WO2015068134A1 - Method to produce a recombinant collagenase to digest collagens - Google Patents

Method to produce a recombinant collagenase to digest collagens 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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Portuguese (pt)
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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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    • C—CHEMISTRY; METALLURGY
    • 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.

Abstract

The present invention relates to a method for producing active recombinant collagenase from Aeromonas hydrophila for use in processes where the digestion of collagens is required, for instance for medical use in the treatment of diseases involving excessive deposition of collagens and collagen-mediated pathologies. Besides clinical and research uses, microbial collagenases have high bioengineering potential in industry, for instance in the food industry for tenderising meat or in the tanning industry, where they contribute to the leather dyeing process. Thus, the present invention aims at providing a method for obtaining and purifying active recombinant collagenase from Aeromonas hydrophila, in which method collagen is digested by collagenase at concentrations that are lower than the cytotoxic concentration of collagenase.

Description

DESCRIÇÃO  DESCRIPTION
MÉTODO PARA PRODUÇÃO DE UMA COLAGENASE RECOMBINANTEMETHOD FOR PRODUCTION OF RECOMBINANT COLLAGENASE
PARA DIGERIR COLAGÉNIOS TO SAY COLLAGEN
Domínio técnico da invenção Technical field of the invention
[0001] A presente invenção diz respeito ao método para produzir colagenase recombinante ativa, proveniente de Aeromonas hydrophila, para aplicação em processos onde a digestão de colagénios é necessária, nomeadamente para aplicação médica no tratamento de doenças que envolvem deposição excessiva de colagénios.  [0001] 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.
Antecedentes da Invenção Background of the Invention
[0002] O colagénio é o constituinte maioritário dos tecidos dos organismos pertencentes ao sub-reino Eumetazoa, estando presente de uma forma geral nos Metazoários (Reino Metazoa ou Animalia), desde organismos mais simples como espongiários até a organismos estruturalmente mais complexos como os mamíferos. Os colagénios constituem uma extensa família de proteínas caracterizada por uma variedade de propriedades químicas e estruturais. São moléculas semi-cristalinas, cuja principal função é suportar as células. Para além da sua função estrutural, através da formação de fibras insolúveis na matriz extracelular, à qual confere força tensora e elasticidade, o colagénio tem funções relacionadas com a atividade e desenvolvimento celulares; tem particular importância em processos associados à remodelação da matriz extracelular, quer em processos fisiológicos quer em processos patológicos.  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.
[0003] Em humanos, o colagénio tem um papel fundamental em processos de cicatrização. Diversos traumatismos da pele, como queimaduras, atos cirúrgicos ou infeções, são frequentemente caracterizados por acumulação excessiva de tecido fibroso rico em vários tipos de colagénios. Várias outras patologias estão associadas à excessiva deposição de colagénios e, por esta razão são frequentemente designadas como patologias mediadas por colagénio. As colagenases, como enzimas capazes de digerir colagénios de forma específica, têm sido utilizadas para tratar diversas patologias mediadas por colagénio. Têm sido aplicadas colagenases de diferentes origens (i.e.: mamíferos, crustáceos, fungos, bactérias) para uso terapêutico. Um exemplo mais frequente de aplicação terapêutica é a utilização de colagenases microbianas no desbridamento enzimático de feridas, para degradação dos tecidos necrosados, tornando mais fácil o processo de cicatrização de queimaduras. Recentemente, a utilização da colagenase produzida por bactérias da espécie Clostridium histolyticum foi licenciada como método não invasivo e aplicada com sucesso no tratamento da doença de Dupuytren, evitando a necessidade de intervenções cirúrgicas recorrentes. [0003] In humans, 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) have been applied for therapeutic use. 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. Recently, 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.
[0004] Muito frequente também é a utilização das colagenases microbianas no estabelecimento e manutenção de culturas de células e tecidos. Tem sido citada a sua aplicação em métodos para o isolamento de células neuronais e células hepáticas, estudos em células estaminais e células epiteliais. Para além da aplicação na clínica e na investigação, as colagenases microbianas têm um potencial biotecnológico elevado ao nível da indústria, nomeadamente na indústria alimentar na tenderização de carnes ou na indústria dos curtumes onde participam no processo de tingimento dos couros. 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.
[0005] Independentemente da aplicação das colagenases microbianas, estas enzimas são normalmente obtidas por processos fermentativos dos microrganismos produtores (como descrito no documento US 2010/0330065) e que requerem o seu isolamento posterior. Esta estratégia de obtenção de enzima apresenta como principais desvantagens a utilização de metodologias que envolvem várias etapas cromatográficas para a sua purificação, com a consequente perda de proteína e de atividade. Outra desvantagem associada a estas colagenases microbianas selvagens é a sua baixa estabilidade ao armazenamento que constitui o maior constrangimento para a eficácia da sua aplicação. [0006] O documento "Clonagem e expressão de uma potencial colagenase de Aeromonas hydrophila" , tese de mestrado de Eliana Cavaleiro, Universidade de Aveiro (EL_2008), divulga a expressão de uma proteína recombinante. Este trabalho consiste numa construção genética que não inclui nenhum tipo de fusão ou marcação; a proteína recombinante obtida não possui qualquer atividade, consequentemente resultando na perda de potencial biotecnológico. A presente invenção apresenta novas abordagens em termos de manipulação genética, nomeadamente pela construção de um gene híbrido que inclui um marcador (cauda de poli-histidinas) e condições de sobrexpressão de proteína recombinante, permitindo a obtenção de colagenase ativa em larga escala e com estabilidade ao armazenamento. Irrespective of the application of microbial collagenases, these enzymes are normally obtained by fermentative processes of the producing microorganisms (as described in US 2010/0330065) and which require their further isolation. This strategy of obtaining enzyme has as main disadvantages the use of methodologies that involve several chromatographic steps for its purification, with the consequent loss of protein and activity. Another disadvantage associated with these wild microbial collagenases is their low storage stability which is the major constraint to the effectiveness of their application. The document "Cloning and expression of a potential collagenase of Aeromonas hydrophila", master's thesis by Eliana Cavaleiro, University of Aveiro (EL_2008), discloses the expression of a recombinant protein. 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.
[0007] O documento "Purification and characterization of a recombinant histidine- tagged microbial collagenase", apresentado no II Workshop de Biotecnologia. "5 anos de Biotecnologia na UA", Aveiro, 2011 , não revela dados sobre a construção genética para expressão da colagenase recombinante ativa, nem faz referência às etapas da sua purificação, mostrando apenas resultados sobre a termoestabilidade associada ao armazenamento da enzima recombinante pura. [0007] The document "Purification and characterization of a recombinant histidine-tagged microbial collagenase" presented at the II Biotechnology Workshop. "5 years of Biotechnology in the AU", Aveiro, 2011, does not disclose data on the genetic construct for expression of active recombinant collagenase, nor does it refer to its purification steps, showing only results on the thermostability associated with the storage of the pure recombinant enzyme.
[0008] O documento "A novel collagenase from Aeromonas sp." apresentado no congresso nacional Microbiotec'll, Braga, 2011, reporta resultados comparativos da caracterização da atividade colagenolítica associada à estirpe AH-3 [Aeromonas piscícola) e o seu mutante nulo para o gene da colagenase. Não é referida qualquer informação relativamente ao nome ou número de acesso da sua sequência, nem são referenciadas quaisquer particularidades inerentes à clonagem e expressão do gene. [0008] The document "A novel collagenase from Aeromonas sp." presented at the national congress Microbiotec'll, Braga, 2011, reports comparative results of the characterization of collagenolytic activity associated with strain AH-3 [Aeromonas piscicultural) and its null mutant for the collagenase gene. No information is given regarding the name or accession number of its sequence, nor are any particularities inherent in cloning and expression of the gene referenced.
[0009] O documento "Pathogenic potential of a collagenase gene from Aeromonas veronii", descrito por Han e colaboradores (HA_2008), refere-se a uma peptidase da família U32 com ~68kDa. O gene que codifica para esta enzima é homólogo ao gene AHA_1043 anotado no genoma de Aeromonas hydrophila ATCC 7966T. No entanto, na presente invenção, a construção baseia-se na sequência da ORF (open reading frame) AHA_0517 (genoma A. hydrophila ATCC 7966T), que codifica para uma potencial colagenase da família M9 (Pfam: PF01752), produto distinto da colagenase descrita por Han e co-autores (HA_2008). The "Pathogenic potential of a collagenase gene from Aeromonas veronii" document, described by Han et al. (HA_2008), refers to a U32 family peptidase with ~ 68kDa. The gene coding for this enzyme is homologous to the AHA_1043 gene annotated in the Aeromonas hydrophila ATCC 7966 T genome. However, in the present invention, 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).
[0010] O documento US3677900 (1972), "METHOD OF PRODUCING COLLAGENASE WITH A SPECIES OF VIBRIO", refere-se a um método de produção de colagenases bacterianas produzidas por bactérias dos géneros Vibrio e Aeromonas. Contudo, a espécie citada - Aeromonas proteolytica - foi posteriormente reclassificada como Vibrio proteolyticus (BA_1980) e o nome da bactéria foi aprovado em 1985 (MO_1985), não se tratando, portanto, de uma patente para produção de colagenases de Aeromonas. [0010] US3677900 (1972), "METHOD OF PRODUCING COLLAGENASE WITH A SPECIES OF VIBRIO", refers to a method of producing bacterial collagenases produced by bacteria of the genera Vibrio and Aeromonas. However, the cited species - Aeromonas proteolytica - was later reclassified as Vibrio proteolyticus (BA_1980) and the name of the bacterium was approved in 1985 (MO_1985) and is therefore not a patent for production of Aeromonas collagenases.
[0011] O documento US2011243920 "Compositions and methods for treating collage- mediated diseases" fundamenta-se no desenvolvimento de um produto e formulações farmacêuticas e consiste na combinação de colagenase I e colagenase II (numa proporção de 1:1) de Clostridium histolyticum para tratamento de doenças que envolvam a deposição de colagénio. US2011243920 "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.
[0012] A presente invenção descreve uma colagenase de origem distinta (Aeromonas hydrophila) das acima citadas, que é produzida de forma pura, ativa e estável, através de um protocolo que envolve um único passo de purificação por cromatografia de afinidade. 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.
Descrição geral da invenção General Description of the Invention
[0013] A presente invenção refere-se a um método para obtenção de uma colagenase, capaz de digerir colagénio, em concentrações inferiores à concentração tóxica para as células, em particular células tipo fibroblasto, ou inferior à concentração usada com outras colagenase. A colagenase descrita na presente invenção, pode ser proveniente de bactérias do género Aeromonas, entre outras. [0014] A colagenase microbiana é produzida de forma recombinante (Fig. 1) e sobreexpressa em bactérias. Dadas as características do produto obtido, nomeadamente a capacidade de digestão de colagénios in vitro, a atividade, em concentrações sub-tóxicas, na expressão de colagénio da matriz extracelular de osteoblastos em cultura e a sua elevada estabilidade ao armazenamento, é possível a sua aplicação em processos onde a digestão de colagénios é pretendida. É aqui reivindicada a sua utilização, assim como de qualquer forma modificada derivada, que mantenha as mesmas propriedades biológicas, quer em âmbito clínico e veterinário, para o tratamento de patologias em que seja pretendida a digestão de colagénios, quer para fins biotecnológicos, para produção de compostos farmacêuticos, na indústria alimentar, ou cosmética. 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. Its use as well as any derivative modified form which maintains the same biological properties, both clinically and veterinarily, for the treatment of conditions in which collagen digestion is sought, or for biotechnological purposes, for production is claimed herein. of pharmaceutical compounds in the food or cosmetic industry.
[0015] Descreve-se um método para obtenção e purificação de colagenase recombinante ativa de Aeromonas compreendendo os seguintes passos A method for obtaining and purifying active recombinant Aeromonas collagenase comprising the following steps is described.
• isolar o DNA genómico de uma cultura de Aeromonas;  • isolate genomic DNA from an Aeromonas culture;
• amplificar uma sequência com um grau de homologia de pelo menos 95 % com a SEQ. ID. N.s 1 ;  • amplify a sequence with a degree of homology of at least 95% with SEQ. ID No. 1;
• integrar, num vector de expressão induzível, a sequência codificante para uma proteína de fusão, em que a referida sequência codificante codifica para um conjunto de histidinas consecutivas ;  Integrating into an inducible expression vector the coding sequence for a fusion protein, wherein said coding sequence codes for a set of consecutive histidines;
• produzir e seleccionar o plasmídeo recombinante desejado;  Producing and selecting the desired recombinant plasmid;
• selecionar e cultivar as referidas células recombinantes e induzir a expressão de colagenase recombinante com indutor apropriado;  Selecting and cultivating said recombinant cells and inducing expression of recombinant collagenase with appropriate inducer;
• recolher e purificar a colagenese recombinante ativa.  • collect and purify active recombinant collagenesis.
[0016] Descreve-se ainda um método para obtenção e purificação de colagenase recombinante, com ainda melhores resultados, que compreende: Further described is a method for obtaining and purifying recombinant collagenase, with even better results, comprising:
• o isolamento do DNA genómico de uma cultura de Aeromonas; • Isolation of genomic DNA from an Aeromonas culture;
• a amplificação do gene da putativa colagenase da referida cultura com uma sequência com um grau de homologia de pelo menos 95 % com a SEQ. I D No. 1 - AHA_0517, de preferência com um grau de homologia de pelo menos 96%, 97%, 98%, ou igual utilizando iniciadores que incluem o local de reconhecimento das enzimas de restrição BspH\ e Hind\ \ \;• amplification of the putative collagenase gene from said culture with a sequence with a degree of homology of at least 95% with the SEQ ID No. 1 - AHA_0517, preferably with a degree of homology of at least 96%, 97%, 98% or equal using primers that include the restriction enzyme recognition site BspH \ and Hind \ \;
• a digestão de um vetor de clonagem, o qual possui um gene marcador de resistência à canamicina, com as enzimas Nco\ e Hind\ \ \, um gene de resistência à canamicina; • digestion of a cloning vector, which has a kanamycin resistance marker gene, with the enzymes Nco \ and Hind \ \ \, a kanamycin resistance gene;
• e que apresente uma sequência de DNA que codifica para um conjunto de histidinas consecutivas (cauda de histidinas);  • and which has a DNA sequence encoding a set of consecutive histidines (histidine tail);
• clonagem do gene amplificado e digerido com as enzimas de restrição BspH\ e Hind\ \ \ no vetor de clonagem selecionado e previamente digerido com as enzimas de restrição Nco\ e Hind\ \ \;  • cloning the amplified gene digested with restriction enzymes BspH \ and Hind \ \ \ into the selected cloning vector and previously digested with restriction enzymes Nco \ and Hind \ \ \;
• a utilização de células hospedeiras, de preferência E. coli, nomeadamente E. coli TOP10F' para transformar e selecionar vetores recombinantes;  The use of host cells, preferably E. coli, namely E. coli TOP10F 'to transform and select recombinant vectors;
• utilização de células de expressão, de preferência E. coli, nomeadamente E. coli BL21 (DE3) para sobreexpressar a proteína pretendida;  Use of expression cells, preferably E. coli, namely E. coli BL21 (DE3) to overexpress the desired protein;
• purificação da proteína com atividade.  • purification of protein with activity.
[0017] A colagenase recombinante obtenível pelo método descrito nesta invenção (e não selvagem), possui um péptido sinal que promove a sua translocação para o meio extracelular (o que facilita o processo de purificação) e uma cauda de histidinas que permite a purificação através de uma cromatografia de afinidade com ião metálico imobilizado. 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.
[0018] A colagenase recombinante para a digestão de colagénio obtenível pelo método descrito nesta invenção, digere o colagénio com concentrações de colagenase inferiores à concentração não tóxica para as células., nomeadamente até 25 μΜ de colagenase, em particular para 100 nM, 150 nM ou 300 nM de colagenase. [0019] Foi ainda demonstrada a sua interação física com colagénio (substrato), que garante uma permanência alargada no local (tecido) onde se pretende ser exercida a sua atividade. 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 μΜ 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.
[0020] Contrariamente à colagenase de Clostridium sp. comercial, esta colagenase produz um padrão hidrolítico mais próximo do padrão produzido por colagenases de humanas o que sugere uma maior potencialidade para a sua utilização médica. Contrary to collagenase of Clostridium sp. commercially, this collagenase produces a hydrolytic pattern closer to the pattern produced by human collagenases suggesting greater potential for its medical use.
[0021] Numa outra realização o método para obtenção e purificação de colagenase compreende os seguintes passos: In another embodiment the method for obtaining and purifying collagenase comprises the following steps:
a) isolar o DNA genómico de uma cultura de Aeromonas de preferência Aeromonas hydrophila ATCC 7966T; (a) isolate genomic DNA from a culture of Aeromonas preferably Aeromonas hydrophila ATCC 7966 T ;
b) amplificar o gene da putativa colagenase da referida cultura com uma sequência com um grau de homologia de pelo menos 95 % com a SEQ. ID. No. 1 - AHA_0517, de preferência com um grau de homologia de pelo menos 96%, 97%, 98%, 99% ou igual, utilizando iniciadores que incluem o local de reconhecimento das enzimas de restrição BspH\ e Hind\\\, de preferência com AHCF_SspHI (SEQ. ID. No. 2) e AHCR_H/'ndlll (SEQ. ID. No. 3), e terem como base a sequência de nucleótidos codificante SEQ. ID. No. 1 do polipeptídeo pre-ColAh, incluindo o péptido-sinal; b) amplifying the putative collagenase gene of said culture with a sequence with a degree of homology of at least 95% with SEQ. ID No. 1 - AHA_0517, preferably with a degree of homology of at least 96%, 97%, 98%, 99% or equal using primers which include the restriction enzyme recognition site BspH \ and Hind \\\, preferably with AHCF_SspHI (SEQ. ID. No. 2) and AHCR_H / ' ndlll (SEQ. ID. No. 3), and based on the nucleotide sequence encoding SEQ. ID No. 1 of pre-ColAh polypeptide, including signal peptide;
c) digerir o referido vetor de clonagem, de preferência que compreende o local para as enzimas Nco\ e Hind\\\, um gene de resistência à canamicina e que apresente uma sequência de DNA que codifica para um conjunto de histidinas consecutivas (cauda de poli-histidinas);  c) digesting said 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);
d) clonar o referido gene da colagenase no referido vetor de clonagem;  d) cloning said collagenase gene into said cloning vector;
e) produzir o plasmídeo recombinante desejado;  e) producing the desired recombinant plasmid;
f) inserir o referido plasmídeo recombinante em células hospedeiras adequadas de E. coli, de preferência E. coli TOP10F', g) transformar o referido plasmídeo recombinante em células de expressão recombinante, de preferência utilizando células hospedeiras compatíveis com o vetor utilizado, como demostrado com E. coli BL21 (DE3) para sobreexpressar a proteína pretendida e ainda pET26b(+); f) inserting said recombinant plasmid into suitable 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 (+);
h) semear, selecionar e cultivar as referidas células recombinantes;  h) sowing, selecting and cultivating said recombinant cells;
i) recolher e purificar a colagenase recombinante activa.  i) collect and purify the active recombinant collagenase.
[0022] Uma outra realização refere-se aos iniciadores, que incluem o local de reconhecimento das enzimas de restrição BspH\ e Hind\\\, que compreendem as seguintes sequências homologas Another embodiment relates to the primers, which include the restriction enzyme recognition site BspH \ and Hind \\\, which comprise the following homologous sequences.
• SEQ. ID No. 2: G G G ATAATC ATG AAC AATCTG G GTACCAG -3';  • SEQ. ID No. 2: G G G ATAATC ATG AAC AATCTG G GTACCAG -3 ';
• SEQ. ID No. 3: TTAAGCTTGTGGGAGGAGTCGTTGGC -3'.  • SEQ. ID No. 3: TTAAGCTTGTGGGAGGAGTCGTTGGC -3 '.
[0023] Numa outra realização do referido método a amplificação pode ocorrer por reações em cadeia pela polimerase realizadas em volumes de 50 μΙ contendo 3 mM de MgCI2, 250 μΜ dNTPs, 0,5 μΜ de cada iniciador (AHCF_SspHI e AHCR_H/'ndlll), 5% DMSO (v/v), 1,5 U Taq DNA polimerase em tampão apropriado e 50-100 ng de DNA. In another embodiment of said method amplification may occur by polymerase chain reactions carried out in 50 μΙ volumes containing 3 mM MgCl 2 , 250 μΜ dNTPs, 0.5 μΜ 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.
[0024] Numa outra realização do referido método a referida amplificação pode ser realizada num termociclador, compreendendo a desnaturação inicial do DNA genómico de Aeromonas spp. à temperatura 94 - 96 °C durante 2 - 5 min; seguido de 40 ciclos de 30 s a 94 - 96 °C, 30 s a 58 °C e 3 min a 70 - 75 °C de preferência 68 ^C; extensão final de 20-30 min a 68-75 °C, de preferência 72 ^C; a temperatura ótima de atividade da Taq polimerase usada, que contudo poderá variar com o fabricante. In another embodiment of said method 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.
[0025] Numa outra realização do referido método pode utilizar-se um vetor de clonagem que permita a aplificação de uma sequência líder (péptido sinal) e uma cauda de histidinas, compatíveis com os terminais do fragmento (gene) a ligar, como demonstrado para o vetor de clonagem ser pET26b(+). [0026] Numa outra forma de realização do referido método a selecção de um clone contendo o plasmídeo recombinante pode ser feita através de cultura em meio seletivo, como demonstrado para o plasmídeo pET26b(+), em que os clones positivos são selecionados em meio suplementado com canamicina. In another embodiment of said method 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 (+). In another embodiment of said method 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.
[0027] Numa outra realização do referido método as células hospedeiras de expressão podem ser transformadas e cultivadas a 26 e 130 rpm durante 18 h. In another embodiment of said method the expression host cells may be transformed and cultured at 26 and 130 rpm for 18 h.
[0028] Numa outra forma de realização do referido método as condições de centrifugação (para obtenção do extrato) podem ser as seguintes: velocidade de 11000 xg, 4 2C; durante 25 minutos. In another embodiment of said method the centrifugation conditions (to obtain the extract) may be as follows: rate of 11000 xg, 4 ° C; for 25 minutes.
[0029] Numa outra realização do referido método a recolha da colagenase recombinante pode ser efetuada por centrifugação refrigerada ou por outro processo de concentração celular, seguida de uma diálise (4 ^c) contra tampão de ligação e filtrado com filtro de seringa (0,2μιη). In another embodiment of said method 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). 2μιη).
[0030] Numa outra forma de realização do referido método a purificação da colagenase recombinante pode ser efetuada por cromatografia de afinidade, de preferência cromatografia de afinidade com ião metálico imobilizado ou por outros processos similares. A cromatografia de afinidade poderá ser realizada de preferência numa coluna acoplada a um sistema de média pressão. In another embodiment of said method 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.
[0031] Descreve-se ainda uma colagenase isolada, obtenível a partir do processo descrito no método anterior, que inclui na sua construção genética um domínio de proteína recombinante sobre-expresso com um marcador de cauda de histidinas. Assim, a colagenase isolada obtenível a partir do processo descrito na presente invenção é capaz de digerir o colagénio em concentrações não tóxicas, muito inferiores às concentrações eficazes com outras colagenases. [0032] Numa outra realização, a referida colagenase isolada inclui na sua construção genética um domínio de proteína recombinante sobre-expresso e um marcador, cauda de histidinas. An 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. Thus, 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. In another embodiment, said isolated collagenase includes in its genetic construct a recombinant overexpressed protein domain and a marker, histidine tail.
[0033] Numa outra de realização isolada obtenível a partir do processo descrito na presente invenção, capaz de digerir o colagénio em concentrações de colagenase inferiores à toxica.  In another isolated embodiment obtainable from the process described in the present invention, capable of digesting collagen at lower than toxic collagenase concentrations.
[0034] Numa outra realização tem a característica da colagenase isolada apresentar capacidade de digestão de colagénio do tipo I, tipo II e tipo III (digestão in vitro, analisada por electroforese). In another embodiment it has the characteristic of isolated collagenase to exhibit type I, type II and type III collagen digestion (in vitro digestion, analyzed by electrophoresis).
[0035] Numa outra realização a colagenase isolada poderá ser usada como medicamento ou em medicina, de preferência no tratamento de tecidos traumatizados, nomeadamente no tratamento de feridas traumáticas, úlceras varicosas e queimaduras e também no tratamento da doença de Dupuytren, uma fibrodisplasia proliferativa da aponevrose palmar; em terapia génica ou eletro-génica; e ainda no tratamento de ciática, nomeadamente em discos intervertebrais herniados. In another embodiment 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.
[0036] É ainda descrita uma composição caracterizada por compreender ou consistir na colagenase descrita anteriormente. A referida colagenase pode ser aplicada em solução, na sua forma liofilizada, ou numa composição compreendida/constituída pela referida colagenase e um biomaterial, nomeadamente o quitosano. Further described is a 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.
[0037] Numa outra realização a referida composição poderá ser uma formulação tópica, uma formulação para administração oral ou parental ou uma formulação injectável. In another embodiment said composition may be a topical formulation, a formulation for oral or parenteral administration or an injectable formulation.
[0038] A presente invenção descreve ainda o uso da referida colagenase obtenível pelo método descrito anteriormente, em técnicas para cultura de células e tecidos animais, tais como isolamento de células endoteliais, células neuronais, cardiomiócitos, hepatócitos e células estaminais; na indústria alimentar, nomeadamente no processamento de carnes para melhoramento das suas propriedades e texturas; e na indústria dos curtumes, nomeadamente para auxiliar no processo de tingimento de couros. 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.
[0039] A presente invenção diz respeito ao processo de clonagem, expressão e purificação de uma colagenase de Aeromonas para digestão de colagénios. The present invention relates to the process of cloning, expression and purification of an Aeromonas collagenase for collagen digestion.
[0040] Um dos objetivos da presente invenção é a produção de colagenase de Aeromonas, seguindo uma metodologia baseada na recombinação genética. Numa realização preferencial, após identificação do gene, foram desenhados iniciadores visando a clonagem dirigida do gene completo que codifica para a colagenase em Aeromonas hydrophila. O gene isolado pode ser clonado num vetor de clonagem contendo uma sequência de DNA que codifica para um conjunto de cinco histidinas consecutivas (cauda de histidinas), que foi posteriormente utilizado para transformar células competentes, de forma a produzir células recombinantes, capazes de expressar a colagenase bacteriana. Após identificação e seleção dos clones positivos, as células recombinantes são induzidas para expressão de colagenase recombinante. O meio extracelular das culturas pode ser separado das células por centrifugação, dialisado e aplicado numa coluna de cromatografia com afinidade para histidinas, para purificação de colagenase recombinante ativa. One object of the present invention is the production of Aeromonas collagenase following a methodology based on genetic recombination. In a preferred embodiment, upon gene identification, 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. After identification and selection of positive clones, 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.
[0041] A presente invenção é útil para aplicação em processos onde se pretende digerir colagénios intersticiais, especificamente para o tratamento de patologias que envolvam a deposição excessiva de matriz extracelular, por exemplo como a Doença de Dupuytren. A colagenase recombinante descrita tem ainda aplicação no estabelecimento de culturas celulares primárias, para as quais é essencial o destacamento dos tecidos de origem e dissociação celular prévia à cultura in vitro. Descrição das Figuras 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
[0042] Para uma mais fácil compreensão da invenção juntam-se em anexo as figuras, as quais, representam realizações preferenciais do invento que, contudo, não pretendem, limitar o objecto da presente invenção.  For easier understanding of the invention, attached are the figures which represent preferred embodiments of the invention which, however, are not intended to limit the scope of the present invention.
Figura 1: Representação esquemática do processo de clonagem e obtenção do plasmídeo recombinante (pCUA). Vetor de clonagem digerido com as enzimas Hind\ \ \/Nco\ (a) e produto de reação em cadeia pela polimerase (PCR) digerido com as enzimas de restrição /-/ /ic/l l l/SspHI (b), gerando terminais complementares para proporcionar a ligação do gene ao vetor (c). 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).
Figura 2: Fluxograma do método para obtenção da colagenase recombinante. Figure 2: Flowchart of the method for obtaining recombinant collagenase.
Figura 3: Diagrama metodológico da expressão e purificação da colagenase recombinante. Figure 3: Methodological diagram of recombinant collagenase expression and purification.
Figura 4: Expressão da colagenase recombinante em E. coli. Sobrexpressão (SDS-PAGE) e atividade proteolítica (zimografia de gelatina) da colagenase nas células recombinantes (R) vs células não transformadas (C). A migração da proteína recombinante corresponde ao peso molecular esperado (~100 kDa) e está assinalada com uma seta. 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.
Figura 5: Purificação da ColAh recombinante por cromatografia de afinidade com ião metálico imobilizado. Figure 5: Purification of recombinant ColAh by immobilized metal ion affinity chromatography.
Figura 6: Digestão de colagénio do tipo I humano com a colagenase recombinante (ColAh). Figure 6: Digestion of human type I collagen with recombinant collagenase (ColAh).
Figura 7: Deteção da interação física entre enzima :substrato (colagenase recombinante:colagénios do tipo I) por imunodeteção. Figura 8: Estabilidade da atividade da coiagenase recombinarrte {CoiAh) ao armazenamento a várias temperaturas (-80 §C; -20 ^C; 4 s e 37 se). 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).
Figura 9: Determinação da viabilidade celular de osteoblastos em cultura expostos ao tratamento com ColAh. Figure 9: Determination of cell viability of cultured osteoblasts exposed to ColAh treatment.
Figura 10: imunodeteção de cotagénio do tipo i nas frações celulares meio extraceiuiar de osteoblastos sujeitos ao tratamento com ColAh durante um ensaio do tipo dose-resposta. figura 11: Distribuição intra e extracelular de colagérsio do tipo i em osteoblastos MC3T3 (15 dias após confluência), analisada por microscopia confocal. 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.
Descrição detalhada da invenção Detailed Description of the Invention
[0043] Como é pretendido nesta secção será festa uma exposição detalhada da invenção. Os métodos revelados sio meramente explicativos e não devem, por isso, ser interpretados como uma restrição da invenção, mas sim como suporte das reivindicações. A invenção será descrita com referência às figuras 1 a 11,  As intended in this section will be held a detailed exhibition of the invention. The disclosed methods are merely explanatory and should not therefore be construed as a restriction of the invention but as a support for the claims. The invention will be described with reference to figures 1 to 11,
[0044] A estratégia básica para aumentar a expressão da proteína ativa passa peia clonagem do gene que codifica para a coiagenase num vetor de expressão e consequente transformação no hospedeiro para expressão heteróloga, 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,
[0045] O vetor de expressão é um vetor de clonagem, construído de forma que o gene aí Inserido seja transcrito/traduzido após a sua introdução nas células hospedeiras. The expression vector is a cloning vector, constructed such that the gene inserted therein is transcribed / translated upon its introduction into host cells.
[0046] Neste trabalho foi utilizado o sistema pET para expressão da coiagenase anotada na ORF AHAJ3517 úe Âero onas hydropbita ATCC 7966T. In this work we used the pET system for expression of coiagenase annotated in ORF AHAJ3517 uero onas hydropbitita ATCC 7966 T.
13 13
FOLHA RETIFICADA (REGRA 91 ) ISA/EP [0047] Como mostra a Fig. 2, a presente invenção refere-se ao método para obtenção de colagenase recombinante que compreende as seguintes etapas: (a) isolamento de DNA genómico de Aeromonas hydrophila ATCC 7966T; (b) amplificação do gene da putativa colagenase por PCR para gerar múltiplas cópias do gene da colagenase (ORF: AHA_0517); (c) clonagem do gene da colagenase de Aeromonas no vetor de clonagem pET26b(+); (d) transformação do vetor recombinante, pCUA, em células competentes (E. coli TOP10F') para produzir um clone de partida; (e) identificação e seleção do clone de partida transformado positivamente; (f) sequenciação do inserto para confirmação da correta fase de leitura do gene no vetor; (g) purificação do plasmídeo recombinante e transformação em células de expressão E. coli BL21(DE3); (h) identificação e seleção das células recombinantes transformadas; (i) indução das células recombinantes para expressão da colagenase recombinante; (j) recolha do meio extracelular da cultura e purificação da colagenase recombinante por cromatografia de afinidade; (k) diálise contra tampão isotónico; (I) ensaio de atividade colagenolítica. RECTIFIED SHEET (RULE 91) ISA / EP As shown in Fig. 2, 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.
[0048] Assim, numa primeira fase do processo, o DNA total da estirpe Aeromonas hydrophila ATCC 7966T foi isolado a partir de culturas líquidas em meio Luria-Bertani (LB) suplementado com o antibiótico ampicilina (50 μg/ml), utilizando o sistema comercial Genomic DNA Purification Kit, seguindo as instruções do fabricante. Posteriormente, o gene que codifica para a putativa colagenase em Aeromonas foi amplificado pela PCR utilizando o seguinte par de iniciadores (sublinhada a sequência de reconhecimento das enzimas de restrição): Thus, in a first stage of the process, 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 μg / 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):
- SEQ. ID No. 2: G G G ATA ATC ATG A AC AATCTG G GTACC AG -3' - SEQ. ID No. 2: G G ATA ATC ATG A AC AATCTG G GTACC AG -3 '
- SEQ. ID No. 3TTAAGCTTGTGGGAGGAGTCGTTGGC -3'  - SEQ. ID No. 3TTAAGCTTGTGGGAGGAGTCGTTGGC -3 '
[0049] Estes iniciadores foram desenhados com base na sequência de nucleótidos codificante (AHA_0517) (SEQ. ID. No. 1) do polipeptídeo pre-ColAh de forma a incluir o péptido-sinal. As reações de PCR foram efectuadas em volumes de 50 μΙ contendo 3 mM de MgCI2, 250 μΜ dNTPs, 0,5 μΜ de cada iniciador (AHCF_SspHI e AHCR_H/'ndlll), 5% DMSO (v/v), 1,5 U Taq DNA polimerase em tampão apropriado e 50-100 ng de DNA. As reações decorreram num termociclador iCycler™ Thermal Cycler nas seguintes condições de amplificação: desnaturação inicial a 94 °C durante 5 min; 40 ciclos de 30s a 94 °C, 30s a 58 °C e 3 min a 72 °C; extensão final de 30 min a 72 °C. These 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 μΙ volumes containing 3 mM MgCl 2 , 250 μΜ dNTPs, 0.5 μΜ 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 ™ 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.
[0050] Como referido anteriormente, as sequências dos iniciadores incluem o local de reconhecimento das enzimas de restrição, BspYW e Hind\\\, (sublinhados na sequência dos iniciadores), indispensáveis para gerar os terminais adequados para posterior ligação ao vetor (Fig. 1). Neste trabalho foram utilizadas as enzimas de restrição BspH\ e Hind\\\ para digerir o produto de PCR e a Nco\ e a Hind\\\ para digerir o vetor, seguindo as instruções do fabricante (tabela 1). As noted above, 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). In this work 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).
Tabela 1 : Digestão wm wm enzim de restrição Reaçâs de digestão: Table 1: Digestion wm wm Restriction Enzyme Digestion Reactions:
* 1/10 cfo vota© final eis tampão de reação (10X)  * 1/10 cfo votes © Final Use Reaction Buffer (10X)
* SO-IQSng de DNA  * SO-IQSng DNA
* 1 IJ d© erafonuetease de restrição  * 1 IJ d © Restriction Eraphonuetease
* Água desl ada até pe fazer o volume §m\ í pi  * Water turned off until volume is set.
Incubar 2-3 horas; temperatura étímada atmdade «izimâtka Incubate 2-3 hours; optimum temperature atmidad «izimâtka
Parar rsagão a -20aC Stop ragaga at -20 to C
[0051] O plasmídeo pET26b(+) foi o vetor escolhido, por conter um gene de resistência à canamicina que permite a seleção posterior dos transformantes. Plasmid pET26b (+) was the vector of choice because it contains a kanamycin resistance gene that allows for subsequent selection of transformants.
[0052] Após digestão, foi efetuada uma precipitação (tabela 2) das reações de digestão do vetor e do inserto (produto de PCR), de modo a eliminar os sais das soluções, que poderiam inibir a reação de ligação. lateia 2; Precipitação das reações de d ges ão Following digestion, precipitation of the vector and insert digestion reactions (PCR product) was performed (Table 2) to eliminate salts of the solutions which could inhibit the binding reaction. late 2; Precipitation of Management Reactions
» Adicionar a cada reação m pi de água destonízada esiéfi e 100 pl <te isepropanoí » Add to each reaction m pi of distilled water esiéfi and 100 pl <te isepropanoí
♦ Incito a - 20¾ 1 hora ♦ Incited at - 20¾ 1 hour
* Cenírffugar 5 m nutos s 14000 r m e descarte r o sotonadanfs  * Cenírffugar 5 m nutos s 14000 r m and discard r sotonadanfs
• Deixar secar a temperatura ambiente  • Allow to dry at room temperature
♦ Ressuspender o DNA em ≠ ás água desàsniza a estéril ♦ Resuspending DNA in water disassociates sterile
[0053] A reação de ligação do fragmento ao vetor foi estabelecida numa razão molar inserto:vetor correspondente a 3:1 (tabela 3), aplicando da seguinte fórmula de cálculo: The binding reaction of the fragment to the vector was established at an insert: vector molar ratio corresponding to 3: 1 (Table 3) by applying the following formula:
Xnq de vetor x tamanho do inserto (kb) x inserto/vetor = ng de vetor tamanho do vetor (kb) Vector xnq x insert size (kb) x insert / vector = ng vector vector size (kb)
TafcaJa 3: Reação de ligação TafcaJa 3: Binding Reaction
* 100 ng DNA (vetor)  * 100 ng DNA (vector)
* 180 r¾do íns8rl©  * 180 noise ¾8rl ©
* 1≠ tampão de figas® |1¾  * 1 fig figas® cap | 1¾
* 1 U 14 DNA Igase  * 1 U 14 DNA Igase
* Água estéril a a perf azer de volume fi i 10 μ!  * Sterile water up to a volume of 10 μ!
Incubar a reacção a SC durante 14-titoras; a reacção pode se Incubate the reaction at S C for 14-theta; the reaction can be
ImaÉHlameníe utilizada ou armazenada a -2õ¾C ImaÉHlameníe used or stored at -2 ° C ¾
[0054] Tendo em conta as suas características, nomeadamente, por serem recA- e endA-, possuírem uma elevada eficiência de transformação e boa produção de plasmídeo, as células TOP10F' foram selecionadas como estirpe hospedeira para gerar um clone de partida (para manter o plasmídeo recombinante). O vetor recombinante obtido, o qual designamos por pCUA, foi utilizado numa fase inicial para transformar a estirpe E. coli TOP10F', segundo o estabelecido na tabela 4. Tabela 4: Transformação de células competentes cie £ cali Given their characteristics, namely, because they are recA- and endA-, have high transformation efficiency and good plasmid production, 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
* Dmm q&hf em gsfe ems afíqusta (50-200 pi) das células * Dmm q & hf in afferent gsfe (50-200 pi) cells
qy;í:m»-compe!sní8S- E crf TOPlOF ou BL2Í(DE¾  qy; í: m »-comp! sni8S- And crf TOPlOF or BL2Í (DE¾
* Adidortar 2-10≠ da reaçlo de ligação as células com etentes* misturando suavemente. Manter em gsfo durante 30 m ufeSc  * Add 2-10 ≠ of the binding reaction to etched cells * by gently mixing. Keep in gsfo for 30 m ufeSc
* Submete? as células a um otoque térmico: 30 se jn os a 4:2SC; 2 msfitÉDS em gelo.. * Submit? the cells at a thermal touch: 30 s and at 4: 2 S C; 2 msfitÉDS in ice ..
* AdÍdanar 2S0≠ de meto SOO. incubar úitmte titara a 3T¾ e a 2S0 rpm.  * Admit 2S0 ≠ of SOO method. incubate only at 3 ° C and 2 rpm.
* Semear 20-100 μΙ da transform ção em placas de meto LÃ  * Seed 20-100 μΙ of transformation into wool boards
suplementado com o 50pgi?ml de eaí amidoa,  supplemented with 50pgi? ml eai starch,
* Incubar as placas a 3'FC ; 16- i Steras, * Incubate plates at 3 ' FC; 16- i Steras,
[0055] A seleção dos transformantes (clones positivos) foi efetuada por PCR usando os iniciadores universais para o vetor pET (T7 Promoter Primer #69348-3, SEQ. ID. 6 - 5'-AATACGACTCACTATAGGG-3' e T7 Terminator Primer #69337-3 SEQ. ID. 7 -5- GCTAGTTATTGCTCAGCGG-3') de acordo com as indicações do fabricante. A avaliação em gel de agarose permitiu a seleção de um clone positivo (contendo o pCUA). Os clones transformados foram criopreservados a -80 ^c, em 15% glicerol (v/v). Selection of transformants (positive clones) was performed by PCR using the universal primers for the pET vector (T7 Promoter Primer # 69348-3, SEQ. ID. 6 - 5'-AATACGACTCACTATAGGG-3 'and T7 Terminator Primer # 69337-3 SEQ ID NO: 7 -5- GCTAGTTATTGCTCAGCGG-3 ') according to the manufacturer's directions. Agarose gel evaluation allowed the selection of a positive clone (containing pCUA). Transformed clones were cryopreserved at -80 ° C in 15% glycerol (v / v).
[0056] A inserção correta do fragmento clonado no pCUA foi confirmada por sequenciação do DNA plasmídico. Para isso, a extracção dos plasmídeos recombinantes foi efetuada utilizando o sistema UltraClean 6 Minute Mini Plasmid Prep Kit, para posterior sequenciação com os iniciadores T7. A sequência nucleotídica obtida (SEQ. ID. No. 4) foi confrontada com sequências depositadas na base de dados do NCBI. Os resultados de sequenciação permitiram ainda confirmar a pauta de leitura da sequência que codifica a cauda de histidinas , presente no vetor pET26b(+). Uma vez confirmada a construção pretendida para o vetor pCUA, procedeu-se à transformação no hospedeiro de expressão - E. coli BL21(DE3) e à selecção de transformantes por PCR, segundo a metodologia descrita anteriormente para seleção do clone de partida (em E. coli TOP10F'). [0057] Para expressar a colagenase recombinante foi seguida a estratégia representada na Fig. 3. Foram utilizados pré-inóculos de um transformante da estirpe E. coli BL21 (DE3) inoculado em meio LB contendo 50 μg/ml canamicina. Uma cultura (50 ml) foi incubada a 37 e 150 rpm até atingir 0,6 unidades de densidade óptica (D. O.) a 600 nm; as células foram induzidas com adição de 0,5 mM isopropil-beta-D- tiogalactopiranosídeo (I PTG). As condições de cultura após indução foram ajustadas para 26 e 130 rpm durante 18 h, no sentido de uma maior expressão de enzima ativa. A sobreexpressão da colagenase recombinante ColAh em E. coli foi avaliada por SDS-PAGE e atividade proteolítica por zimografia de gelatina (Fig. 4). Correct insertion of the cloned fragment into pCUA was confirmed by plasmid DNA sequencing. For this, extraction of the recombinant plasmids was performed using the UltraClean 6 Minute Mini Plasmid Prep Kit system for subsequent sequencing with T7 primers. The nucleotide sequence obtained (SEQ. ID. No. 4) was compared to sequences deposited in the NCBI database. The sequencing results also confirmed the reading pattern of the histidine tail coding sequence present in the pET26b (+) vector. Once the desired construct for the pCUA vector was confirmed, the E. coli BL21 (DE3) expression host was transformed and PCR transformant selected according to the methodology described above for starting clone selection (in E . coli TOP10F '). 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 µg / 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).
[0058] O meio extracelular foi recolhido após centrifugação a 11000 xg, 4 ^c, durante 25 minutos e dialisado (4^ C, 1 h) contra tampão de ligação e filtrado com filtro de seringa (0,2 μιη), antes do passo de purificação por cromatografia de afinidade. A cromatografia foi realizada numa coluna de afinidade com ião metálico imobilizado em particular HisTrap™ FF, acoplada a um sistema de média pressão em particular AKTA Basic. Assim, após equilíbrio com 10 volumes de coluna em tampão de ligação (20 mM fosfato de sódio, 0,5 M NaCI, 5 mM imidazol, pH 7,4), a amostra (10 ml) foi aplicada à coluna e eluída em 10 % (v/v) de tampão de eluição (20 mM fosfato de sódio, 0,5 M NaCI, 500 mM imidazol, pH 7,4), que corresponde a 50 mM de imidazol (Fig. 5). A fração recolhida foi analisada em zimografia de gelatina (0,1%) e por SDS-PAGE, para confirmar a sua atividade e o seu estado de pureza. A colagenase recombinante foi ainda testada em relação à sua capacidade para digerir colagénio do tipo I (Fig. 6), de acordo com o protocolo descrito por Duarte e colaboradores (DU_2005) e a sua interação física com este substrato (Fig. 7). Foi também confirmada a estabilidade ao armazenamento da sua atividade por incubação da ColAh em diferentes temperaturas (-80 9C, -20 9C, 4 5C e 37 ^C) e tempos de armazenamento (Fig. 8). Extracellular medium was collected after centrifugation at 11000 xg, 4Â ° C for 25 minutes and dialyzed (4Â ° C, 1 h) against binding buffer and filtered with syringe filter (0.2 μιη) prior to affinity chromatography purification step. Chromatography was performed on a particular HisTrap ™ 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).
FORMAS DE REALIZAÇÃO [0059] O procedimento para obtenção da solução de colagenase recombinante para digestão de colagénios será mais facilmente compreendido em contexto dos exemplos seguintes. São possíveis realizações da invenção e, por isso, não deverão ser interpretados como limitações da invenção. WAYS OF CARRYING OUT The procedure for obtaining the recombinant collagenase solution for collagen digestion will be more readily understood in the context of the following examples. Embodiments of the invention are possible and therefore should not be construed as limitations of the invention.
[0060] Uma forma de realização compreende a utilização de doses sub-tóxicas de colagenase recombinante para redução de colagénios em osteoblastos em cultura. One embodiment comprises the use of sub-toxic doses of recombinant collagenase for collagen reduction in cultured osteoblasts.
[0061] A obtenção de colagenase recombinante de Aeromonas compreende as etapas descritas anteriormente e que se encontram esquematicamente representadas na Fig. 2. Assim, após isolamento de DNA genómico de Aeromonas hydrophila ATCC 7966T, são utilizados os iniciadores AHCF_SspHI e AHCR_/-/ /idll l, especificamente concebidos para permitirem, simultaneamente, selecionar a ORF AHA_0517 e introduzir, em cada uma das extremidades do gene, as sequências de reconhecimento das respetivas enzimas de restrição. Estas sequências são especificamente reconhecidas e clivadas pelas enzimas de restrição BspH\ e Hind\ \\ (tabela 1) para gerar terminais coesivos compatíveis com os terminais do vetor pET26b(+) digerido pelas enzimas Nco\ e Hind\ \ \, como representado na Fig. 1 e seguindo as indicações para a reação de ligação (tabela 3). Após ligação do inserto ao vetor, o plasmídeo recombinante é utilizado para transformar células competentes (E. coli TOP10F') e produzir um clone de partida, seguindo o protocolo descrito na tabela 4. U ma vez selecionado como clone positivo em placas de meio LB contendo 50 μg/ml canamicina (antibiótico que permite selecionar as células transformadas pelo plasmídeo), procede-se à purificação do plasmídeo e a sequência do inserto é determinada por sequenciação automática de modo a confirmar se o gene foi clonado na fase de leitura correta, o que permite a expressão da proteína recombinante ativa contemplando a cauda de histidinas (SEQ. I D. No. 5) para posterior purificação por cromatografia de afinidade. Após confirmação, o plasmídeo recombinante é então usado para transformar células de expressão E. coli BL21(DE3) (tabela 4). À semelhança do que foi descrito anteriormente, os clones positivos são selecionados em placas contendo canamicina e a presença do inserto é confirmada por PCR utilizando os iniciadores específicos para o vetor e posterior determinação da sequência pelo método de Sanger (SA_1977). Uma vez confirmado, o clone positivo é inoculado em meio LB (suplementado com 50 μg/ml canamicina) para produzir um pré-inóculo que é usado para estabelecimento da cultura para sobrexpressão de colagenase recombinante. Como representado no esquema da Fig. 3, 1 ml de pré-inóculo é utilizado para inocular 50 ml de meio contendo canamicina, que é incubado a 37 ^c, sob agitação (150 rpm). Quando atinge a densidade ótica 0,6, medida a 600nm, a cultura é induzida com 500 mM de IPTG e incubada durante 18 h, a 26 ^c e sob agitação controlada (130 rpm). O meio extracelular da cultura é recolhido, após sedimentação das células por centrifugação (11000 xg, 25 minutos, 4 ^c), posteriormente dialisado (4 ^c) contra tampão de ligação (20 mM fosfato de sódio, 0,5 M NaCI, 5 mM imidazol, pH 7,4) sob ligeira agitação. A amostra é filtrada com filtro de seringa (0,2 μιη) e aplicada a uma coluna de afinidade com ião metálico imobilizado (HisTrap™ FF), acoplada a um sistema de média pressão, de preferência AKTA Basic. Assim, após equilíbrio com 10 volumes de coluna em tampão de ligação, a amostra (10 ml) é aplicada e a corrida é estabelecida a um fluxo constante (1 ml/min). A colagenase recombinante é eluída da coluna em 10% de tampão de eluição (20 mM fosfato de sódio, 0,5 M NaCI, 0,5 M imidazol, pH 7,4), que corresponde a 50 mM de imidazol (Fig. 5). A fração recolhida é analisada em zimografia de gelatina (0,1 %) e por SDS-PAGE, para confirmar a sua atividade e o seu estado de pureza. Obtaining recombinant Aeromonas collagenase comprises the steps described above and shown schematically in Fig. 2. Thus, after isolation of genomic DNA from Aeromonas hydrophila ATCC 7966 T , 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. These sequences are specifically recognized and cleaved by the restriction enzymes BspH \ and Hind \ \\ (table 1) to generate cohesive termini compatible with the termini of the pET26b (+) vector digested by the enzymes Nco \ and Hind \ \ \, as depicted in Fig. 1 and following the indications for the binding reaction (table 3). After binding of the insert to the vector, the recombinant plasmid is used to transform competent cells (E. coli TOP10F ') and produce a starting clone following the protocol described in table 4. Once selected as positive clone in LB medium plates containing 50 μg / ml kanamycin (antibiotic for selecting cells transformed by the plasmid), 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). As described above, 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 μg / 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). 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, 4Â ° C), then dialyzed (4Â ° 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 μιη) and applied to an immobilized metal ion affinity column (HisTrap ™ FF) coupled to a medium pressure system, preferably AKTA Basic. Thus, after equilibration with 10 column volumes in binding buffer, 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.
[0062] A amostra é quantificada utilizando o kit de quantificação de proteínas Micro BCA, segundo as indicações do fabricante. The sample is quantified using the Micro BCA Protein Quantitation Kit as directed by the manufacturer.
[0063] O objetivo desta aplicação é demonstrar o efeito da colagenase recombinante de Aeromonas na proliferação e diferenciação de células de mamíferos em cultura. Para isso é utilizada a linha celular MC3T3-E1 (ATCC), uma linha celular de osteoblastos de ratinho. Estas células encontram-se na superfície óssea e são diretamente responsáveis pela formação dos ossos. Foram particularmente selecionadas porque durante o processo de diferenciação as células precursoras dos osteoblastos alteram a sua morfologia e a expressão de marcadores funcionais, começando por segregar proteínas da matriz óssea, como colagénio do tipo I, que representa cerca de 90% da matriz orgânica e providencia a estrutura para a posterior mineralização do osso. The purpose of this application is to demonstrate the effect of recombinant Aeromonas collagenase on proliferation and differentiation of cultured mammalian cells. For this the 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.
[0064] Assim, a cultura da linha celular MC3T3-E1 foi estabelecida de acordo com o descrito (Pl_2010). Resumidamente, as células MC3T3-E1 foram mantidas a 37^C em atmosfera húmida, 5 % C02, em meio mínimo essencial contendo 2 mM glutamina numa solução salina equilibrada EBSS (Eagle's Balanced Salt Solution), suplementado com 10 % (v/v) soro bovino fetal (FBS), 1 % (v/v) de uma solução com penicilina (100 U/ml) e estreptomicina (100 mg/ml) e 3,7 g/l de NaHC03. Culturas sub-confluentes (80 - 90 % de confluência) são divididas 1:5 com 0,25 % solução de tripsina/EDTA a 5 % C02, 375C. Thus, culture of the MC3T3-E1 cell line was established as described (Pl_2010). Briefly, 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.
[0065] Para determinação da viabilidade celular dependente da dose e avaliação do efeito da ColAh na proliferação, lxlO5 células são semeadas em poços de 35 mm, em placas de cultura de 6 poços (numa densidade de lxlO4 células/cm2) e cultivadas nas condições anteriormente referidas. O meio é renovado a cada 2 dias de cultura. Após 18 dias em cultura, que corresponde ao 15^ dia após confluência, o meio de cultura é substituído por meio suplementado com 100, 150 e 300 nM de colagenase pura, e as células são incubadas durante 16 horas. Depois deste período de exposição, o meio é removido por aspiração e substituído por meio fresco contendo 10% de uma solução de resazurina 0,1 mg/ml para determinação da viabilidade celular. Após 4 h de incubação a 37 2C, em atmosfera húmida com 5 % C02, 1 ml de meio é recolhido e a densidade ótica (DO) a 570 e 600nm é medida para todas as amostras. A DO final (DOf) resulta da subtração entre a razão D057o/D06oo de cada amostra e a razão D057o/D06oo do controlo negativo (meio contendo 10% de uma solução 0,1 mg/ml resazurina). Como controlo positivo, a DOf das células incubadas em 0 nM de colagenase recombinante é considerada 100% e a viabilidade celular é calculada como percentagem relativa deste valor controlo. Como está representado no gráfico da Fig. 9, as concentrações de ColAh recombinante testadas, e obtidas pelo método da presente invenção não afetam a viabilidade das células MC3T3. For dose-dependent cell viability determination and evaluation of the effect of ColAh on proliferation, 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. After 4 h incubation at 37 ° C in a humidified atmosphere with 5% CO 2 , 1 ml of medium is collected and the optical density (OD) at 570 and 600 nm is measured for all samples. The final OD (DOF) of the subtraction result between reason 5 7 D0 / D0 6 oo of each sample and the ratio 5 7 D0 / D0 6 oo of the negative control (medium containing 10% of a solution 0.1 mg / ml resazurin ). As a positive control, the OD of cells incubated in 0 nM recombinant collagenase is considered 100% and cell viability is calculated as a relative percentage of this control value. As depicted in the graph of Fig. 9, ColAh concentrations recombinant cells tested and obtained by the method of the present invention do not affect the viability of MC3T3 cells.
[0066] No sentido de determinar o efeito da ColAh na proliferação celular dos osteoblastos em cultura, é avaliado o perfil de expressão e segregação de colagénio do tipo I, utilizando metodologias baseadas em imunodeteção. In order to determine the effect of ColAh on the cell proliferation of cultured osteoblasts, the collagen type I expression and segregation profile is evaluated using immunodetection based methodologies.
[0067] Assim, após ensaio de exposição à ColAh, o meio de cultura (1 ml) de cada condição é recolhido para microtubos contendo 10% dodecil sulfato de sódio (SDS). As respetivas células são cuidadosamente lavadas com tampão fosfato salino (PBS1) e recolhidas com uma solução 1% SDS (m/v). As amostras são fervidas durante 10 minutos em banho-maria (100 ^c) e é determinada a concentração de proteína total (BCA Protein Assay Reagent, Pierce). Seguidamente, a mesma quantidade de proteína total (60 μg), para ambos os tipos de frações celulares obtidos (células e meio de cultura), foi resolvida em gel 6,5% SDS-PAGE em tampão tris:glicina. As proteínas separadas por eletroforese foram eletrotransferidas para uma membrana de nitrocelulose para posterior imunodeteção com anticorpo primário anti-colagénio tipo I, após bloqueamento da membrana com solução 5% (m/v) leite magro em TBS-T (10 mM Tris-HCI, pH 8,0, 150 mM NaCI, 0,5% Tween) (DU_2005). A deteção da ligação proteína-anticorpo é efetuada através de quimioluminescência, com anticorpo secundário anti-IgGs de coelho, conjugado com peroxidase de rábano e recorrendo ao Kit ECL, de acordo com as instruções do fabricante e reveladas em câmara escura, em chapas auto-radiográficas. Thus, after ColAh exposure assay, culture medium (1 ml) from each condition is collected into microtubes containing 10% sodium dodecyl sulfate (SDS). The respective cells are carefully washed with phosphate buffered saline (PBS 1 ) and collected with a 1% SDS (w / v) solution. The samples are boiled for 10 minutes in a water bath (100Â ° C) and the total protein concentration (BCA Protein Assay Reagent, Pierce) is determined. Then, the same amount of total protein (60 μg) for both types of cell fractions obtained (cells and culture medium) was resolved on 6.5% SDS-PAGE gel in tris: glycine buffer. 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). 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.
[0068] Os resultados mostram, claramente, a redução de colagénio em ambas as frações - células e meio extracelular, demostrando o efeito da colagenase na expressão The results clearly show the reduction of collagen in both fractions - cells and extracellular medium, demonstrating the effect of collagenase on expression
1 PBS (lx): Dissolver 8g NaCI, 0,2g KCI, l,44g Na2HP04, 0,24g of KH2P04 em 800ml de água destilada; ajustar o pH a 7,4; perfazer 1L e esterilizar em autoclave (latm, 121^C, 20min) da matriz extracelular por parte dos osteoblastos (Fig. 10), o mesmo efeito é observado em concentrações de colagenase não tóxicas. 1 PBS (1x): Dissolve 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 , 0.24g of KH 2 PO 4 in 800ml distilled water; adjust the pH to 7.4; make 1L and autoclave (latm, 121 ^ C, 20min) extracellular matrix by osteoblasts (Fig. 10), the same effect is observed at non-toxic collagenase concentrations.
[0069] A distribuição celular do colagénio foi analisada por imunocitoquímica de acordo com Pl_2010. À semelhança com a descrição anterior as culturas foram estabelecidas em placas de 35 mm de diâmetro contendo lamelas e foram tratadas segundo as mesmas condições de exposição à colagenase purificada. No fim do teste, as lamelas foram fixadas em 4% (v/v) de formaldeído em PBS e permeabilizadas com metanol. Os locais não específicos foram bloqueados por incubação (1 h) com 3 % (m/v) albumina do soro bovina (BSA) em PBS e a imunocitoquímica foi realizada com anticorpo anti- colagénio do tipo I diluído em 3% BSA-PBS. Depois de 3 lavagens em PBS, as células fixadas foram incubadas com uma solução de anticorpo secundário ligado ao fluorocromo fluoresceína iso-tiocianato (FICT). As lamelas foram montadas em lâminas de microscopia em meio de montagem di-hidrocloreto de 4',6-diamidino-2'-fenilindol (DAPI) com reagente de Vectashield (para reduzir o fotodecaimento da fluorescência). As imagens foram adquiridas num microscópio confocal LSM 510 META com um laser Árgon (488nm) de forma a excitar o conjugado FITC e um laser Diode 405-430 para avaliar a fluorescência DAPI. The cellular distribution of collagen was analyzed by immunocytochemistry according to Pl_2010. As with the above description the cultures were set up in 35 mm diameter plates containing coverslips and were treated under the same conditions of exposure to purified collagenase. At the end of the test, 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. After 3 washes in PBS, fixed cells were incubated with a fluorochromo fluorescein isothiocyanate (FICT) bound secondary antibody solution. The 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.
[0070] As imagens adquiridas mostram que o tratamento das células secretoras de colagénio com a colagenase recombinante promove uma resposta dependente da dose, refletindo-se numa maior proliferação de osteoblastos para a maior concentração de colagenase utilizada no ensaio (Fig. 11). É também evidente a redução da deposição de colagénio do tipo I na matriz extracelular das células em cultura. 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.
[0071] Os resultados obtidos comprovam que a ColAh recombinante induz propriedades fisiologicamente relevantes nos osteoblastos, mesmo em concentrações sub-tóxicas para as células. De facto, a primeira fase da formação óssea é caracterizada por segregação massiva de colagénios, sendo o colagénio do tipo I a proteína mais abundante do osso. [0072] Em conclusão, as propriedades biológicas associadas à colagenase obtida pelo método descrito, sugerem que esta nova colagenase bacteriana tem potencial biotecnológico, nomeadamente para aplicação médica no tratamento de doenças que envolvem deposição excessiva de colagénios. The results obtained show that recombinant ColAh induces physiologically relevant properties in osteoblasts, even at sub-toxic concentrations to cells. In fact, the first phase of bone formation is characterized by massive collagen segregation, with type I collagen being the most abundant protein in bone. In conclusion, the biological properties associated with collagenase obtained by the described method suggest that this new bacterial collagenase has biotechnological potential, namely for medical application in the treatment of diseases involving excessive collagen deposition.
[0073] Numa outra forma de realização possível da presente invenção a colagenase recombinante, ColAh, à semelhança do que se conhece para outras colagenases microbianas, pode ter aplicações diversas, com a vantagem de possuir uma elevada estabilidade ao armazenamento, p.ex., a sua potencial aplicação a desbridação enzimática de tecidos, pela sua capacidade degradativa de colagénios facilitando a regeneração e cicatrização, nomeadamente no tratamento de feridas traumáticas, úlceras varicosas e queimaduras. In another possible embodiment of the present invention recombinant collagenase, ColAh, as known for other microbial collagenases, 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.
[0074] A doença de Dupuytren é uma fibrodisplasia proliferativa da aponevrose palmar, caracterizada por deposição excessiva de colagénio na matriz extracelular, uma patologia prevalente em populações como a Britânica, a Norte-Americana, e a Australiana (SY_2012). Para tratamento recorre-se frequentemente à fasciectomia cirúrgica que não limita a recorrência da patologia e que, por ser um método invasivo, conduz ao aumento da morbilidade dos doentes. Uma nova estratégia terapêutica não invasiva foi recentemente implementada e baseia-se na utilização de Colagenase de Clostridium histolyticum (CCH) Xiaflex® (com ratio 1 :11 constante para as colagenases CoIG e CoIH, respetivamente). 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). For treatment, 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).
[0075] A publicação recente dos resultados sobre o efeito da CCH Xiaflex® a nível molecular e celular (SY_2012) fundamenta e reforça a potencial aplicação da colagenase recombinante ColAh no tratamento da doença de Dupuytren, numa das formas preferidas de realização da presente invenção. [0076] Numa outra forma de realização possível da presente invenção é aplicada a utilização da colagenase recombinante em metodologias de transformação/transfecção de células tumorais em processos relacionados com a terapia genica ou eletro-génica. The recent publication of results on the molecular and cellular effect of CCH Xiaflex ® (SY_2012) underlies and reinforces the potential application of ColAh recombinant collagenase in the treatment of Dupuytren's disease in one of the preferred embodiments of the present invention. In another possible embodiment of the present invention the use of recombinant collagenase in tumor cell transformation / transfection methodologies in methods related to gene or electro-gene therapy is applied.
[0077] Numa outra forma de realização possível da presente invenção é aplicada a utilização da ColAh recombinante para o tratamento de ciática, nomeadamente em discos intervertebrais herniados. In another possible embodiment of the present invention the use of recombinant ColAh is applied for the treatment of sciatica, namely in herniated intervertebral discs.
[0078] Numa outra forma de realização possível da presente invenção é aplicada a utilização da colagenase recombinante ColAh em técnicas que envolvem culturas de células e tecidos animais (isolamento de células endoteliais, células neuronais, cardiomiócitos, hepatócitos, células estaminais, entre outras). In another possible embodiment of the present invention the use of recombinant ColAh collagenase is applied in techniques involving cell and animal tissue cultures (endothelial cell isolation, neuronal cells, cardiomyocytes, hepatocytes, stem cells, among others).
[0079] Numa outra forma de realização possível da presente invenção é aplicada a utilização da colagenase recombinante ColAh na indústria alimentar, nomeadamente no processamento de carnes para incremento das suas propriedades e texturas. In another possible embodiment of the present invention the use of recombinant ColAh collagenase is applied in the food industry, namely in the processing of meat to increase its properties and textures.
[0080] Numa outra forma de realização possível da presente invenção é aplicada a utilização da colagenase recombinante ColAh na indústria associada aos curtumes, nomeadamente para auxiliar no processo de tingimento de couros. [0080] In another possible embodiment of the present invention the use of the recombinant ColAh collagenase in the tannery industry is applied, namely to aid in the leather dyeing process.
[0081] Numa outra forma de realização possível da presente invenção é aplicada a utilização da colagenase recombinante ColAh para a extração e purificação de colagénios. [0081] In another possible embodiment of the present invention the use of recombinant ColAh collagenase for collagen extraction and purification is applied.
[0082] Lista de sequências de nucleotidos (SEQ. ID. No. 1 - 4). As sequências de nucleotidos encontram-se descritas através do código de uma letra de nucleotidos. A legenda dos nucleotidos é a que se segue (código de uma letra): A - adenina, T - timina, G - guanidina, C - citosina. SEQ. I D. No. 1 - Sequência nucleótidica do gene da colagenase (AHA_0517) de Aeromonas hydrophila ATCC 7966T List of nucleotide sequences (SEQ. ID. No. 1-4). 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)
ATG AAC AATCTG G GTACC AG GTTG CTG CTG CTG G C AG C ACCG G G CCTGTTTG CC ACTTCG G C ATG AAC AATCTG G GTACC AG GTTG CTG CTG CTG G C AG C ACCG G G CCTGTTTG CC ACTTCG G C
G CTG G CCAACTACTCCCCCTCCG ACTG G C AAC AGTATC AACTC AAAGGTG AATCC AG CCGTC AG CTG G CCAACTACTCCCCCTCCG ACTG G C AAC AGTATC AACTC AAAGGTG AATCC AG CCGTC A
GCTGGGAGATCGTCTCACCGAAGTGACCTACGAGCTGAGCGCCCGCAGCGGCGGCGCCCCCGCTGGGAGATCGTCTCACCGAAGTGACCTACGAGCTGAGCGCCCGCAGCGGCGGCGCCCCC
TATCAGCAGCTGAGGGTGTATCGCCGCTTCGACTGGAGCGATGCCAACCTGGCTGCGCTGGCTATCAGCAGCTGAGGGTGTATCGCCGCTTCGACTGGAGCGATGCCAACCTGGCTGCGCTGGC
AGAACAGCAGTGCGGCGAGCCGCAGCTCAAGATCGAGCTGGGCTGGCAGATCCGCTACCTCAGAACAGCAGTGCGGCGAGCCGCAGCTCAAGATCGAGCTGGGCTGGCAGATCCGCTACCTC
AG CTGTG AAG AG GTG GTG CCG G CTGG C AAG G CCGTTCCG G CCAG C AG CTATG ACTATG G CTAG CTGTG AAG AG GTG GTG CCG G CTGG C AAG G CCGTTCCG G CCAG C AG CTATG ACTATG G CT
ACGGCATGAAGCAGGGCCGCTGGGAGCCACTGGCAGGCACCCCGACCGCGCCGCGTCAGGACGGCATGAAGCAGGGCCGCTGGGAGCCACTGGCAGGCACCCCGACCGCGCCGCGTCAGG
ATCGTCTG CCG CTG G CG G AG CG GGTC ATCCTG G GTC AC AG CG AG C AG G AACTG G ATCG CTGATCGTCTG CCG CTG G CG G AG CG GGTC ATCCTG G GTC AC AG CG AG C AG G AACTG G ATCG CTG
CGAGCTCAACGCCGAAGGCCGCTGCGCCGAGCAGGCATGGCAGTACCAGCCGCAAAACTGGCGAGCTCAACGCCGAAGGCCGCTGCGCCGAGCAGGCATGGCAGTACCAGCCGCAAAACTGG
CAGCAGTTGAAGGTGCTGGAAGAGACCCCCAATGAACGGGATGGCCGCCTCGAGCAGATCTCAGCAGTTGAAGGTGCTGGAAGAGACCCCCAATGAACGGGATGGCCGCCTCGAGCAGATCT
TCTTCCG CCTG C AG CCC ATCGTG G GC AG CC AG G CCG CC AAG C AG GTG AG CG AG CTG CACGTCTCTTCCG CCTG C AG CCC ATCGTG G GC AG CC AG G CCG CC AAG C AG GTG AG CG AG CTG CACGTC
TGGCGCCAGTACACCTGGTTGCTGGAACAAGCCAAGACCCAGCAAGAGTGCGATGAACCCCATGGCGCCAGTACACCTGGTTGCTGGAACAAGCCAAGACCCAGCAAGAGTGCGATGAACCCCA
GACCCGTCAGGAAGGGGACAAGACCATCAGCTACCGGGTGTGCCGCCAGACCCTGCCCGCCGACCCGTCAGGAAGGGGACAAGACCATCAGCTACCGGGTGTGCCGCCAGACCCTGCCCGCC
G G C AG CG AAGTG C AG GTG GTCTTG AAG G AC ACCG G CTATC AATACCCG GTG G G G G G C AG CGG G C AG CG AAGTG C AG GTG GTCTTG AAG G AC ACCG G CTATC AATACCCG GTG G G G G G C AG CG
AATGGCAGACCCTGCCGGAAACAACCGAGTGGCAGGAGAGCCGGGTGCTCAACCACCCCATAATGGCAGACCCTGCCGGAAACAACCGAGTGGCAGGAGAGCCGGGTGCTCAACCACCCCAT
CGTGCTGGCCAGCAAGGAAGAGCAGCTCGACTGTCGCCGTGCCGACGGCCGCGCCTGTTCCCGTGCTGGCCAGCAAGGAAGAGCAGCTCGACTGTCGCCGTGCCGACGGCCGCGCCTGTTCC
GAGCCAGATCTGCCGGGCACCGAGCTGCTGGACGCCGAAGCGGCCAAGATAGTGCAGGATGGAGCCAGATCTGCCGGGCACCGAGCTGCTGGACGCCGAAGCGGCCAAGATAGTGCAGGATG
CCAGTGGCCAGCCTGCGCCGGTCTGGCAGGAGAACTATGGTCACGATGACACCAAGCTGCTGCCAGTGGCCAGCCTGCGCCGGTCTGGCAGGAGAACTATGGTCACGATGACACCAAGCTGCTG
GCCGTGTCGCGCGGCATCCAGAGCCTGCTGGCGGCCAATCAGCCGGCCCATCCAGCCATGAAGCCGTGTCGCGCGGCATCCAGAGCCTGCTGGCGGCCAATCAGCCGGCCCATCCAGCCATGAA
GCTGCTGCTGGAATACGTGCGTGCCCACAACTACCACAACTACGGCAAGCACAAGGAAGACGGCTGCTGCTGGAATACGTGCGTGCCCACAACTACCACAACTACGGCAAGCACAAGGAAGACG
GCCCGGCCGCCGCCGAGGCGCTGGCCGAGGCGTTGACGGCGCTGGGCGCCCATCCGCTGCTGCCCGGCCGCCGCCGAGGCGCTGGCCGAGGCGTTGACGGCGCTGGGCGCCCATCCGCTGCT
CTTCCCGGAGCAGGCCAGCGACGAGGTTGGTGCAGTCATGGGTGCCTGGAGCATCGCCCTGCTTCCCGGAGCAGGCCAGCGACGAGGTTGGTGCAGTCATGGGTGCCTGGAGCATCGCCCTG
C ACG GTCAGTTC AAG AG CCC AG C AGTGC AG AG CCG CTTTG G C ACCCTG CTCG G CG AGTTC AAC ACG GTCAGTTC AAG AG CCC AG C AGTGC AG AG CCG CTTTG G C ACCCTG CTCG G CG AGTTC AA
CCAG ATG CTG G CCTAC AG C ACCCG CC ATG CCAG CG AG ATC AACG GTC AG C ACG CCTGG G C AACCAG ATG CTG G CCTAC AG C ACCCG CC ATG CCAG CG AG ATC AACG GTC AG C ACG CCTGG G C AA
CCG G CCTGTTCG ATCTG CTCAACTTCCTCG ACTTCG CCAG CG ACTAC AG CG ACCCCTTCG CCAACCG G CCTGTTCG ATCTG CTCAACTTCCTCG ACTTCG CCAG CG ACTAC AG CG ACCCCTTCG CCAA
CG ACTTCCG CC AAC AG G ACG GTG AG CTG CG C AAG C AG CTG C ACG CCCTCG G C ATG AG CG AACG ACTTCCG CC AAC AG G ACG GTG AG CTG CG C AAG C AG CTG C ACG CCCTCG G C ATG AG CG AA
CTCGCGTTGTGGAAGGGACGGGATGGCGCCGATCTGTTCCTGCTCAACAACGTGCTGGATGCCTCGCGTTGTGGAAGGGACGGGATGGCGCCGATCTGTTCCTGCTCAACAACGTGCTGGATGC
CTACACCCGCCTCTACCGGGTCGCCCGCTATACCCGCCCGGACGAGCTCGACGGCTATCGCAA ACTGTTG G ATG ACTCCGTC ATCG C ACTGGTTCG CCACC AC AACCTG ATCCCCG GTG GCCAG C ACTACACCCGCCTCTACCGGGTCGCCCGCTATACCCGCCCGGACGAGCTCGACGGCTATCGCAA ACTGTTG G ATG ACTCCGTC ATCG C ACTGGTTCG CCACC AC AACCTG ATCCCCG GTG GCCAG CA
GAGTCAGGATCTGCTGGAAGACATGTCACTGACCCTCTCCACCTACTACCTGACCTACACGGAGAGTCAGGATCTGCTGGAAGACATGTCACTGACCCTCTCCACCTACTACCTGACCTACACGGA
CCGCACCAGCGAGGCCTGCATCAGCGGTGACTTCGCCGGGCTCTGCACCCCTGTCCGGGTGGCCGCACCAGCGAGGCCTGCATCAGCGGTGACTTCGCCGGGCTCTGCACCCCTGTCCGGGTGG
AGGACGTGCTGCCGTTCGAGCACACCTGCTCGCCGACCCTGCGCCTGCGGGCCCAGGACCTGAGGACGTGCTGCCGTTCGAGCACACCTGCTCGCCGACCCTGCGCCTGCGGGCCCAGGACCTG
ACCATGGATCAGGCCGAAGGGATCTGCCGTGAACTGGGTGCCGAAGAGCAGCAGTTCCATCACCATGGATCAGGCCGAAGGGATCTGCCGTGAACTGGGTGCCGAAGAGCAGCAGTTCCATC
AACAGATGGAGACCGGCTGGCAGCCGGTGGCGGACGATCACAACGAGGCGCTGGAACTGGAACAGATGGAGACCGGCTGGCAGCCGGTGGCGGACGATCACAACGAGGCGCTGGAACTGG
TG GTCTTC AACTCCTCCG CCG ACTG G AAACG CTATG G CAGTG CTCTGTTCG G CG G CGTCTCC ATG GTCTTC AACTCCTCCG CCG ACTG G AAACG CTATG G CAGTG CTCTGTTCG G CG G CGTCTCC A
CCGACAACGGCGGCATCTACCTCGAAGGGGATCCGGCTCGCCCCGGCAACCAGGCCCGCTTCCCGACAACGGCGGCATCTACCTCGAAGGGGATCCGGCTCGCCCCGGCAACCAGGCCCGCTTC
TTCGCCTACGAGGCGGAGTGGAAGCGCCCAGCGTTCCAGGTGTGGAACCTGCGCCACGAGTTTCGCCTACGAGGCGGAGTGGAAGCGCCCAGCGTTCCAGGTGTGGAACCTGCGCCACGAGT
ATGTG C ACTACCTG G ACG G CCG CTTCAACC AGTACG G C AG CTTCGG CC ACTACCCG CTC AACCATGTG C ACTACCTG G ACG G CCG CTTCAACC AGTACG G C AG CTTCGG CC ACTACCCG CTC AACC
GCACCACCTGGTGGTCGGAAGGGCTGGCGGAGTTCATCGCCCACGGCCAGTGCTTCGCCCGCGCACCACCTGGTGGTCGGAAGGGCTGGCGGAGTTCATCGCCCACGGCCAGTGCTTCGCCCGC
GGTCTGGACAACGTCGCCGGCCGTCCTGCCAGTGATCGTCCGGCCTTGGCCGACATCCTGCAGGTCTGGACAACGTCGCCGGCCGTCCTGCCAGTGATCGTCCGGCCTTGGCCGACATCCTGCA
CCTGGATTACGACAAGGGTGGCGAGATGGTCTACTCCTGGTCCTACACGGTGCACCGCTTCCTCCTGGATTACGACAAGGGTGGCGAGATGGTCTACTCCTGGTCCTACACGGTGCACCGCTTCCT
GAACGAAACCGGTCGCGGCGCCAGCTGGCTGGCCATGGCCCAGGCCCTGCGCAACCCGGATGAACGAAACCGGTCGCGGCGCCAGCTGGCTGGCCATGGCCCAGGCCCTGCGCAACCCGGAT
CAGCAGCAGGCCATGAGCACCTTCGAAGCCGAGCTGGACCAGCTGATTGCCAATGACAGCGCAGCAGCAGGCCATGAGCACCTTCGAAGCCGAGCTGGACCAGCTGATTGCCAATGACAGCG
AG G CCTACC AG CAGTG G CTCG G CCG CG AG CTG CTG CCCTGGTG GGAAGCCAACAAGGACTCAG G CCTACC AG CAGTG G CTCG G CCG CG AG CTG CTG CCCTGGTG GGAAGCCAACAAGGACTC
CGACGAGTGCAAGGCCAACGACTCCTCCCACTGA CGACGAGTGCAAGGCCAACGACTCCTCCCACTGA
SEQ. I D. No. 2 - Sequência nucleótidica do iniciador AHCF_SspHI 5'- G G G ATA ATC ATG A AC A ATCTG G GTACC AG -3' SEQ I.D. No. 2 - AHCF_SspHI 5'-G Primer Nucleotide Sequence ATC ATG ATCA A ATCG G GTACC AG -3 '
SEQ. I D. No. 3 - Sequência nucleótidica do iniciador tkHCR_Hind\ \ \ 5'- TTAAGCTTGTGGGAGGAGTCGTTGGC -3' SEQ I D. No. 3 - Primer nucleotide sequence tkHCR_Hind \ \ \ 5'- TTAAGCTTGTGGGAGGAGTCGTTGGC -3 '
SEQ. I D. No. 4 - Sequência nucleótidica obtida por sequenciação com os iniciadores universais T7 promotor primer e T7 terminator primer SEQ I D. No. 4 - Nucleotide sequence obtained by sequencing with T7 primer promoter and T7 terminator primer universal primers
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT TAACTTTAAGAAGGAGATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTG CTCCTCGCTGCCCAGCCGGCGATGGCCATGAACAATCTGGGTACCAGGTTGCTGCTGCTGGC AG C ACCGG G CCTGTTTG CC ACTTCG GCG CTG G CC AACTACTCCCCCTCCG ACTG G CAACAGTA TCAACTCAAAGGTGAATCCAGCCGTCAGCTGGGAGATCGTCTCACCGAAGTGACCTACGAGCTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTT TAACTTTAAGAAGGAGATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTG CTCCTCGCTGCCCAGCCGGCGATGGCCATGAACAATCTGGGTACCAGGTTGCTGCTGCTGGC ACCGG AG C CC G CCTGTTTG ACTTCG CTG GGC G CC G AACTACTCCCCCTCCG ACTG CAACAGTA TCAACTCAAAGGTGAATCCAGCCGTCAGCTGGGAGATCGTCTCACCGAAGTGACCTACGAGC
TGAGCGCCCGCAGCGGCGGCGCCCCCTATCAGCAGCTGAGGGTGTATCGCCGCTTCGACTGGTGAGCGCCCGCAGCGGCGGCGCCCCCTATCAGCAGCTGAGGGTGTATCGCCGCTTCGACTGG
AGCGATGCCAACCTGGCTGCGCTGGCAGAACAGCAGTGCGGCGAGCCGCAGCTCAAGATCGAGCGATGCCAACCTGGCTGCGCTGGCAGAACAGCAGTGCGGCGAGCCGCAGCTCAAGATCG
AGCTGGGCTGGCAGATCCGCTACCTCAGCTGTGAAGAGGTGGTGCCGGCTGGCAAGGCCGTAGCTGGGCTGGCAGATCCGCTACCTCAGCTGTGAAGAGGTGGTGCCGGCTGGCAAGGCCGT
TCCGGCCAG CAG CTATG ACTATG G CTACGG CATGAAG CAG GGCCG CTG GGAGCCACTG G C ATCCGGCCAG CAG CTATG ACTATG G CTACGG CATGAAG CAG GGCCG CTG GGAGCCACTG G C A
GGCACCCCGACCGCGCCGCGTCAGGATCGTCTGCCGCTGGCGGAGCGGGTCATCCTGGGTCGGCACCCCGACCGCGCCGCGTCAGGATCGTCTGCCGCTGGCGGAGCGGGTCATCCTGGGTC
ACAGCGAGCAGGAACTGGATCGCTGCGAGCTCAACGCCGAAGGCCGCTGCGCCGAGCAGGCACAGCGAGCAGGAACTGGATCGCTGCGAGCTCAACGCCGAAGGCCGCTGCGCCGAGCAGGC
ATGGCAGTACCAGCCGCAAAACTGGCAGCAGTTGAAGGTGCTGGAAGAGACCCCCAATGAAATGGCAGTACCAGCCGCAAAACTGGCAGCAGTTGAAGGTGCTGGAAGAGACCCCCAATGAA
CGGGATGGCCGCCTCGAGCAGATCTTCTTCCGCCTGCAGCCCATCGTGGGCAGCCAGGCCGCCGGGATGGCCGCCTCGAGCAGATCTTCTTCCGCCTGCAGCCCATCGTGGGCAGCCAGGCCGC
CAAG CAG GTGAGCGAG CTG C ACGTCTG G CG CC AGTAC ACCTG GTTG CTGGAACAAGCCAAGCAAG CAG GTGAGCGAG CTG C ACGTCTG G CG CC AGTAC ACCTG GTTG CTGGAACAAGCCAAG
ACCCAGCAAGAGTGCGATGAACCCCAGACCCGTCAGGAAGGGGACAAGACCATCAGCTACCACCCAGCAAGAGTGCGATGAACCCCAGACCCGTCAGGAAGGGGACAAGACCATCAGCTACC
GGGTGTGCCGCCAGACCCTGCCCGCCGGCAGCGAAGTGCAGGTGGTCTTGAAGGACACCGGGGGTGTGCCGCCAGACCCTGCCCGCCGGCAGCGAAGTGCAGGTGGTCTTGAAGGACACCGG
CTATCAATACCCGGTGGGGGGCAGCGAATGGCAGACCCTGCCGGAAACAACCGAGTGGCAGCTATCAATACCCGGTGGGGGGCAGCGAATGGCAGACCCTGCCGGAAACAACCGAGTGGCAG
GAGAGCCGGGTG CTC AACC ACCCCATCGTG CTG GCCAG CAAG GAAGAG CAG CTCG ACTGTCGAGAGCCGGGTG CTC AACC ACCCCATCGTG CTG GCCAG CAAG GAAGAG CAG CTCG ACTGTC
GCCGTGCCGACGGCCGCGCCTGTTCCGAGCCAGATCTGCCGGGCACCGAGCTGCTGGACGCGCCGTGCCGACGGCCGCGCCTGTTCCGAGCCAGATCTGCCGGGCACCGAGCTGCTGGACGC
CGAAGCGGCCAAGATAGTGCAGGATGCCAGTGGCCAGCCTGCGCCGGTCTGGCAGGAGAACCGAAGCGGCCAAGATAGTGCAGGATGCCAGTGGCCAGCCTGCGCCGGTCTGGCAGGAGAAC
TATGGTCACGATGACACCAAGCTGCTGGCCGTGTCGCGCGGCATCCAGAGCCTGCTGGCGGCTATGGTCACGATGACACCAAGCTGCTGGCCGTGTCGCGCGGCATCCAGAGCCTGCTGGCGGC
CAATCAGCCG GCCCATCCAGCCATGAAG CTG CTG CTG G AATACGTGCGTG CCC AC AACTACCCAATCAGCCG GCCCATCCAGCCATGAAG CTG CTG CTG G AATACGTGCGTG CCC AC AACTACC
ACAACTACGGCAAGCACAAGGAAGACGGCCCGGCCGCCGCCGAGGCGCTGGCCGAGGCGTACAACTACGGCAAGCACAAGGAAGACGGCCCGGCCGCCGCCGAGGCGCTGGCCGAGGCGT
TGACGGCGCTGGGCGCCCATCCGCTGCTCTTCCCGGAGCAGGCCAGCGACGAGGTTGGTGCTGACGGCGCTGGGCGCCCATCCGCTGCTCTTCCCGGAGCAGGCCAGCGACGAGGTTGGTGC
AGTCATG G GTGCCTGGAG C ATCG CCCTG C ACG GTC AGTTC AAG AGCCC AG CAGTG CAGAGCCAGTCATG G GTGCCTGGAG C ATCG CCCTG C ACG GTC AGTTC AAG AGCCC AG CAGTG CAGAGCC
G CTTTG G CACCCTG CTCG G CG AGTTCAACC AG ATG CTG GCCTACAG CACCCGCCATGCCAG CG CTTTG G CACCCTG CTCG G CG AGTTCAACC AG ATG CTG GCCTACAG CACCCGCCATGCCAG C
GAGATCAACGGTCAGCACGCCTGGGCAACCGGCCTGTTCGATCTGCTCAACTTCCTCGACTTCGAGATCAACGGTCAGCACGCCTGGGCAACCGGCCTGTTCGATCTGCTCAACTTCCTCGACTTC
GCCAGCGACTACAGCGACCCCTTCGCCAACGACTTCCGCCAACAGGACGGTGAGCTGCGCAAGCCAGCGACTACAGCGACCCCTTCGCCAACGACTTCCGCCAACAGGACGGTGAGCTGCGCAA
GCAGCTGCACGCCCTCGGCATGAGCGAACTCGCGTTGTGGAAGGGACGGGATGGCGCCGATGCAGCTGCACGCCCTCGGCATGAGCGAACTCGCGTTGTGGAAGGGACGGGATGGCGCCGAT
CTGTTCCTGCTC AAC AACGTG CTG G ATGCCTAC ACCCG CCTCTACCG G GTCG CCCG CTATACCCCTGTTCCTGCTC AAC AACGTG CTG G ATGCCTAC ACCCG CCTCTACCG G GTCG CCCG CTATACCC
G CCCG G ACG AG CTCG ACG G CTATCG CAAACTGTTG G ATG ACTCCGTC ATCG CACTG GTTCG CG CCCG G ACG AG CTCG ACG G CTATCG CAAACTGTTG G ATG ACTCCGTC ATCG CACTG GTTCG C
CACCACAACCTGATCCCCGGTGGCCAGCAGAGTCAGGATCTGCTGGAAGACATGTCACTGACCACCACAACCTGATCCCCGGTGGCCAGCAGAGTCAGGATCTGCTGGAAGACATGTCACTGAC
CCTCTCCACCTACTACCTGACCTACACGGACCGCACCAGCGAGGCCTGCATCAGCGGTGACTTCCTCTCCACCTACTACCTGACCTACACGGACCGCACCAGCGAGGCCTGCATCAGCGGTGACTT
CGCCGGGCTCTGCACCCCTGTCCGGGTGGAGGACGTGCTGCCGTTCGAGCACACCTGCTCGC CGACCCTGCGCCTGCGGGCCCAGGACCTGACCATGGATCAGGCCGAAGGGATCTGCCGTGA ACTGGGTGCCGAAGAGCAGCAGTTCGCCGGGCTCTGCACCCCTGTCCGGGTGGAGGACGTGCTGCCGTTCGAGCACACCTGCTCGC CGACCCTGCGCCTGCGGGCCCAGGACCTGACCATGGATCAGGCCGAAGGGATCTGCCGTGA ACTGGGTGCCGAAGAGCAGCAGTT
CCATCAACAGATGGAGACCGGCTGGCAGCCGGTGGCGGACGATCACAACGAGGCGCTGGAACCATCAACAGATGGAGACCGGCTGGCAGCCGGTGGCGGACGATCACAACGAGGCGCTGGAA
CTGGTG GTCTTC AACTCCTCCG CCG ACTG G AAACG CTATG G CAGTG CTCTGTTCG G CG G CGTCCTGGTG GTCTTC AACTCCTCCG CCG ACTG G AAACG CTATG G CAGTG CTCTGTTCG G CG G CGTC
TCCACCGACAACGGCGGCATCTACCTCGAAGGGGATCCGGCTCGCCCCGGCAACCAGGCCCGTCCACCGACAACGGCGGCATCTACCTCGAAGGGGATCCGGCTCGCCCCGGCAACCAGGCCCG
CTTCTTCGCCTACGAGGCGGAGTGGAAGCGCCCAGCGTTCCAGGTGTGGAACCTGCGCCACGCTTCTTCGCCTACGAGGCGGAGTGGAAGCGCCCAGCGTTCCAGGTGTGGAACCTGCGCCACG
AGTATGTG CACTACCTG G ACG G CCG CTTCAACC AGTACG G CAG CTTCGG CC ACTACCCG CTC AAGTATGTG CACTACCTG G ACG G CCG CTTCAACC AGTATG G CAG CTTCGG CC ACTACCCG CTC A
ACCGCACCACCTGGTGGTCGGAAGGGCTGGCGGAGTTCATCGCCCACGGCCAGTGCTTCGCCACCGCACCACCTGGTGGTCGGAAGGGCTGGCGGAGTTCATCGCCCACGGCCAGTGCTTCGCC
CGCGGTCTGGACAACGTCGCCGGCCGTCCTGCCAGTGATCGTCCGGCCTTGGCCGACATCCTCGCGGTCTGGACAACGTCGCCGGCCGTCCTGCCAGTGATCGTCCGGCCTTGGCCGACATCCT
G C ACCTGG ATTACG AC AAG GGTG G CG AG ATG GTCTACTCCTG GTCCTAC ACG GTG C ACCG CTG C ACCTGG ATTACG AC AAG GGTG G CG AG ATG GTCTACTCCTG GTCCTAC ACG GTG C ACCG CT
TCCTGAACGAAACCGGTCGCGGCGCCAGCTGGCTGGCCATGGCCCAGGCCCTGCGCAACCCTCCTGAACGAAACCGGTCGCGGCGCCAGCTGGCTGGCCATGGCCCAGGCCCTGCGCAACCC
GGATCAGCAGCAGGCCATGAGCACCTTCGAAGCCGAGCTGGACCAGCTGATTGCCAATGACAGGATCAGCAGCAGGCCATGAGCACCTTCGAAGCCGAGCTGGACCAGCTGATTGCCAATGACA
GCGAGGCCTACCAGCAGTGGCTCGGCCGCGAGCTGCTGCCCTGGTGGGAAGCCAACAAGGAGCGAGGCCTACCAGCAGTGGCTCGGCCGCGAGCTGCTGCCCTGGTGGGAAGCCAACAAGGA
CTCCGACGAGTGCAAGGCCAACGACTCCTCCCACAAGCTTGCGGCCGCACTCGAGCACCACCCTCCGACGAGTGCAAGGCCAACGACTCCTCCCACAAGCTTGCGGCCGCACTCGAGCACCACC
ACCACCACCTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACACCACCACCTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCAC
CGCTGAGCAATAACTAGC CGCTGAGCAATAACTAGC
T7 Promoter Primer #69348-3 T7 Promoter Primer # 69348-3
SEQ. I D. No. 6 - 5'-AATACGACTCACTATAGGG-3' ;  SEQ I.D. No. 6 - 5'-AATACGACTCACTATAGGG-3 ';
T7 Terminator Primer #69337-3 T7 Terminator Primer # 69337-3
SEQ. I D. No. 7 - 5'-G CTAGTTATTG CTC AG CG G -3' .  SEQ I.D. No. 5 - 5'-G CTAGTTATTG CTC AG CG G -3 '.
T7 direto: SEQ. I D. No. 8- 5'- TAATACGACTCACTATAGGG - 3'; T7 straight: SEQ. I.D. No. 8- 5'-TAATACGACTCACTATAGGG - 3 ';
T7 Reverso: SEQ. I D. No. 7 - 5'- G CTAGTTATTG CTC AG CG G - 3'; Sequência líder:  T7 Reverse: SEQ. I.D. No. 7 - 5'-G CTAGTTATTG CTC AG CG G - 3 '; Leader Sequence:
sublinhado; Região codificante: negrito underlined; Coding Region: Bold
[0083] Sequência da proteína recombinante ativa da colagenase (colAh) de Aeromonas hydrophila ATCC 7966T (SEQ. I D. No. 5) sobreexpressa e purificada nesta invenção. A sequência de proteína recombinante ativa encontra-se descrita através do código de uma letra de aminoácidos. A legenda dos aminoácidos é a que se segue (código de uma letra): Histidina - H, Arginina - R, Lisina - K, Isoleucina - I, Fenilalanina - F, Leucina - L, Triptofano - W, Alanina - A, Metionina - M, Prolina - P, Valina - V, Cisteína - C, Asparagina - N, Glicina - G, Serina - S, Glutamina - Q, Tirosina - Y, Treonina - T, Ácido aspártico - D, Ácido glutâmico - E. Sequence of Aeromonas hydrophila ATCC 7966 T recombinant collagenase (colAh) active protein (SEQ. I D. No. 5) overexpressed and purified in this invention. 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.
SEQ. I D. No. 5 -SEQ I D. No. 5 -
MNNLGTRLLLLAAPGLFATSALANYSPSDWQQYQLKGESSRQLGDRLTEVTYELSARSGGAPYQQMNNLGTRLLLLAAPGLFATSALANYSPSDWQQYQLKGESSRQLGDRLTEVTYELSARSGGAPYQQ
LRVYRRFDWSDAN LAALAEQQCGEPQLKI ELGWQI RYLSCEEVVPAGKAVPASSYDYGYGMKQGLRVYRRFDWSDAN LAALAEQQCGEPQLKI ELGWQI RYLSCEEVVPAGKAVPASSYDYGYGMKQG
RWEPLAGTPTAPRQDRLPLAERVI LGHSEQELDRCELNAEGRCAEQAWQYQPQNWQQLKVLEERWEPLAGTPTAPRQDRLPLAERVI LGHSEQELDRCELNAEGRCAEQAWQYQPQNWQQLKVLEE
TPNERDGRLEQIFFRLQPIVGSQAAKQVSELHVWRQYTWLLEQAKTQQECDEPQTRQEGDKTISYTPNERDGRLEQIFFRLQPIVGSQAAKQVSELHVWRQYTWLLEQAKTQQECDEPQTRQEGDKTISY
RVCRQTLPAGSEVQVVLKDTGYQYPVGGSEWQTLPETTEWQESRVLNHPIVLASKEEQLDCRRADRVCRQTLPAGSEVQVVLKDTGYQYPVGGSEWQTLPETTEWQESRVLNHPIVLASKEEQLDCRRAD
GRACSEPDLPGTELLDAEAAKIVQDASGQPAPVWQENYGHDDTKLLAVSRGIQSLLAANQPAHPGRACSEPDLPGTELLDAEAAKIVQDASGQPAPVWQENYGHDDTKLLAVSRGIQSLLAANQPAHP
AMKLLLEYVRAHNYHNYGKHKEDGPAAAEALAEALTALGAHPLLFPEQASDEVGAVMGAWSIALAMKLLLEYVRAHNYHNYGKHKEDGPAAAEALAEALTALGAHPLLFPEQASDEVGAVMGAWSIAL
HGQFKSPAVQSRFGTLLGEFNQMLAYSTRHASEI NGQHAWATGLFDLLNFLDFASDYSDPFANDFHGQFKSPAVQSRFGTLLGEFNQMLAYSTRHASEI NGQHAWATGLFDLLNFLDFASDYSDPFANDF
RQQDGELRKQLHALGMSELALWKGRDGADLFLLNNVLDAYTRLYRVARYTRPDELDGYRKLLDDSRQQDGELRKQLHALGMSELALWKGRDGADLFLLNNVLDAYTRLYRVARYTRPDELDGYRKLLDDS
VIALVRHHNLI PGGQQSQDLLEDMSLTLSTYYLTYTDRTSEACISGDFAGLCTPVRVEDVLPFEHTCSVIALVRHHNLI PGGQQSQDLLEDMSLTLSTYYLTYTDRTSEACISGDFAGLCTPVRVEDVLPFEHTCS
PTLRLRAQDLTMDQAEGICRELGAEEQQFHQQMETGWQPVADDHNEALELVVFNSSADWKRYPTLRLRAQDLTMDQAEGICRELGAEEQQFHQQMETGWQPVADDHNEALELVVFNSSADWKRY
GSALFGGVSTDNGGIYLEGDPARPGNQARFFAYEAEWKRPAFQVWNLRHEYVHYLDGRFNQYGGSALFGGVSTDNGGIYLEGDPARPGNQARFFAYEAEWKRPAFQVWNLRHEYVHYLDGRFNQYG
SFGHYPLNRTTWWSEGLAEFIAHGQCFARGLDNVAGRPASDRPALADI LHLDYDKGGEMVYSWSSFGHYPLNRTTWWSEGLAEFIAHGQCFARGLDNVAGRPASDRPALADI LHLDYDKGGEMVYSWS
YTVHRFLNETGRGASWLAMAQALRNPDQQQAMSTFEAELDQLIANDSEAYQQWLGRELLPWWYTVHRFLNETGRGASWLAMAQALRNPDQQQAMSTFEAELDQLIANDSEAYQQWLGRELLPWW
EANKDSDECKANDSSHKLAAALEHHHHH . EANKDSDECKANDSSHKLAAALEHHHHH.
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Baumann P, Baumann L, Bang SS, Woolkalis MJ (1980) Reevaluation of the taxonomy of Vibrio, Beneckea, and Photobacterium : abolition of the genus Beneckea. Curr. Microbiol. 4:127-132.  Baumann P, Baumann L, Bang SS, Woolkalis MJ (1980) Reevaluation of the taxonomy of Vibrio, Beneckea, and Photobacterium: Abolition of the Beneckea genus. Curr. Microbiol. 4: 127-132.
CE 2012 Cemazar M, Golzio M, Sersa G, Escoffre JM, Coer A, Vidic S, Teissie J (2012). Hyaluronidase and collagenase increase the transfection efficiency of gene electrotransfer in various murine tumors. Hum Gene Ther. 23(l):128-37. CE 2012 Cemazar M, Golzio M, Sersa G, Escoffre JM, Coer A, Vidic S, Teissie J (2012). Hyaluronidase and collagenase increase the transfection efficiency of electrotransfer gene in various murine tumors. Hum Gene Ther. 23 (1): 128-37.
DU_2005 DU_2005
Duarte AS, Pereira AO, Cabrita AM, Moir AJ, Pires EM, Barros MM (2005). The characterisation of the collagenolytic activity of cardosin A demonstrates its potential application for extracellular matrix degradative processes. Curr Drug Discov Technol. 2(l):37-44.  Duarte AS, Pereira AO, Goat AM, Moir AJ, Pires EM, Barros MM (2005). The characterization of the collagenolytic activity of cardosin A demonstrates its potential application for extracellular matrix degradative processes. Curr Drug Discov Technol. 2 (1): 37-44.
EL_2008 EL_2008
Eliana Cavaleiro, Tese de Mestrado, Universidade de Aveiro (2008) http://biblioteca.sinbad.ua.pt/teses/2009000353  Eliana Cavaleiro, Master Thesis, University of Aveiro (2008) http://bibl libraries.sinbad.ua.pt/theses/2009000353
HA_2008  HA_2008
Han HJ, Taki T, Kondo H, Hirono I, Aoki T (2008) Pathogenic potential of a collagenase gene from Aeromonas veronii. Can J Microbiol. 54(1):1-10.  Han HJ, Taki T, Kondo H, Hiron I, Aoki T (2008) Pathogenic potential of a collagenase gene from Aeromonas veronii. Can J Microbiol. 54 (1): 1-10.
KA_2008  KA_2008
Kanth S, Venba R, Madhan B, Chandrababu NK, Sadulla S (2008). Studies on the influence of bacterial collagenase in leather dyeing. Dyes and Pigments, 76: 338-447 MO_1985  Kanth S, Venba R, Madhan B, Chandrababu NK, Sadulla S (2008). Studies on the influence of bacterial collagenase in leather dyeing. Dyes and Pigments, 76: 338-447 MO_1985
Moore W, Cato E, Moore LV (1985) Index of the bacterial and yeast nomenclatural changes published in the International Journal of Systematic Bacteriology since the 1980 Approved Lists of bacterial names (1 January 1980 to 1 January 1985). Int. J. Syst. Bacteriol. 35:382-407.  Moore W, Cato E, Moore LV (1985) Index of the bacterial and nomenclatural yeast changes published in the International Journal of Systematic Bacteriology since the 1980 Approved Lists of bacterial names (1 January 1980 to 1 January 1985). Int. J. Syst. Bacteriol. 35: 382-407.
MO_2008 MO_2008
Mostafaie A, Bidmeshkipour A, Shirvani Z, et al. (2008) Kiwifruit actinidin: A proper new collagenase for isolation of cells from different tissues. Appl Biochem Biotech 144 (2): 123-131.  Mostafaie A, Bidmeshkipour A, Shirvani Z, et al. (2008) Kiwifruit actinidin: A proper new collagenase for isolation of cells from different tissues. Appl Biochem Biotech 144 (2): 123-131.
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Pina S, Vieira SI, Rego P, Torres PM, da Cruz e Silva OA, da Cruz e Silva EF, Ferreira JM. (2010) Biological responses of brushite-forming Zn- and ZnSr- substituted beta- tricalcium phosphate bone cements. Eur Cell Mater. 20:162-77. SA_1977 Pina S, Vieira SI, Rego P, PM Torres, Cruz e Silva OA, Cruz e Silva EF, Ferreira JM. (2010) Biological responses of brushite-forming Zn- and ZnSr-substituted beta-tricalcium phosphate bone cements. Eur Cell Mater. 20: 162-77. SA_1977
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Suphatharaprateep W, Cheirsilp B, Jongjareonrak A. (2011) Production and properties of two collagenases from bactéria and their application for collagen extraction. New Biotechnology. 28(6): 649-655.  Suphatharaprateep W, Cheirsilp B, Jongjareonrak A. (2011) Production and properties of two collagenases from bacteria and their application for collagen extraction. New Biotechnology. 28 (6): 649-655.
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Syed F, Thomas AN, Singh S, Kolluru V, Emeigh Hart SG, Bayat A. (2012) In vitro study of novel collagenase (XIAFLEX®) on Dupuytren's disease fibroblasts displays unique drug related properties. PLoS One. 7(2):e31430. Syed F, Thomas AN, Singh S, Kolluru V, Emeigh Hart SG, Bayat A. (2012) In vitro study of novel collagenase (XIAFLEX ® ) on Dupuytren's disease fibroblasts displays unique drug related properties. PLoS One. 7 (2): e31430.
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Wu Z, Wei LX, Li J, Wang Y, Ni D, Yang P, Zhang Y (2009). Percutaneous treatment of non-contained lumbar disc herniation by injection of oxygen-ozone combined with collagenase. Eur J Radiol. 72(3):499-504.  Wu Z, Wei LX, Li J, Wang Y, Ni D, Yang P, Zhang Y (2009). Percutaneous treatment of non-contained lumbar disc herniation by injection of oxygen-ozone combined with collagenase. Eur J Radiol. 72 (3): 499-504.
[0084] As realizações preferenciais acima descritas são combináveis entre si. As seguintes reivindicações definem adicionalmente realizações preferenciais da presente invenção. The preferred embodiments described above are combinable with each other. The following claims further define preferred embodiments of the present invention.
[0085] A presente invenção não é, naturalmente, de modo algum restrita às realizações descritas neste documento e uma pessoa com conhecimentos médios da área poderá prever muitas possibilidades de modificação da mesma e de substituições de características técnicas por outras equivalentes, dependendo dos requisitos de cada situação, tal como definido nas reivindicações anexas. The present invention is, of course, not in any way restricted to the embodiments described herein and a person of ordinary skill in the art may envisage many possibilities for modification thereof and substitution of technical characteristics for other equivalents, depending on the requirements of the invention. each situation as defined in the appended claims.
7 de Novembro de 2014 November 7, 2014

Claims

REIVINDICAÇÕES
Método para obtenção e purificação de colagenase recombinante ativa de Aeromonas compreendendoos seguintes passos: Method for obtaining and purifying active recombinant collagenase from Aeromonas comprising the following steps:
isolar o DNA genómico de uma cultura de Aeromonas; isolate genomic DNA from an Aeromonas culture;
amplificar uma sequência com um grau de homologia de pelo menos 95 % com a SEQ. I D. N.s 1; amplify a sequence with a degree of homology of at least 95% with SEQ. I D. N.s 1;
integrar, num vector de expressão induzível a sequência codificante para uma proteína de fusão, em que a referida sequência codificante codifica para um conjunto de histidinas consecutivas; integrating into an inducible expression vector the coding sequence for a fusion protein, said coding sequence coding for a set of consecutive histidines;
produzir e seleccionar o plasmídeo recombinante desejado; producing and selecting the desired recombinant plasmid;
selecionar e cultivar as referidas células recombinantes e induzir a expressão de colagenase recombinante com indutor apropriado; selecting and cultivating said recombinant cells and inducing the expression of recombinant collagenase with an appropriate inducer;
recolher e purificar a colagenese recombinante. collect and purify recombinant collagenase.
Método para obtenção e purificação de colagenase recombinante ativa de Aeromonas compreendendoos seguintes passos: Method for obtaining and purifying active recombinant collagenase from Aeromonas comprising the following steps:
a) isolar o DNA genómico de uma cultura de Aeromonas; a) isolate genomic DNA from an Aeromonas culture;
b) amplificar o gene da putativa colagenase da referida cultura com uma sequência com um grau de homologia de pelo menos 95 % com a SEQ. I D. N. b) amplify the putative collagenase gene from said culture with a sequence with a degree of homology of at least 95% with SEQ. I D.N.
2 1 - AHA_0517 utilizando iniciadores que incluem o local de reconhecimento das enzimas de restrição BspH\ e Hind\ \ \; 2 1 - AHA_0517 using primers that include the recognition site for the restriction enzymes BspH\ and Hind\ \ \;
c) digerir um vetor de clonagem que compreende o local para as enzimas Nco\ e Hind\ \ \; c) digest a cloning vector that comprises the site for the Nco\ and Hind\ \ \ enzymes;
d) clonar o referido gene da colagenase no referido vetor de clonagem; e) produzir e seleccionar o plasmídeo recombinante desejado; d) cloning said collagenase gene into said cloning vector; e) producing and selecting the desired recombinant plasmid;
f) inserir o referido plasmídeo recombinante em células hospedeiras adequadas, de preferência E. coli, nomeadamente E. coli TOP10F' para transformar e selecionar vetores recombinantes; g) transformar o referido plasmídeo recombinante em células de expressão recombinante, de preferência E. coli, nomeadamente E. coli BL21 (DE3); h) selecionar e cultivar as referidas células de expressão recombinantes e induzir a expressão de colagenase recombinante; f) inserting said recombinant plasmid into suitable host cells, preferably E. coli, namely E. coli TOP10F' to transform and select recombinant vectors; g) transforming said recombinant plasmid into recombinant expression cells, preferably E. coli, namely E. coli BL21 (DE3); h) selecting and cultivating said recombinant expression cells and inducing expression of recombinant collagenase;
i) recolher e purificar a colagenase recombinante. i) collect and purify the recombinant collagenase.
3. Método de acordo com as reivindicações anteriores em que as referidas Aeromonas são microrgaismos da espécie Aeromonas hydrophila ATCC 7966T. 3. Method according to previous claims wherein said Aeromonas are microorganisms of the species Aeromonas hydrophila ATCC 7966 T.
4. Método de acordo com qualquer uma das reivindicações anteriores, em que os referidos iniciadores compreendem o local de reconhecimento das enzimas de restrição SEQ. ID No. 2 ou SEQ. ID No. 3 e terem ainda como base a sequência de nucleotidos codificante SEQ. ID No. 1 do polipeptídeo pre-ColAh, incluindo o péptido-sinal. 4. Method according to any one of the preceding claims, wherein said primers comprise the SEQ restriction enzyme recognition site. ID No. 2 or SEQ. ID No. 3 and are also based on the coding nucleotide sequence SEQ. ID No. 1 of the pre-ColAh polypeptide, including the signal peptide.
5. Método de acordo com qualquer uma das reivindicações anteriores, em que os referidos iniciadores compreendem as seguintes sequências homólogas: 5. Method according to any one of the preceding claims, wherein said primers comprise the following homologous sequences:
SEQ. ID No. 2, SEQ. ID No. 3. SEQ. ID No. 2, SEQ. ID No. 3.
6. Método de acordo com qualquer uma das reivindicações anteriores, em que a referida amplificação ocorre por reações em cadeia pela polimerase. 6. Method according to any one of the preceding claims, wherein said amplification occurs by polymerase chain reactions.
7. Método de acordo com a reivindicação anterior, em que as referidas reações em cadeia pela polimerase são realizadas em volumes de 50 μΙ contendo 3 mM de MgCI2, 250 μΜ dNTPs, 0,5 μΜ dos referidos iniciadores - SEQ. ID No. 2, SEQ. ID No. 3 -, 5% DMSO (v/v), 1,5 U Taq DNA polimerase em tampão apropriado e 50-100 ng de DNA. 7. Method according to the previous claim, wherein said polymerase chain reactions are carried out in volumes of 50 μΙ containing 3 mM MgCl 2 , 250 μΜ dNTPs, 0.5 μΜ of said primers - SEQ. ID No. 2, SEQ. ID No. 3 -, 5% DMSO (v/v), 1.5 U Taq DNA polymerase in appropriate buffer and 50-100 ng DNA.
8. Método de acordo com qualquer uma das reivindicações anteriores, em que a referida amplificação é realizada num termociclador, compreendendo a desnaturação inicial do DNA genómico de Aeromonos spp. à temperatura 94 - 96 °C durante 2 - 5 min; seguido de 40 ciclos de 30 s a 94 - 96 °C, 30 s a 58 °C e 3 min a 70 - 75 °C de preferência 68 ^C; extensão final de 20-30 min a 68-75 °C, de preferência 72 ^c. 8. Method according to any one of the preceding claims, wherein said amplification is carried out in a thermocycler, comprising initial denaturation of the genomic DNA of Aeromonos spp. at temperature 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.
9. Método de acordo com as reivindicações anteriores, em que o vetor de clonagem é pET26b(+). 9. Method according to previous claims, wherein the cloning vector is pET26b(+).
10. Método de acordo com qualquer uma das reivindicações anteriores, em que as células hospedeiras para amplificação do vector recombinante são células de E. coli, de preferência E. coli TOP10F'. 10. Method according to any one of the preceding claims, wherein the host cells for amplification of the recombinant vector are E. coli cells, preferably E. coli TOP10F'.
11. Método de acordo com qualquer uma das reivindicações anteriores, em que as células hospedeiras para a sobreexpressão da proteína recombinante são células de E. coli, de preferência E. coli BL21 (DE3). 11. Method according to any one of the preceding claims, wherein the host cells for overexpression of the recombinant protein are E. coli cells, preferably E. coli BL21 (DE3).
12. Método de acordo com a reivindicação anterior, em que as referidas células hospedeiras são cultivadas a 25 - 37 ^c, e 100-150 rpm durante 16-18 h, de preferência 26 ^C, 130^0, 130 rpm. 12. A method according to the preceding claim, wherein said host cells are cultured at 25 - 37 °C, and 100-150 rpm for 16-18 h, preferably 26 °C, 130 °C, 130 rpm.
13. Método de acordo com as reivindicações anteriores, em que a seleção de um clone do passo d) é feita através de cultura em meio seletivo, em que os clones positivos são selecionados em meio suplementado com canamicina. 13. Method according to the previous claims, in which the selection of a clone from step d) is done through culture in selective medium, in which positive clones are selected in medium supplemented with kanamycin.
14. Método de acordo com as reivindicações anteriores, em que a recolha da colagenase recombinante é por centrifugação refrigerada, seguida de diálise do sobrenadante a 4 contra tampão de ligação e posterior filtração. 14. Method according to the previous claims, in which the recombinant collagenase is collected by refrigerated centrifugation, followed by dialysis of the supernatant at 4°C against binding buffer and subsequent filtration.
Método de acordo com as reivindicações anteriores, em que a referida purificação da colagenase recombinante é por cromatografia de afinidade, de preferência com ião metálico imobilizado. Method according to the previous claims, wherein said purification of the recombinant collagenase is by affinity chromatography, preferably with immobilized metal ion.
Colagenase isolada obtenível a partir do processo descrito em qualquer uma reivindicações 1 - 15 para a digestão de colagénio, em que o colagénio é digerido pela colagenase com concentrações de colagenase inferiores à concentração tóxica da colagenase para as células. Isolated collagenase obtainable from the process described in any one of claims 1 - 15 for the digestion of collagen, wherein the collagen is digested by collagenase with concentrations of collagenase lower than the concentration toxic to the cells of the collagenase.
Colagenase de acordo com a reivindicação 16 capaz de digerir colagénio do tipo I, tipo II, tipo III ou suas misturas. Collagenase according to claim 16 capable of digesting type I, type II, type III collagen or mixtures thereof.
Colagenase isolada de acordo com a reivindicação 16 para uso como medicamento ou em medicina. Isolated collagenase according to claim 16 for use as a medicament or in medicine.
Colagenase isolada de acordo com a reivindicação 15 para o uso no tratamento de tecidos traumatizados, nomeadamente no tratamento de feridas traumáticas, úlceras varicosas e queimaduras ou também no tratamento outras patologias associadas à excessiva deposição de colagénios como por exemplo a doença de Dupuytren. Isolated collagenase according to claim 15 for use in the treatment of traumatized tissues, in particular in the treatment of traumatic wounds, varicose ulcers and burns or also in the treatment of other pathologies associated with excessive collagen deposition, such as Dupuytren's disease.
Colagenase isolada de acordo com a reivindicação 16 para o uso em terapia génica ou eletro-génica. Isolated collagenase according to claim 16 for use in gene or electrogenic therapy.
Colagenase isolada acordo com a reivindicação 16 para uso no tratamento de ciática, nomeadamente em discos intervertebrais herniados. Isolated collagenase according to claim 16 for use in the treatment of sciatica, particularly in herniated intervertebral discs.
Composição caracterizada por compreender a colagenase descrita na reivindicação 16. Composition characterized by comprising the collagenase described in claim 16.
Composição de acordo com a reivindicação anterior em que a colagenase é liofilizada. Composition according to the previous claim in which collagenase is lyophilized.
24. Composição de acordo com as reivindicações 22-23 compreendo ainda um biomaterial, em particular quitosano. 24. Composition according to claims 22-23 further comprising a biomaterial, in particular chitosan.
25. Composição de acordo com as reivindicações 22-24 em que a composição é uma formulação tópica, uma formulação por administração oral ou parental ou uma formulação injectável. 25. A composition according to claims 22-24 wherein the composition is a topical formulation, a formulation for oral or parental administration or an injectable formulation.
26. Uso da colagenase recombinante descrita nas reivindicações 16-21 ou obtida pelo método descrito de acordo com as reivindicações 1-15 em técnicas para cultura de células e tecidos animais tais como isolamento de células endoteliais, células neuronais, cardiomiócitos, hepatócitos e células estaminais. 26. Use of the recombinant collagenase described in claims 16-21 or obtained by the method described in accordance with claims 1-15 in techniques for culturing animal cells and tissues such as isolation of endothelial cells, neuronal cells, cardiomyocytes, hepatocytes and stem cells .
27. Uso da colagenase recombinante descrita nas reivindicações 16-21 ou obtida pelo método descrito de acordo com as reivindicações 1-15 na indústria alimentar, nomeadamente no processamento de carnes para melhoramento das suas propriedades e texturas. 27. Use of the recombinant collagenase described in claims 16-21 or obtained by the method described in accordance with claims 1-15 in the food industry, particularly in the processing of meat to improve its properties and textures.
28. Uso da colagenase recombinante descrita nas reivindicações 16-21 ou obtida pelo método descrito de acordo com as reivindicações 1-15 na indústria dos curtumes, nomeadamente para auxiliar no processo de tingimento de couros. de Novembro de 2014 28. Use of the recombinant collagenase described in claims 16-21 or obtained by the method described in accordance with claims 1-15 in the tanning industry, in particular to assist in the leather dyeing process. November 2014
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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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