separated by DNA electrophoresis.
  • the DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically.
  • the vector was linearised by the same set of restriction enzymes. The incorporation of the gene of interest was achieved via DNA ligation. Ten ng of the insert was incubated with 100 ng of linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C.
  • the reaction product was used for heat-shock transformation of E. coli DHIOp chemo-competent cells. Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the plasmid was confirmed by DNA sequencing.
  • Plasmid transformation Vector plasmid of the invention containing the gene of interest coding sequence was used to transform production strain E. coli BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42° C/60 sec.). Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petri dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1 %).
  • the inoculum was cultivated at 37°C for 16 hours under constant agitation at 220 rpm.
  • 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose).
  • composition for IL 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose.
  • the cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm.
  • the TEV protease processing can be used for leader protein removal.
  • Purified protein was dialysed against the same buffer as used for purification except the imidazole (e.g. 100 mM Tris-HCl pH 8, 300 mM NaCl, 0.1 % Tween 20 (v/v)). Dialysed protein was incubated with TEV protease in the 1:30 ratio (w/w) for 16 hours at 4°C. Cleaved mixture was loaded on the IMAC column and the unbound protein (where the leader protein including his-tag was cleaved) is collected.
  • imidazole e.g. 100 mM Tris-HCl pH 8, 300 mM NaCl, 0.1 % Tween 20 (v/v)
  • the stability of the plasmid having the sequence SEQ ID NO: 1 in E. coli genome was verified.
  • the test for plasmid presence was performed as follows. 10 pl of liquid culture was used to inoculate 10 ml of liquid broth with antibiotics, and 20 pl was seeded onto the agar plate with antibiotics. The optical density and colony counting were performed after 16 h of cultivation at 37°C. Five subsequent passages were done, and the colony counting, and optical density measurements were performed for each of them.
  • a combination of plasmid DNA isolation methods, cell number counting and quantitative polymerase chain reaction (qPCR) was used to determine the copy number of plasmid DNA in the low copy, medium copy or high copy plasmid range. Copy number was determined against standard high copy (pUC57) and low copy (pET28) plasmids. All tests described below were performed in technically and biologically independent triplicates.
  • the primers used for amplification are identical to the primers used for amplification:
  • Reverse primer (-) SEQ ID NO: 17: 5 ' - TCTCGCTTTTATACAGGGTG - 3 '
  • Both primers define a specific binding region of 248 base pairs in the gene encoding the lysostaphin protein.
  • the reaction temperature profile was composed of an initial denaturation of 95°C for 10 min, 45 cycles alternating 95°C for 10 s, 60°C for 20 s and 72°C for 20 s.
  • the melting point of the PCR products was analysed with an increment of 0.5 °C in the range of 60-95 °C for 30 minutes. Samples were plated in duplicate for all DNA dilutions, for a total of 9 x 240 samples. PCR assay and melting point analysis were performed by detecting a change in fluorescence in the reaction mixture containing SybrGreen intercalation dye.
  • Plasmid DNA copy number was used based on pET28 low copy ( ⁇ 15 - 20) and pUC57 high copy (-500- 700) standards in cases where at all dilutions the melting point analysis corresponded to a single PCR product of 82.5°C, which corresponds to the expected product.
  • Apxla is related to RTX toxins (APXI - APXIV) of Actinobacilus pleuropneumonia.
  • APXla is a strongly cytotoxic and haemolytic pore-forming factor. Together with capsular polysaccharides and mural lipo-polysaccharides, Apxla is an essential factors of virulence and thus responsible for fibrinhaemorrhagic pleuropneumonia in swine.
  • the DNA sequence coding for Apxla C -terminal domain sequence with E. colt optimized codon usage distribution was synthesized and inserted into the expression vector via restriction enzyme digestion and subsequent ligation. The produced protein is of 27 kDa apparent molecular weight.
  • Plasmid of the invention with an inserted Apxla gene (SEQ ID NO: 18):
  • Gene encoding Apxla was processed by restriction enzymes Ndel/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C.
  • the restriction mixture was separated using DNA electrophoresis.
  • the DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically.
  • the vector was linearised by the same procedure. The incorporation of the gene of interest was achieved via DNA ligation. 10 ng of the linearised insert was incubated with 100 ng of linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C. The reaction product was used for heat-shock transformation of E.
  • Vector plasmid of the invention containing the gene of interest coding sequence was used to transform production strain E. coli BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42°C/60 sec.) in the water bath. Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petry dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1%). The inoculum was cultivated at 37°C for 12 hours under constant agitation at 220 rpm.
  • 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose).
  • composition for IL 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose.
  • the cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm.
  • the protein production was verified by protein SDS- PAGE electrophoresis and subsequent Coomasie Brilliant blue staining of whole cell lysates. For three proteins, the production rate was analysed in more detail.
  • Plasmid pUbExlOO improves the subsequent purification process and makes its production much more efficient.
  • Bst 5.9 polymerase is a chimeric enzyme consisting of the main scaffold of Bst polymerase from Thermus aquations Taq polymerase. A short loop from Bst polymerase from Geobacillus stearothermophilus was introduced. Bst 5.9 polymerase is used in the isothermal amplification reaction. The coding sequence with E. coli optimized codon usage distribution was synthesized and inserted into the expression vector via restriction enzyme digestion and subsequent ligation. The produced protein is of 72 kDa apparent molecular weight.
  • the reaction product was used for heat-shock transformation of E. coli DH10 ⁇ chemo-competent cells. Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the vector plasmid of the invention was confirmed by DNA sequencing.
  • Vector plasmid of the invention containing the gene of interest coding sequence was used to transform production strain E. coli BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42°C/60 sec.) in the water bath. Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petri dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1 %). The inoculum was cultivated at 37°C for 12 hours under constant agitation at 220 rpm.
  • 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose).
  • composition for IL 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose.
  • the cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm.
  • the protein production was verified by protein SDS- PAGE electrophoresis and subsequent Coomasie Brilliant blue staining of whole cell lysates. For three proteins, the production rate was analysed in more detail.
  • Bst 5.9 protein was compared with the commonly used laboratory expression plasmid pET.
  • the difference is most pronounced in the first 24 hours, when the plasmid of the invention-Bst5.9 has a density of only 3.5 McFarland and pET28-Bst5.9 has a density of 25 McFarland.
  • the density of pET28-Bst5.9 increased to 35 McFarland and pUbExlOO-Apxla to 42 McFarland.
  • the resulting pUbExl00-Bst5.9 pellet had a higher yield (14.5 g/L) than pET28-Bst5.9 (8.75 g/L).
  • the final protein production was 3.3-fold higher with the new plasmid of the invention-Apxla. Due to the relatively higher production of protein per unit of biomass, the plasmid of the invention improves the subsequent purification process and makes its production significantly more efficient. Production of lysostaphin using the plasmid of the invention.
  • Lysostaphin is a 27 kDa metalloendopeptidase, the antimicrobial lytic enzyme that is produced by Staphylococcus simulans. Lysostaphin is highly active against Staphylococcus aureus strains. The bacteriolytic activity of lysostaphin is due to its ability to hydrolyse the penta-glycine cross bridge of .S' aureus peptidoglycan. The coding sequence with E. coli-optimized codon usage distribution was synthesized and inserted into the expression vector via restriction enzyme digestion and subsequent ligation.
  • Plasmid of the invention with an inserted Lysostaphine gene (SEQ ID NO: 20):
  • Gene for lysostaphin was processed by restriction enzymes Ndel/Notl in CutSmart restriction Buffer (New England Biolabs, USA) for 1 hour at 37°C.
  • the restriction mixture was separated using DNA electrophoresis.
  • the DNA fragment of appropriate size was excised and isolated by isolation kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The DNA concentration was determined spectrophotometrically.
  • the vector was linearised by the same procedure. The incorporation of the gene of interest was achieved via DNA ligation. Ten ng of the linearised insert was incubated with 100 ng of linearised vector plasmid of the invention in the presence of T4 DNA ligase for 12 hours at 16°C. The reaction product was used for heat-shock transformation of E.
  • coli DH10 ⁇ chemo-competent cells Cultivation on agar plates with kanamycin selection was used to select cells carrying the plasmid. The presence of the gene of interest in the plasmid of the invention was confirmed by DNA sequencing.
  • the plasmid of the invention containing lysostaphin gene coding sequence was used to transform production strain E. colt BL21 (DE3). 50 ng of plasmid DNA was added to chemo -competent cells, which were transformed by heat shock (42°C/60 sec.) in the water bath. Positive clones were selected using seeding on LB agar plates with kanamycin. Fifteen colonies from the Petri dish were transferred to a liquid LB medium with kanamycin (100 pg/ml) and glucose (1 %). The inoculum was cultivated at 37°C for 12 hours under constant agitation at 220 rpm.
  • 1 ml of cell culture was used to inoculate 100 ml of sterile autoinduction media (composition for IL: 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose).
  • composition for IL 10 g Tryptone; 5 g Yeast extract; 3.3 g ammonium sulphate; 50 mM phosphate buffer pH 6.7; 0.5 g glucose; 2 g lactose.
  • the cultivation was kept for 48 hours at 22°C under constant agitation at 220 rpm.
  • the protein production was verified by protein SDS- PAGE electrophoresis and subsequent Coomasie Brilliant blue staining of whole cell lysates. For three proteins, the production rate was analysed in more detail. Comparison of expression of lysostaphin.
  • lysostaphin protein was compared with the commonly used laboratory expression plasmid pET.
  • the difference is most pronounced in the first 24 hours, when of the invention-Lysostaphin has a density of only 5 McFarland and pET28-Lysostaphin has a density of 13 McFarland.
  • the density of pET28- Lysostaphin increased to 22 McFarland and of the invention-Lysostaphin to 25 McFarland.
  • the resulting plasmid of the invention-Lysostaphin pellet had a lower yield (8.25 g/L) than pET28- Lysostaphin (13.75 g/L). However, the final protein production was 4.96x higher with the pUbExlOO- Lysostaphin. Due to the relatively higher production of protein per unit of biomass, the plasmid of the invention improves the subsequent purification process and makes its production significantly more efficient.

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Abstract

La présente invention concerne un nouveau vecteur d'expression (plasmide) contenant une séquence ORI nouvellement conçue. Le nouveau vecteur d'expression est plus stable et permet une production élevée de protéine recombinante par biomasse totale.
EP22799844.0A 2022-10-03 2022-10-03 Vecteur d'expression pour la production de protéines recombinantes dans des cellules hôtes procaryotes Pending EP4599068A1 (fr)

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PCT/CZ2022/050100 WO2023193837A1 (fr) 2022-10-03 2022-10-03 Vecteur d'expression pour la production de protéines recombinantes dans des cellules hôtes procaryotes

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EP4599068A1 true EP4599068A1 (fr) 2025-08-13

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