WO2015068134A1 - Méthode de production d'une collagénase recombinante pour digérer les collagènes - Google Patents

Méthode de production d'une collagénase recombinante pour digérer les collagènes Download PDF

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WO2015068134A1
WO2015068134A1 PCT/IB2014/065877 IB2014065877W WO2015068134A1 WO 2015068134 A1 WO2015068134 A1 WO 2015068134A1 IB 2014065877 W IB2014065877 W IB 2014065877W WO 2015068134 A1 WO2015068134 A1 WO 2015068134A1
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collagenase
recombinant
cells
seq
collagen
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António Carlos MATIAS CORREIA
Ana Sofia DIREITO DOS SANTOS DUARTE
Ana Cristina DE FRAGA ESTEVES
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Universidade De Aveiro
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24003—Microbial collagenase (3.4.24.3)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • 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

Definitions

  • the present invention relates to the method for producing active recombinant collagenase from Aeromonas hydrophila for application in processes where collagen digestion is required, particularly for medical application in the treatment of diseases involving excessive collagen deposition.
  • Collagen is the major constituent of the tissues of organisms belonging to the Eumetazoa sub-kingdom, and is generally present in the Metazoan Kingdoms (Kingdom Metazoa or Animalia), from simpler organisms such as sponges to structurally more complex organisms such as mammals. .
  • Collagens are a large family of proteins characterized by a variety of chemical and structural properties. They are semi-crystalline molecules whose main function is to support the cells. In addition to its structural function, through the formation of insoluble fibers in the extracellular matrix, which confers tensile strength and elasticity, collagen has functions related to cellular activity and development; It is of particular importance in processes associated with extracellular matrix remodeling, both in physiological and pathological processes.
  • collagen plays a key role in healing processes.
  • Various skin trauma, such as burns, surgical acts, or infections are often characterized by excessive accumulation of fibrous tissue rich in various types of collagen.
  • Several other conditions are associated with excessive collagen deposition and for this reason are often referred to as collagen-mediated conditions.
  • Collagenases as enzymes capable of digesting collagen specifically, have been used to treat various collagen-mediated conditions.
  • Collagenases of different origins ie mammals, crustaceans, fungi, bacteria
  • a more frequent example of therapeutic application is the use of microbial collagenases in enzymatic wound debridement to degrade necrotic tissue, making the healing process of burns easier.
  • the use of collagenase produced by bacteria of the species Clostridium histolyticum has been licensed as a noninvasive method and successfully applied in the treatment of Dupuytren's disease, avoiding the need for recurrent surgical interventions.
  • microbial collagenases Very common is also the use of microbial collagenases in the establishment and maintenance of cell and tissue cultures. Its application has been cited in methods for isolating neuronal and liver cells, stem cell and epithelial cell studies. In addition to clinical and research application, microbial collagenases have a high biotechnological potential at industry level, particularly in the food industry in meat tenderization or in the tanning industry where they participate in the leather dyeing process.
  • This work is a genetic construct that does not include any fusion or labeling;
  • the obtained recombinant protein has no activity, consequently resulting in loss of biotechnological potential.
  • the present invention provides novel approaches in terms of genetic manipulation, namely by constructing a hybrid gene that includes a marker (polyhistidine tail) and recombinant protein overexpression conditions, allowing stable and large-scale active collagenase to be obtained. to storage.
  • the gene coding for this enzyme is homologous to the AHA_1043 gene annotated in the Aeromonas hydrophila ATCC 7966 T genome.
  • the construct is based on the sequence of the open reading frame (ORF) AHA_0517 (A. hydrophila ATCC 7966 T genome), which codes for a potential M9 family collagenase (Pfam: PF01752), a distinct product of collagenase described by Han and coauthors (HA_2008).
  • compositions and methods for treating collage-mediated diseases is based on the development of a product and pharmaceutical formulations and consists of the combination of collagenase I and collagenase II (1: 1 ratio) of Clostridium histolyticum to treatment of diseases involving collagen deposition.
  • the present invention describes a collagenase of different origin (Aeromonas hydrophila) from the above, which is produced in a pure, active and stable manner by a protocol involving a single affinity chromatography purification step.
  • the present invention relates to a method for obtaining a collagenase capable of digesting collagen at concentrations below the toxic concentration for cells, in particular fibroblast-like cells, or below the concentration used with other collagenase.
  • the collagenase described in the present invention may come from bacteria of the genus Aeromonas, among others. Microbial collagenase is recombinantly produced (Fig. 1) and overexpressed in bacteria. Given the characteristics of the product obtained, namely the in vitro collagen digestion capacity, the activity at sub-toxic concentrations in the expression of collagen in the extracellular matrix of cultured osteoblasts and its high storage stability, its application is possible. in processes where collagen digestion is desired.
  • a method for obtaining and purifying active recombinant Aeromonas collagenase comprising the following steps is described.
  • host cells preferably E. coli, namely E. coli TOP10F 'to transform and select recombinant vectors;
  • E. coli namely E. coli BL21 (DE3) to overexpress the desired protein
  • Recombinant collagenase obtainable by the method described in this invention (and not wild), has a signal peptide that promotes its translocation to the extracellular medium (which facilitates the purification process) and a histidine tail that allows purification through of an immobilized metal ion affinity chromatography.
  • Recombinant collagenase for collagen digestion obtainable by the method described in this invention digests collagen with collagenase concentrations below the non-toxic concentration for cells, namely up to 25 ⁇ collagenase, in particular to 100 nM, 150 nM or 300 nM collagenase. It has also been demonstrated its physical interaction with collagen (substrate), which guarantees an extended stay in the place (tissue) where its activity is to be exercised.
  • cloning vector preferably comprising the site for the enzymes Nco ⁇ and Hind ⁇ , a kanamycin resistance gene and having a DNA sequence encoding a set of consecutive histidines (tail of polyhistidines);
  • E. coli host cells preferably E. coli TOP10F '
  • g transforming said recombinant plasmid into recombinant expression cells, preferably using host cells compatible with the vector used, as demonstrated with E. coli BL21 (DE3) to overexpress the desired protein and further pET26b (+);
  • primers which include the restriction enzyme recognition site BspH ⁇ and Hind ⁇ , which comprise the following homologous sequences.
  • amplification may occur by polymerase chain reactions carried out in 50 ⁇ volumes containing 3 mM MgCl 2 , 250 ⁇ dNTPs, 0.5 ⁇ from each primer (AHCF_SspHI and AHCR_H / ' ndlll ), 5% DMSO (v / v), 1.5 U Taq DNA polymerase in appropriate buffer and 50-100 ng DNA.
  • said amplification may be performed on a thermocycler comprising the initial denaturation of the genomic DNA of Aeromonas spp. at 94 - 96 ° C for 2 - 5 min; followed by 40 cycles of 30 s at 94 - 96 ° C, 30 s at 58 ° C and 3 min at 70 - 75 ° C preferably 68 ° C; final extension 20-30 min at 68-75 ° C, preferably 72 ° C; the optimum activity temperature of the Taq polymerase used, which may however vary by manufacturer.
  • a cloning vector may be used which allows the application of a leader sequence (signal peptide) and a histidine tail, compatible with the terminals of the fragment (gene) to be ligated, as shown for the cloning vector is pET26b (+).
  • the selection of a clone containing the recombinant plasmid may be done by culture in selective medium, as shown for plasmid pET26b (+), wherein positive clones are selected in supplemented medium. with kanamycin.
  • the expression host cells may be transformed and cultured at 26 and 130 rpm for 18 h.
  • the centrifugation conditions may be as follows: rate of 11000 xg, 4 ° C; for 25 minutes.
  • the collection of recombinant collagenase may be carried out by refrigerated centrifugation or other cell concentration process, followed by dialysis (4c) against binding buffer and syringe filter filtration (0 ° C). 2 ⁇ ).
  • the purification of recombinant collagenase may be effected by affinity chromatography, preferably immobilized metal ion affinity chromatography or other similar processes.
  • affinity chromatography may preferably be performed on a column coupled to a medium pressure system.
  • isolated collagenase obtainable from the process described in the previous method, which includes in its genetic construct a recombinant protein domain overexpressed with a histidine tail marker is further described.
  • isolated collagenase obtainable from the process described in the present invention is capable of digesting collagen at non-toxic concentrations, well below effective concentrations with other collagenases.
  • said isolated collagenase includes in its genetic construct a recombinant overexpressed protein domain and a marker, histidine tail.
  • it has the characteristic of isolated collagenase to exhibit type I, type II and type III collagen digestion (in vitro digestion, analyzed by electrophoresis).
  • collagenase alone may be used as a medicine or medicine, preferably in the treatment of traumatized tissues, namely in the treatment of traumatic wounds, varicose ulcers and burns and also in the treatment of Dupuytren's disease, a proliferative fibrodysplasia of the palmar aponevrose; in gene or electro-gene therapy; and also in the treatment of sciatica, namely in herniated intervertebral discs.
  • composition comprising or consisting of the collagenase described above.
  • Said collagenase may be applied in solution in its lyophilized form or in a composition comprised of said collagenase and a biomaterial, namely chitosan.
  • said composition may be a topical formulation, a formulation for oral or parenteral administration or an injectable formulation.
  • the present invention further describes the use of said collagenase obtainable by the method described above in techniques for culturing animal cells and tissues such as isolation of endothelial cells, neuronal cells, cardiomyocytes, hepatocytes and stem cells; in the food industry, particularly in the meat processing to improve its properties and textures; and in the tanning industry, in particular to assist in the leather dyeing process.
  • the present invention relates to the process of cloning, expression and purification of an Aeromonas collagenase for collagen digestion.
  • One object of the present invention is the production of Aeromonas collagenase following a methodology based on genetic recombination.
  • primers were designed for directed cloning of the complete gene encoding collagenase in Aeromonas hydrophila.
  • the isolated gene can be cloned into a cloning vector containing a DNA sequence encoding a set of five consecutive histidines (histidine tail), which was later used to transform competent cells to produce recombinant cells capable of expressing the bacterial collagenase.
  • recombinant cells are induced for expression of recombinant collagenase.
  • Extracellular culture medium can be separated from cells by centrifugation, dialyzed and applied to a histidine affinity chromatography column for purification of active recombinant collagenase.
  • the present invention is useful for application in processes where it is intended to digest interstitial collagen, specifically for the treatment of conditions involving excessive extracellular matrix deposition, for example as Dupuytren's Disease.
  • the described recombinant collagenase has further application in the establishment of primary cell cultures, for which the detachment of tissues of origin and cell dissociation prior to in vitro culture is essential. Description of the Figures
  • Figure 1 Schematic representation of the cloning and recombinant plasmid (pCUA) process.
  • Cloning vector digested with Hind ⁇ ⁇ ⁇ / Nco ⁇ (a) enzymes and restriction enzyme polymerase chain reaction (PCR) product digested with restriction enzymes / - / / ic / lll / SspHI (b), generating complementary termini to provide binding of the gene to vector (c).
  • Figure 2 Flowchart of the method for obtaining recombinant collagenase.
  • Figure 3 Methodological diagram of recombinant collagenase expression and purification.
  • Figure 4 Expression of recombinant collagenase in E. coli. Overexpression (SDS-PAGE) and proteolytic activity (gelatin zymography) of collagenase in recombinant (R) vs untransformed (C) cells. Migration of the recombinant protein corresponds to the expected molecular weight ( ⁇ 100 kDa) and is marked with an arrow.
  • Figure 5 Purification of recombinant ColAh by immobilized metal ion affinity chromatography.
  • Figure 6 Digestion of human type I collagen with recombinant collagenase (ColAh).
  • Figure 7 Detection of physical interaction between enzyme: substrate (recombinant collagenase: type I collagen) by immunodetection.
  • Figure 8 Stability of recombinarrte ⁇ CoiAh collagenase activity) at various storage temperatures (-80 C ⁇ ; -20 ° C; 4 it is 37).
  • Figure 9 Determination of cell viability of cultured osteoblasts exposed to ColAh treatment.
  • Figure 10 Type I cotagen immunodetection in the extracellular half-cell fractions of osteoblasts subjected to ColAh treatment during a dose response assay.
  • Figure 11 Intra and extracellular distribution of type i collagen in MC3T3 osteoblasts (15 days after confluence), analyzed by confocal microscopy.
  • the basic strategy for increasing expression of the active protein is by cloning the gene encoding for coiagenase into an expression vector and consequent transformation in the host to heterologous expression,
  • the expression vector is a cloning vector, constructed such that the gene inserted therein is transcribed / translated upon its introduction into host cells.
  • the present invention relates to the method for obtaining recombinant collagenase comprising the following steps: (a) isolating Aeromonas hydrophila ATCC 7966 T genomic DNA; (b) amplification of the putative collagenase gene by PCR to generate multiple copies of the collagenase gene (ORF: AHA_0517); (c) cloning of the Aeromonas collagenase gene into pET26b (+) cloning vector; (d) transforming the recombinant vector, pCUA, into competent cells (E.
  • coli TOP10F to produce a starting clone; (e) identification and selection of the positively transformed starting clone; (f) insert sequencing to confirm the correct reading phase of the gene in the vector; (g) purification of the recombinant plasmid and transformation into E. coli BL21 (DE3) expression cells; (h) identification and selection of transformed recombinant cells; (i) induction of recombinant cells for expression of recombinant collagenase; (j) collecting extracellular culture medium and purifying recombinant collagenase by affinity chromatography; (k) dialysis against isotonic buffer; (I) collagenolytic activity assay.
  • the total DNA of the Aeromonas hydrophila ATCC 7966 T strain was isolated from liquid cultures in Luria-Bertani (LB) medium supplemented with the ampicillin antibiotic (50 ⁇ g / ml) using the Genomic DNA Purification Kit commercial system following the manufacturer's instructions. Subsequently, the gene encoding the putative collagenase in Aeromonas was amplified by PCR using the following pair of primers (underlining the restriction enzyme recognition sequence):
  • primers were designed based on the coding nucleotide sequence (AHA_0517) (SEQ. ID. No. 1) of the pre-ColAh polypeptide to include the signal peptide.
  • PCR reactions were performed in 50 ⁇ volumes containing 3 mM MgCl 2 , 250 ⁇ dNTPs, 0.5 ⁇ from each primer (AHCF_SspHI and AHCR_H / ' ndlll), 5% DMSO (v / v), 1.5 U Taq DNA polymerase in appropriate buffer and 50-100 ng DNA.
  • Reactions were performed on an iCycler TM Thermal Cycler thermocycler under the following amplification conditions: initial denaturation at 94 ° C for 5 min; 40 cycles of 30s at 94 ° C, 30s at 58 ° C and 3 min at 72 ° C; 30 min final extension at 72 ° C.
  • the primer sequences include the restriction enzyme recognition site, BspYW and Hind ', (underlined in the primer sequence), indispensable for generating the appropriate termini for subsequent binding to the vector (Fig. 1).
  • the restriction enzymes BspH ⁇ and Hind ⁇ were used to digest the PCR product and Nco ⁇ and Hind ⁇ were used to digest the vector, following the manufacturer's instructions (Table 1).
  • Plasmid pET26b (+) was the vector of choice because it contains a kanamycin resistance gene that allows for subsequent selection of transformants.
  • TOP10F 'cells have been selected as a host strain to generate a starting clone (to maintain the recombinant plasmid).
  • the obtained recombinant vector which we call pCUA, was used at an early stage to transform E. coli strain TOP10F 'as set forth in table 4.
  • Table 4 Transformation of competent cali cells
  • Fig. 3 In order to express recombinant collagenase, the strategy shown in Fig. 3 was followed. Pre-inocula of an E. coli BL21 (DE3) strain transformant inoculated into LB medium containing 50 ⁇ g / ml kanamycin were used. A culture (50 ml) was incubated at 37 and 150 rpm until it reached 0.6 optical density (OD) units at 600 nm; the cells were induced with the addition of 0.5 mM isopropyl beta-D-thiogalactopyranoside (I PTG). The culture conditions after induction were adjusted to 26 and 130 rpm for 18 h for greater active enzyme expression. Overexpression of recombinant ColAh collagenase in E. coli was assessed by SDS-PAGE and proteolytic activity by gelatin zymography (Fig. 4).
  • Extracellular medium was collected after centrifugation at 11000 xg, 4 ⁇ ° C for 25 minutes and dialyzed (4 ⁇ ° C, 1 h) against binding buffer and filtered with syringe filter (0.2 ⁇ ) prior to affinity chromatography purification step. Chromatography was performed on a particular HisTrap TM FF immobilized metal ion affinity column coupled to a particular medium pressure AKTA Basic system. Thus, after equilibration with 10 column volumes in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4), the sample (10 ml) was applied to the column and eluted at 10 ° C.
  • % (v / v) elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4), which corresponds to 50 mM imidazole (Fig. 5).
  • the collected fraction was analyzed by gelatin zymography (0.1%) and by SDS-PAGE to confirm its activity and purity.
  • Recombinant collagenase was further tested for its ability to digest type I collagen (Fig. 6) according to the protocol described by Duarte et al. (DU_2005) and its physical interaction with this substrate (Fig. 7). Storage stability of its activity was also confirmed by incubating ColAh at different temperatures (-80 ° C, -20 ° C, 45 ° C and 37 ° C) and storage times (Fig. 8).
  • One embodiment comprises the use of sub-toxic doses of recombinant collagenase for collagen reduction in cultured osteoblasts.
  • Aeromonas collagenase comprises the steps described above and shown schematically in Fig. 2.
  • the primers AHCF_SspHI and AHCR _ / - / are used.
  • / idll 1, specifically designed to simultaneously allow ORF AHA_0517 to be selected and to introduce at each end of the gene the recognition sequences of the respective restriction enzymes.
  • the plasmid is purified and the sequence of the insert is determined by automatic sequencing to confirm that the gene has been cloned at the correct reading stage, which allows expression of the active recombinant protein contemplating the histidine tail (SEQ. I D. No. 5) for further purification by affinity chromatography. Upon confirmation, the recombinant plasmid is then used to transform E. coli BL21 (DE3) expression cells (Table 4).
  • positive clones are selected on kanamycin-containing plates and the presence of the insert is PCR-confirmed using the vector-specific primers and subsequent sequence determination by the Sanger method (SA_1977). Once confirmed, the positive clone is inoculated into LB medium (supplemented with 50 ⁇ g / ml kanamycin) to produce a pre-inoculum that is used for culture establishment for recombinant collagenase overexpression. As depicted in the scheme of Fig. 3, 1 ml of pre-inoculum is used to inoculate 50 ml of kanamycin-containing medium, which is incubated at 37 ° C under shaking (150 rpm).
  • the culture When it reaches the optical density 0.6, measured at 600nm, the culture is induced with 500 mM IPTG and incubated for 18 h at 26 ° C under controlled shaking (130 rpm). Extracellular culture medium is harvested after cell pelletization by centrifugation (11000 xg, 25 minutes, 4 ⁇ ° C), then dialyzed (4 ⁇ ° C) against binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4) under slight agitation. The sample is filtered with a syringe filter (0.2 ⁇ ) and applied to an immobilized metal ion affinity column (HisTrap TM FF) coupled to a medium pressure system, preferably AKTA Basic.
  • a medium pressure system preferably AKTA Basic.
  • the sample (10 ml) is applied and the run is established at a constant flow (1 ml / min).
  • Recombinant collagenase is eluted from the column in 10% elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4), which corresponds to 50 mM imidazole (Fig. 5 ).
  • the collected fraction is analyzed by gelatin zymography (0.1%) and by SDS-PAGE to confirm its activity and purity.
  • the sample is quantified using the Micro BCA Protein Quantitation Kit as directed by the manufacturer.
  • the purpose of this application is to demonstrate the effect of recombinant Aeromonas collagenase on proliferation and differentiation of cultured mammalian cells.
  • MC3T3-E1 (ATCC) cell line is used, a mouse osteoblast cell line. These cells are on the bone surface and are directly responsible for bone formation. They were particularly selected because during the differentiation process the osteoblast precursor cells alter the its morphology and the expression of functional markers, beginning by secreting bone matrix proteins such as type I collagen, which represents about 90% of the organic matrix and provides the structure for subsequent bone mineralization.
  • MC3T3-E1 cells were maintained at 37 ° C in a humidified, 5% CO 2 atmosphere in minimal essential medium containing 2 mM glutamine in a 10% (v / v) EBSS (Eagle's Balanced Salt Solution) balanced salt solution. ) fetal bovine serum (FBS), 1% (v / v) solution with penicillin (100 U / ml) and streptomycin (100mg / ml) and 3.7 g / l NaHC0 3. Sub-confluent cultures (80 - 90% confluence) are split 1: 5 solution with 0.25% trypsin / EDTA 5% C0 2, 375C.
  • FBS fetal bovine serum
  • penicillin 100 U / ml
  • streptomycin 100mg / ml
  • Sub-confluent cultures 80 - 90% confluence
  • 1x10 5 cells are seeded in 35 mm wells in 6-well culture plates (at a density of 1x10 4 cells / cm 2 ) and grown under the above conditions.
  • the medium is renewed every 2 days of culture. After 18 days in culture, which corresponds to the 15th day after confluence, the culture medium is replaced with medium supplemented with 100, 150 and 300 nM pure collagenase, and the cells are incubated for 16 hours. After this exposure period, the medium is removed by aspiration and replaced with fresh medium containing 10% of a 0.1 mg / ml resazurin solution for determination of cell viability.
  • the collagen type I expression and segregation profile is evaluated using immunodetection based methodologies.
  • culture medium (1 ml) from each condition is collected into microtubes containing 10% sodium dodecyl sulfate (SDS).
  • SDS sodium dodecyl sulfate
  • the respective cells are carefully washed with phosphate buffered saline (PBS 1 ) and collected with a 1% SDS (w / v) solution.
  • PBS 1 phosphate buffered saline
  • SDS w / v
  • the samples are boiled for 10 minutes in a water bath (100 ⁇ ° C) and the total protein concentration (BCA Protein Assay Reagent, Pierce) is determined.
  • BCA Protein Assay Reagent Pierce
  • Proteins separated by electrophoresis were electrotransferred to a nitrocellulose membrane for further immunodetection with anti-collagen type I primary antibody, following blocking of the membrane with 5% (w / v) solution milk in TBS-T (10 mM Tris-HCI, pH 8.0, 150 mM NaCl, 0.5% Tween) (DU_2005).
  • TBS-T 10 mM Tris-HCI, pH 8.0, 150 mM NaCl, 0.5% Tween
  • Protein-antibody binding is detected by chemiluminescence with horseradish peroxidase-conjugated rabbit anti-IgGs secondary antibody using the ECL Kit according to the manufacturer's instructions and revealed in darkroom on self-adhesive plates. radiographic images.
  • the cellular distribution of collagen was analyzed by immunocytochemistry according to Pl_2010.
  • the cultures were set up in 35 mm diameter plates containing coverslips and were treated under the same conditions of exposure to purified collagenase.
  • the coverslips were fixed in 4% (v / v) formaldehyde in PBS and permeabilized with methanol. Non-specific sites were blocked by incubation (1h) with 3% (w / v) bovine serum albumin (BSA) in PBS and immunocytochemistry was performed with type I anti-collagen antibody diluted in 3% BSA-PBS.
  • BSA-PBS bovine serum albumin
  • FICT fluorochromo fluorescein isothiocyanate
  • coverslips were mounted on microscopy slides in 4 ', 6-diamidino-2'-phenylindole dihydrochloride (DAPI) mounting medium with Vectashield reagent (to reduce fluorescence photoblot). Images were acquired on an LSM 510 META confocal microscope with an Argon (488nm) laser to excite the FITC conjugate and a Diode 405-430 laser to assess DAPI fluorescence.
  • DAPI 6-diamidino-2'-phenylindole dihydrochloride
  • the acquired images show that the treatment of collagen-secreting cells with recombinant collagenase promotes a dose-dependent response, reflecting increased proliferation of osteoblasts to the highest concentration of collagenase used in the assay (Fig. 11). Reduction of the deposition of type I collagen in the extracellular matrix of cultured cells is also evident.
  • recombinant collagenase, ColAh may have diverse applications, with the advantage of having high storage stability, e.g. Its potential application to enzymatic debridement of tissues, due to its degradative capacity of collagen facilitating regeneration and healing, namely in the treatment of traumatic wounds, varicose ulcers and burns.
  • Dupuytren's disease is a proliferative fibrodysplasia of palmar aponevrosis, characterized by excessive deposition of collagen in the extracellular matrix, a prevalent condition in populations such as the British, North American, and Australian (SY_2012).
  • surgical fasciectomy is often used, which does not limit the recurrence of the pathology and, as an invasive method, leads to increased patient morbidity.
  • a new noninvasive therapeutic strategy has recently been implemented and is based on the use of Clostridium histolyticum (CCH) Xiaflex ® Collagenase (with constant 1: 11 ratio for CoIG and CoIH collagenases, respectively).
  • ColAh collagenase is applied in techniques involving cell and animal tissue cultures (endothelial cell isolation, neuronal cells, cardiomyocytes, hepatocytes, stem cells, among others).
  • ColAh collagenase is applied in the food industry, namely in the processing of meat to increase its properties and textures.
  • nucleotide sequences are described by one letter nucleotide code. The nucleotide legend is as follows (one letter code): A - adenine, T - thymine, G - guanidine, C - cytosine. SEQ I D. No. 1 - Aeromonas hydrophila ATCC 7966 T Collagenase Gene Nucleotide Sequence (AHA_0517)
  • TCTTCCG CCTG C AG CCC ATCGTG G GC AG CC AG G CCG CC AAG C AG GTG AG CG AG CTG CACGTC
  • CTGTTCCTGCTC AAC AACGTG CTG G ATGCCTAC ACCCG CCTCTACCG G GTCG CCCG CTATACCC
  • T7 straight SEQ. I.D. No. 8- 5'-TAATACGACTCACTATAGGG - 3 ';
  • T7 Reverse SEQ. I.D. No. 7 - 5'-G CTAGTTATTG CTC AG CG G - 3 '; Leader Sequence:
  • Aeromonas hydrophila ATCC 7966 T recombinant collagenase (colAh) active protein SEQ. I D. No. 5
  • the active recombinant protein sequence is described by the one letter amino acid code.
  • the amino acid label is as follows (one letter code): Histidine - H, Arginine - R, Lysine - K, Isoleucine - I, Phenylalanine - F, Leucine - L, Tryptophan - W, Alanine - A, Methionine - M, Proline - P, Valine - V, Cysteine - C, Asparagine - N, Glycine - G, Serine - S, Glutamine - Q, Tyrosine - Y, Aspartic Acid - D, Glutamic acid - E.

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Abstract

La présente invention concerne une méthode pour produire une collagénase recombinante active, provenant d'Aeromonas hydrophila, en vue d'une application dans des processus où la digestion de collagènes est nécessaire, notamment en vue d'une application médicale dans le traitement de maladies impliquant un dépôt excessive de collagènes et des pathologies médiées par le collagène. Outre leur application en milieu clinique et à des fins de recherche, les collagénases microbiennes présentent un potentiel biotechnologique élevé au niveau industriel, notamment dans l'industrie alimentaire pour l'attendrissement de viandes ou dans l'industrie de la tannerie, où elles contribuent au processus de coloration des cuirs. Ainsi, la présente invention vise à fournir une méthode pour l'obtention et la purification d'une collagénase recombinante active d'Aeromonas hydrophila, méthode selon laquelle le collagène est digéré par la collagénase avec des concentrations de collagénase inférieures à la concentration cytotoxique de la collagénase.
PCT/IB2014/065877 2013-11-07 2014-11-07 Méthode de production d'une collagénase recombinante pour digérer les collagènes WO2015068134A1 (fr)

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PT107276A PT107276B (pt) 2013-11-07 2013-11-07 Método para produção de colagenase recombinante para digestão de colagénios
PT107276 2013-11-07

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0430635A1 (fr) * 1989-11-28 1991-06-05 Suntory Limited Gène de collagénase bactérien
EP2363461A1 (fr) * 2008-11-19 2011-09-07 Meiji Seika Pharma Co., Ltd. Collagénase de fusion à laquelle un marqueur d'affinité est fixé et son procédé de fabrication
WO2013106510A2 (fr) * 2012-01-12 2013-07-18 Auxilium Pharmaceuticals, Inc. Enzymes de clostridium histolyticum et procédés pour les utiliser
WO2013156525A1 (fr) * 2012-04-18 2013-10-24 Fidia Farmaceutici S.P.A. Nouveau procédé de production et de purification de l'enzyme collagénase issue de vibrio alginolyticus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0430635A1 (fr) * 1989-11-28 1991-06-05 Suntory Limited Gène de collagénase bactérien
EP2363461A1 (fr) * 2008-11-19 2011-09-07 Meiji Seika Pharma Co., Ltd. Collagénase de fusion à laquelle un marqueur d'affinité est fixé et son procédé de fabrication
WO2013106510A2 (fr) * 2012-01-12 2013-07-18 Auxilium Pharmaceuticals, Inc. Enzymes de clostridium histolyticum et procédés pour les utiliser
WO2013156525A1 (fr) * 2012-04-18 2013-10-24 Fidia Farmaceutici S.P.A. Nouveau procédé de production et de purification de l'enzyme collagénase issue de vibrio alginolyticus

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 13 August 2012 (2012-08-13), "Aeromonas piscicola strain AH-3 collagenase (colAh) gene, complete cds.", XP002737018, retrieved from EBI accession no. EMBL:JQ639076 Database accession no. JQ639076 *
DATABASE UniProt [online] 12 December 2006 (2006-12-12), "SubName: Full=Collagenase family {ECO:0000313|EMBL:ABK38430.1};", XP002737017, retrieved from EBI accession no. UNIPROT:A0KFM4 Database accession no. A0KFM4 *
EIJI TAMAI ET AL: "High-level expression of his-tagged clostridial collagenase in Clostridium perfringens", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 80, no. 4, 16 July 2008 (2008-07-16), pages 627 - 635, XP019623735, ISSN: 1432-0614 *
ELIANA MARISA DOSSANTOS CAVALEIRO: "Clonagem e expressão de uma potencial colagenasede Aeromonas hydrophila", 2008, XP002737016, Retrieved from the Internet <URL:http://core.ac.uk/download/pdf/15561007.pdf> *
MANDL I: "BACTERIAL COLLAGENASES AND THEIR CLINICAL APPLICATIONS", ARZNEIMITTEL FORSCHUNG. DRUG RESEARCH, ECV EDITIO CANTOR VERLAG, AULENDORF, DE, vol. 32, no. 10A, 1 January 1982 (1982-01-01), pages 1381 - 1384, XP000857741, ISSN: 0004-4172 *
R. SESHADRI ET AL: "Genome Sequence of Aeromonas hydrophila ATCC 7966T: Jack of All Trades", JOURNAL OF BACTERIOLOGY, vol. 188, no. 23, 15 September 2006 (2006-09-15), pages 8272 - 8282, XP055174766, ISSN: 0021-9193, DOI: 10.1128/JB.00621-06 *

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