PT107276B - METHOD FOR PRODUCTION OF RECOMBINANT COLAGENASE FOR DIGESTION OF COLLAGEN - Google Patents

Abstract

The present invention relates to a method for producing active recombinant cologenase derived from aoromones HYDROPHILA for application in processes where cologenesis is required, especially for medical application in the treatment of diseases involving excessive deposition of cologenes and pathologies mediated by cologenesis. BEYOND APPLICATION IN CLINICS AND RESEARCH, MICROBIAL COLLAGENASES HAVE A HIGH BIOTECHNOLOGICAL POTENTIAL AT THE LEVEL OF THE INDUSTRY, ESPECIALLY IN THE FOOD INDUSTRY IN THE TENDERIZATION OF MEAT OR IN THE INDUSTRY OF CURTUMES WHERE THEY PARTICIPATE IN THE PROCESS OF DYEING OF THE LEATHER. In this manner, the present invention aims to provide a method for the collection and purification of active recombinant colagenase from HYDROPHILA AEROMONES wherein COLLAGENASE is DIGESTED BY COLAGENASE WITH COLLAGENASE CONCENTRATIONS UNDER THE TOXIC CONCENTRATION OF COLLAGENASE TO THE CELLS.

Description

RECOMBINANT COIAGENASE FOR COLLAGEN DIGESTION, ITS PRODUCTION METHOD AND ITS USES

Technical Field of the Invention The present invention relates to the method for producing recombinant activating oolagenase derived from stewardesses hydrochia for application in processes where collagen digestion is necessary, particularly for medical application in the treatment of diseases involving excessive collagen deposition. .

Background of the Invention Collagen is the major constituent of the tissues of organisms belonging to the Eumecazoa sub-kingdom, being present in one. Methasoa or Animalla, 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. 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; particular importance in processes associated with extracellular matrix remodeling, whether in physiological or pathological processes,

In humans, collagen plays a key role in healing processes. Several skin trauma, such as burns, surgical acts or infections, are often characterized by excessive accumulation of fibrous tissue rich in various types of coiagenes. Several other conditions are associated with excessive deposition of collagen and for this reason are often referred to as collagen-mediated conditions. Collagenases, as enzymes capable of specifically digesting collagen, have been used to treat various collagen-mediated conditions. collagen. 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, for degradation of necrotic tissues, making them worse. The healing process of burns is easy. Recently, the use of collagenase produced by bacteria of the species Ctostridium histolyticum has been licensed as a noninvasive method and successfully applied in the treatment of Dupuytren's disease, avoiding <3 the need for recurrent surgical interventions.

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 cells and liver cells, studies on stem cells and epithelial cells. In addition to clinical and research application, microbial collagenases have a high level of technological potential at industry level, namely: in the food industry in meat tendencing or in the tanning industry where they participate in the process of faking leathers.

Apart from the application of microbial collagen, these enzymes are normally obtained by means of favorable processes of the producing microorganisms (as described in US 2010/0330065) © which require their further isolation. This strategy of obtaining the above presents as main disadvantages the use of methodologies that involve several eromatographic steps. your purification with. the consequent loss of prov.ei.na and activity. Another disadvantage associated with these microbial collagenases is that their storage stability is low, and their constitution is the constraint for the effectiveness of their application.

0006;

The document Cloning and: expression of a potential collagenase thesis of Meromenas hydrophiia Masters of Sliana Cavaleiro> University of ãveiro (ET t_.2 0 08}, recombinant diurase This expression of a protein work consists of a genetic construct which does not include any kind of fusion or labeling, the recombinant protein obtained has no activity, therefore resulting in loss of biotechnological potential.The present invention presents novel approaches in terms of genetic manipulation, notably by the construction of a hybrid gene that includes a marker (polyhistidine tail) and recombinant protein overexpression conditions, allowing for large-scale active collagenase with storage stability.

[0007.]; The document " ⁵ Urification and Characterization of a Microbial Reedombinant Tagged." collagenase, presented at the II Biotechnology Workshop. * 5 years of Biotechnology at. UM, Aveiro, 2011, does not disclose data on the genetic construct for expression of recombinant collagenase, nor does it refer to the stages of its purification results., Showing only results About the thermoesibility of the recombinant enzyme associated weapons Do it.

[0008] 0 document The novel collagenase from Aeromonas sp, presented at the National Congress Mi.crobi.otec '11, Braga, 201.1, repo rt r e rs a nd comparisons of the characterization of. activity, collagenolytic associated with striper AS-3 (Aeromonas piicola) and its null mutant for the coiagenase gene. No information is given for the name or. sequence number, · nor are any particularities inherent in the. cloning and gene expression, [0009] The potential pathogenesis of a gene gene from Veronil stewards, described by Han et al. {HA .... 2QQ8}, refers to an IJ32 family peptidase with ~ 68fcDa . The gene coding for this enzyme is homologous to the AHA gene .... 1043 noted: in the stewardess hydi'opdil & ATCG 7966 * genome. However, in the present invention, the construct is based on the GRF sequence. (open reading) AHA .... 0517 (genome A, xCpdropbiia: ATCC 7966), which codes for a potential M9 family coiagenase (Pfami: PF01752) ·, a distinct product from the coiagenase described by Kan eco-authors (K A .... 20 08).

[0010] US3677900 (1972 ;, METROD QF PRODUC1NG COLLAGENASE W1TK SPFCXEF QF VISRXO, refers to a method of production of bacterial collagenases produced by bacteria of the genera Vibrio and Stewardesses. It was later classified as Ariobrio proteoíttious (BA_1980) and the name of the bacterium was approved in 1985 (MO-1985) and is therefore not a patent for the production of stewardesses collagenases.

[Ο 01 1] o do oamen t S D S 2 0112439 2 0 * Compositions and methods for treafing collagen-mediated diseases undamerität in the development of a product and formulations. pharmaceuticals and consists of the combination of collagenase I and collagenase II to give a 1: 1 ratio of Ciostridium hlarolyticum for the treatment of diseases involving collagen deposition.

The present invention describes a collagenase of different origin (Aeromonas hydropáila) from the above, which is produced in a pure, active and stable manner by a protocol involving a single affinity chromatography purification step.

General Description of the Invention This invention relates to a method for obtaining a collagenase capable of digesting collagen at concentrations below the toxic concentration for cells, in particular fibrobasto-like cells, or below the concentration used with other cells. collagenase. The collagenase described in the present invention may come from bacteria of the genus Aeromonas, among others.

(0014] Microbial collagenase is recombinantly produced (Fig. 1} and overexpressed in bacteria. Given the characteristics of the product obtained, namely the capacity for in vitro collagen digestion, the activity in sub-toxic Î ± -coricentrations in expression of collagen in the exfraceiular matrix of cultured osteoblasts and their high storage stability, their application is possible in processes where collagen digestion is intended for use herein, as well as any modified derivative thereof, which maintain the same biological properties, both clinically and veterinarily, to treat pathologies that would require the digestion of eolagens, either for biotechnological purposes, for the production of pharmaceutical compounds, in the food or cosmetic industry.

A method for obtaining and purifying Aercsonas active recorabinant collagenase comprising the following steps is described * isolating genomic DNA from a stewardess culture;

• amplify an blush sequence a degree of time at least 95% blush â € 3KQ. Also iv 1; * to integrate, on one vector of induced expression,

coding sequence for a fusion protein, wherein said coding sequence codes for a set of consecutive histidines;

• Produce and select the desired teeomtoinant plasmid;

Selecting and culturing said recombinant cells and inducing expression of recorabinant collagenase with the appropriate inducer;

• collect and purify the active recombinant oologenesis.

Further described is a method for obtaining and purifying recorabinant collagenase with even better results, comprising:

* Isolation of genomic DNA from a culture of

Aoremovas;

The application of the putative collagenase gene of said culture to a sequence with a degree of homology of at least 55% with SEQ. Preferably, it has a degree of homology of at least 56%, 97%, 98%, or the like using primers which inolute the site. recognition of restriction enzymes iccni and HindiΐI;

Digestion of a chlorination vector which has a kanamycin resistance marker gene, such as the enzymes Icol and Hindi! _I: A kanamycin resistance gene;

• and which has a DKA sequence encoding a set of consecutive histidines (histidine tail)?

• cloning of the amplified genet and digested with restriction enzymes SspBi: and Hindiii in the selected cloning vector and previously digested with the restriction enzymes dcol and Bindl1;

The use of host cells, preferably coli, namely

TOP10F 'to transform and select reciprocating vectors;

Use of expression cells, preferably coli, namely B, c1 BL21 (DE3) to overexpress the desired protein;

«Purification of protein with activity.

[Q017 (collagenase re obtainable by the method described in this invention (non-wild)), it has a peptide signal that promotes its transiocation to the extracellular medium (which facilitates the purification process) and a histidine tail that allows purification through a immobilized metal ion affinity chromatography.

[GG18] Collagenase recomhihahte for the digestion of Î ±.iagen obtainable by the method described: in this invention, collagen digests collagenase concentrations below the non-toxic concentration for cells, up to 25 μΜ collagenase, in particular to 100: nM, ISO nM or 300 nM oelageuase, [0019] Their physical interaction with collagen (substrate) has also been demonstrated, which ensures extended stay in the site (tissue) where their activity is to be performed, [00201 Gontrarily to Commercial collagenase collagenase collagenase, this collagenase produces a nearer hydrohytic pattern than that produced by human collagenases suggesting greater potential for its medical use.

In another embodiment the method for obtaining and purifying collagenase comprises the following steps:

(a) isolate the gendamic DMA from a culture of Aeromcaas preferably ATCC 7966 · Stewardesses;

fo) amplifying said culture collagenase putative gene sequence with a degree anchor pole least 95% homology to SEQ _ate:. XD- No, 1 aha_G51'7, preferably with a degree of homology of at least 96%, 97%, 98%, 99% or equal, using primers which include the enzyme recognition site of; restriction AspHX and HindiXX, preferably with AHCF_.SapHI (SSQ, 1D, No. 2) and AHGR-H.indiXI (SEQ. XD. No, 3). and based on the nucleotide sequence encoding you SEQ-XD. Pollpeptide-1 and pre-ColAh No. 1, including the signal peptide;

c) digesting said cloning vector, preferably comprising the site for the enzymes Neol and Sindill, a tircinic resistance gene, and having a sequence, of SNA encoding a set of consecutive histidines (polyhistom tail). idinas)?

d) cloning said collagenase gene into said cloning vector;

e) producing the desired recombinant plasmid;

f) inserting said recombinant plasmid into suitable E. coli host cells, preferably. X. coii TOPlQFd

g) transforming said recombinant plasmid into recombinant expression cells, preferably using host cells compatible with the vector used, as shown with? c BL21 (DE3; to overexpress the desired protein, plus pFT2Sb (+};

h) sowing, selecting and cultivating said recombining cells;

f) collecting and purifying the active recombinant collagenase.

Another embodiment relates to primers, which include the recognition site of the restriction enzymes Fépílil and Hindi XX, which comprise the following horno1ogas • SSQ sequences. ID No. 2: GGGAT.AATC ATG AAC AATCTGGGTACC AG - 3 ';

• SSQ. XD No. 3: TTAAGCTTGTGGGAGGAGTCGTTGGC -3 ‘.

In another embodiment of said method amplification may occur by polymerase chain reactions carried out in 50 μΐ volumes containing 3 mN NgClr, 250 uN dNWs, 0.5 μΜ from each primer (AHGF_BspHl and

(RCI (NMRI)), 5% pMSG (v / v), 1.5 U Tag 'DMA ppl.iiterase in appropriate buffer and 50-100 ng of DNA.

In another embodiment of said method said amplification may be performed on a thermocycler,

understanding the initial denaturation of Genomic DNA of It was s pp, . at a temperature of 94 - 96 ° C for 2 - 5min, · followed by 40 30 s cycles at 94 - 96 ' ^C C, 30s at 58 ^G C and: 3min to ~ 0 - 7 C, preferably 5 ° C 68 ^S; final extension of 2Q ~ 3Qmin to 68 -75 ° C, preferably 72 ° C ^and C; the temperature

optimal activity of the tag polymerase used, which however may vary by manufacturer.

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 termini of the fragment (gene) to be ligated as shown. for the cloning vector to be pST26b (+), In another embodiment of said method selection of a clone containing the recombinant plasmid may be done by culture in selective medium, as shown for plasmid pET26b (+). ) _; wherein the positive clones are selected in medium supplemented with cancinic acid.

In another embodiment of said method the expression host cells may be transformed and cultured at 26Â ° C and 130 rpm for 18h. In another embodiment of said method the centrifugation conditions: (to obtain extract ) can; be as follows: velocity 10000g, ^4S; W; for 25 horns.

In another embodiment of said method the collection of eolagenase reco.m.bina.nr.e may: be performed by refrigerated centrifugation or by another cell concentration process, followed by dialysis (4 ^S C) against buffer of binding is filtered with syringe filter (0.2 pm).

In another embodiment of said method the purification of recombinant cholageiasis may be effected by affinity chromatography, preferably chromatography. affinity for immobilized metal ion or other similar processes. Affinity chromatography may preferably be performed on a column coupled to a medium pressure system, an eolagenase is further described. isolated from the process described in the above method which includes in its genetic construct a recombinant protein domain expressed and expressed with a histidine tail marker. Thus, isolated eolagenase obtainable from the process described in the present invention is capable of digesting collagen at non-toxic concentrations, much lower than the effective concentrations with other collagenases. In another embodiment, said isolated collagenase includes in its genetic construct an over-expressed recombinant protein domain and a marker, histidine tail.

In another isolated embodiment obtainable from the process described in the present invention capable of digesting collagen at lower than toxic collagenase concentrations, In another embodiment it has the characteristic of isolated collagenase to have the ability to digest Type I, Type II, and Type III collagen (vitreous digestion, analyzed by electrothorax) <

In another embodiment collagenase alone may be used as a medicine or medicine, preferably for the treatment of traumatized tissues, namely for the treatment of traumatic wounds, varicose ulcers and burns and also for the treatment of Dupuytren's disease, a proliferative fibrous dysplasia of aponevrosis. palmar; in gene or electro-gene therapy; and in the treatment of sciatica, namely in intervertebral discs, there is further described a face composition comprising or consisting of the collagenase described above. applied in solution, in its lyophilized form, or in a composition comprised of said collagenase and a biomaterial, namely guitosan, In another embodiment said composition may be a topical formulation, a formulation for oral or parenteral administration or a injectable formulation ', [003 8 j A present in The invention further discloses 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, cortiomyocytes, hepatoeites and stem cells; There is a food industry, namely in meat processing: to improve its properties and textures; and in the tanning industry, in particular to assist in the leather dyeing process.

[0039] The present invention relates to the process of cloning, expression and purification of an Aeromonan collagenase for coiagen digestion. [0040] One of the objects of the present invention is the production of flight attendant collagenase following a methodology based on Genetic combination. In a preferred embodiment, upon identification of the gene, primers were designed for directed cloning of the complete gene encoding collagenase in Ânmr.u npdrophiia. The isolated gene can be cloned into a cloning vector containing a UNA sequence coding for a set of five consecutive histidines (histidine tail), which was later used to transform competent cells to produce recombinant cells. capable of expressing bacferian collagenase After identification and selection of positive clones, recombinant cells expressing reeombinant collagenase.

The extracellular medium of the centrifugation cultures can be separated from the cells by dialysate and applied to a histidine affinity column for active reeombinant collagenase.

The present invention is useful for application in processes where it is intended to digest interstitial coiagenes, specifically for the treatment of conditions involving excessive extracellular matrix deposition, for example as Bapuytren's Disease. The described recombinant collagenase has further application in the establishment of primary cell cultures, for which detachment of tissues of origin and prior cell dissociation is essential in vitro culture.

DESCRIPTION OF THE FIGURES t0042] For an 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.

Figure 1: Schematic representation of the cloning process and obtaining recombinant plasmid (pCAA). Cloning vector digested with //.iodiir/Vcoí (a) and polymerase chain reaction product (PCR) digested with restriction enzymes RindIIl / fispHl (fc)> generating complementary termini to provide gene binding to vector (c) where:

IA - represents the digested piasmid;

1B - represents the digested RGB product;

1C - represents the binding reaction.

Figure 2: Methodological diagram of recombinant collagenase expression and purification.

Figure: 3t Expression of collagenase: recombinant in B. colí. SDS overexpression (PAGE) and proteolytic activity (gelatin zymography} of collagenase in recombinant (R) cells vs untransformed cells (G). Migration of the recombinant protein corresponds to the expected molecular weight (−1,100 kDa) and is marked with a drought.

Figure 4: Purification gives recombinant ColAh by immobilized metal ion affinity chromatography.

Figure 5: Digestion of human type 1 coiagene with recorabinant coiagenase (ColAh :),

Figure 6: Detection of physical interaction between enzyme; substrate (recorabinant collagenase: type X collagen) by immunodetection ...

Figure 7: r e c a nab r e (-80 ° C; -20

Stability of collagenase (ColAh) activity at storage at various temperatures * C; 4 ^? C and 37 ^V C).

Figure 8: Determination of viability osteoblasts in culture exposed to ColAh cell treatment.

Figure 9: T-type coiagen immunodetection in cell fractions and extreeluary media of osteoblasts subjected to ColAh treatment during a test-type assay.

Figure 10: Intra and extracellular distribution of type I coiagen in MC3T3 osteoblasts (15 days after confluence): analyzed by confocal microscopy,

Detailed Description of the Invention {0043] As is required: in this section will be _an: detailed explanation of the invention. The disclosed methods are purely 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 1.9, The basic strategy for increasing: expression of the active protein by cloning the coding gene for coiagenase into an expression vector and consequent transformation in the host for expression. hetergIoga, [0045] The expression vector is one. cloning vector, const.ru gone form. that the gene inserted therein is transcribed / translated and after its introduction into host cells.

[90461 In this work a pET system was used for expression of the coiagenase noted in Aeromonas úydíOph / Je ORA ARA_0517 ATCC 966 ⁷ , [0047] The present invention relates to the method for obtaining recombinant coiagenase comprising the following steps: ( a) isolation of genomic DNA from Aeromonas hydrophila ΛΤ-CC 7966¾ (b) amplification of the putative coiagenase gene by PCR. to generate multiple copies of the coiagenase gene (ORE: AHA_0S17) (c) cloning the Aeromonas coiagenase gene into the pET26toÇi} cloning vector; (d) transformation of recombinant vector, pcUA. was competent cells (E. coli TOP10E ') to produce a starting cl, One; (e) identification and selection of the positivamerite transformed starting chlorine; - insertion tracking for confirmation of the correct reading phase of the gene in the vector; (g) purification of the recomirinance plasmid and transformation into E. coli B1-21 DK3 expression cells; (h) identification and selection of transformed recombinant cells; (i) induction of recombinant cells for expression of recombinant collagenase; (j) collection of medium ex trace 1 υ 3. air purification culture: from collagenase:

affinity chromatography recombinant; (k) dialysis against collagenolytic buffer.

isotonic;

Thus, in a first step of the process, the total DNA of the strain Aeranonas iydfopdia ATCC 79 6 6 'was isolated from liquid cultures in supplemented iuria-Bertani (LB) medium to the ampullin antibiotic ( 50 pgAml} using the Genomle MA Purification Kit following the manufacturer's instructions Subsequently, the gene: encoding the putative collagenase in Aaromcoas was amplified by the PCB using the following pair of primers (underlining the enzyme recognition sequence restriction} ::

- SEQ. 1D NO, 2: GGGATAÃTGArGAACAATCIGGGTACCAG-3 '

SECTION 1D No. iWAAGGT ^ GTGGGAGGIGTCGWGGG -3 'These primers were designed based on the coding nucleotide sequence. (AHA-05.17). 1D. Kc. 1} of the pre-ColAh polypeptide to include the signal peptide. PCB reactions were performed in 50 µl volumes containing 3 ml of MgGla, 250 pM dNTPs, 0.5 pN of each primer (AhCF ... EspHl and AHCR (Kndndri), 5%

DMSO (v / vi 1.5 U DNA pol Τα imerase in an appropriate buffer and 50-100 ng of DNA The reactions took place in a donor T ermoci iCycled Thermai Cyder amplification under the following conditions :: initial denaturation at 94 ⁰ C for Smin; 40 cycles of 30s at 94 C, 3 0s 5® ^Q G and d 3 min 72 30 min final extension at 72 ° C ^:::

As noted above, the primer sequences include the re-recognition site of the SspHI and HindXII restriction teaches (underlined in the primer sequence), indispensable for generating the associated termini for subsequent binding to the vector (Fig. 1 In this work the restriction enzymes BspHl and Hindu I were used to digest the RCR product, and dcol and fiadili to digest the vector, following the manufacturer's instructions (Table 1).

Table 1: Digestion with a fesirition enzyme

Digestion Reaction:

• final volume t / w ide reaction buffer (10X) »50-180ng DMA • 1 U restriction endfonuclease • Distilled water to fine volume! 1Dpi

Incubate 2-3 hours: Temperature of enzyme activity Stop reaction at -20 * C [0051j Contain selection

Plasmid © pET26fe (+) was the vector of choice for a kanamycin resistance gene that allows the transformants to be posterior.

After digestion (Table 2) of the (PCR product) reactions, a veto was made so as to eliminate precipitating salts and insert solutions that could inhibit the binding reaction. .

Table 2; Predisposition of the reaction reactions * Add to each street 30 μ; de-agonized: de-assembled and isopropanol incubated with SCFC; 1 hour · * Oeatníaga? 5 mindiós to 14 000? P? A 'è discard the soferensdanía * Let? dry at room temperature * Respertiferous ó: DNA in 20 pi of sterile water DesiótMaadá [0053] The fragmentation-vector binding reaction was established at an insert: vector molar ratio of 3 ri (Table 3), applying the following calculation formula :

Vector xng x insert size (kb) x insert / vector ~ ng vector size (kb)

Table 3: Stopper reaction ♦ all DNA DNA custod * 180 ug: da Insert t pi buffer: isgase · d Oh * t U 14. DMA hgase

- Water eoténi pa? A gerbes? or fly íní 10 pi

Incubate reacaãu M ^S G for 14-i Oheras; The reaction may be immediately used or stored at 0 ° C in view of its particular characteristics. rsoA- and e.hi, having a high transformation efficiency and good px: piasmid oduction, TOPIOF 'cells were selected as a host strain to generate a starting clone (to keep the recombinant piasmid). The obtained recombinant vector, hereinafter referred to as pCOA, was useful in one phase: initial to transform strain E. coli TOPIOF 'as set forth in table i,

Tafoeís 4; Transformation of the competent cells • Defrost in an aliquot {50-200 μ!} Of the chemotompetent cells - £ cof / TGPWF 'or 8L2KDE3) »Add 2-t 0 μΙ of the binding reaction to the competent cells, mixing gently. Keep on ice for 30 minutes, the cells • Give a check íérroscc 30 segundes C at 42 ^{° C;} 2 minutes on goal.

• Add 250 µl of SOC medium. Incubate for 3 hours? ^SC and at 250 rpm.

• Seed 2.0-100 pl of transformation into LA medium plates supplemented with canamlcine SQpg / mi.

• incubate the plates at 37 ° C; 16- f Shcras.

[55] The selection of the transformants (positive ions) was. performed by PGR using the universal primers for the pET vector (T7 Prorer Priraer # 6.9348-3, SSQ. ID, 6 ~ 5'-AATACGACTCAGTATAGGG-3 · 'and T7 Termini tor Primei; # 69337-3 SEQ-. ID 7 -5-GCTAGTTATTGGTCAGCGG-3 ') according to manufacturer's indications. Evaluation on agarose gel allowed selection of a positive clone (containing pCEA) * Transformed cyions were cryopreserved at -80 ^S C at 1.5 % Glycerol (v / v), The correct insertion of the pCUA-cloned fragment was confirmed by sequencing of the plasmid DMA. To this end, the extraction of the recombinant plasmids was performed using the UltraClean 6 Minute Mini Plasmid Prep Kit. further sequencing with the T 'primers. The nucleotide sequence obtained (SEQ, 1D. No, 4) was compared with sequences deposited in the NCBI database. The sequencing results allowed us to confirm the reading pattern of the sequence encoding the bistidine tail, present in the vector pET26hít. once confirmed the intended construction for, the pCUA vector, proceeded to. transformation into the expression host BL2II (DE3) - and selection of pdx1 PCB transformants according to the methodology described above for selection of the starting elone (in Flolioli topiQF ').

(0057} To express recorabinant coiagenase, the strategy shown in Fig. 2 was followed. Pre-inocula were used from a transformant of the F. coli BD21 (DE 3) strain inoculated into BB medium containing 50 pg / ml kanamycin, One culture ( 50 ml) was incubated at 37 ° C and 150 rpm to 0.5 optical density (OD) units at 600 nm, cells were induced with the addition of 0.5 mM isopropyl beta-D-thiogalsdtopitanoside (IPTG). Culture conditions after induction were adjusted to 26 ^S C and 130 rpm for 18 h for increased expression of active enzyme A. Overexpression of recombinant ColAh coiagenase in F, coli was assessed by GQS-RAGB and proteolytic activity by gelatin zymography (Fig. 3).

580058.] Extracellular medium was collected after centrifugation at 110 ° Cg, 4 ° C for 25 minutes and dialyzed (4 ° C, 1 h) against binding buffer. and filtered with a syringe filter (0.2: gm) prior to the affinity chromatography purification step. Chromatography was performed on a particular HisTrap ” ⁵ 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 mmol imidazole, pH 7.4), the sample (10 ml) was applied to the column and poured into 10:% (v / vj of elution buffer: (20 sodium phosphate phosphate, 0.5 M FfaCl, 500 mP imidazole, pH 7.4), which corresponds to 50 mHe: imidazole (Fig. 4). The collected fraction was analyzed by gelatin zymography (0.1%) and by SDS-PAGE to confirm its activity and its purity.The recombinant collagen was further removed for its ability to digest the genius type. X; Fig. 5), according to the protocol described by Duarte et al. (1) 1) .... 2005} and its physical interaction with this substrate (Fig. 6). its activity by incubation at different temperatures · Colah; ·· ^'O 0 C, -20 C ^s, and C 1 -C 37 ^s , and storage time (Fig. 7).

EMBODIMENTS The procedure for obtaining recombinant eolagenase 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.

[00603 FOMA _form: embodiment comprises the use of sub-toxic doses of recombinant collagens eolagenase to reduction in osteoblasts in culture.

[0061]

Thus, after tiydropih i 1 to AHC.F__BspHI. allow,: enter ir, restriction sequences. I recognized

HindiXI (Table 1} for AICC 7966b Insulation

Obtaining eolagenase comprising the described steps of genbmoid DMA are used and specifically, AKCR-Hindi II selects at each of the recognition ends thereof. These sequences are cleaved by the recombinant restriction enzymes from above. of Aeromonas the primers designed for gene ORF AHÃ05® 17 ©, the enzymes of specifically BspHX, and generate cohesive termini compatible with the termini of the pET26b (+) vector digested by the enzymes and HindIIT, as depicted in Fig. 1 and following the indications for the binding reaction (table 3). After binding of the insert to the vector, the reeombinant plasmid is used to transform competent cells (F, coli TQPÍW 'and produce a starting cione following the protocoib: described in table 4. once selected as positive cione in half-lb plates. containing 50 µg / mi kanamycin (antibiotic to select for transformed cells: by plasmid), plasmid is purified and the sequence of the insert determined by automatic sequencing to confirm that the gene was cloned at the correct reading stage. , which allows expression of the active reeombinant protein contemplating the histidine tail (SEQ, ID, NO. 5} for further purification by affinity chromatography. Upon confirmation, the reeombinant plasmid is then used to transform B <coil BL21 expression cells. (DE3) (table. 4), As described above, positive clones are selected on kanamycin-containing plates and the prosthesis Insert is confirmed by PCB. using primers specific for the vector and subsequent sequence determination by the Sanger method (SA__1977). Once confirmed, positive cyione is inoculated into LB medium (supplemented with 50 pg / ml canaminic acid) to produce a pre-inoculum that is used for crop establishment for overexpression reeombinante collagenase As shown in the layout of FIG, 2, 1 ml of pre-inoculum is used to inoculate 50 ml medium containing kanamycin, which is incubated at 37 ^& C. under agitation (150 rpm ), When it reaches the optical density 0.6, measured at 600 nm, the culture is induced with 500 mM IPTG and incubated for 18 h at 26 ° C and under controlled shaking (130 rpm). . culture is collected after sedimentation of cells by centrifugation (llQOOxg, 25 minutes :, ^S 4 C), dialyzed posfceriormenfce (1 ^{I C!)} against binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4) under slight agitationThe sample is filtered with a syringe filter (0.2 µm) and applied to an immobilized metal ion affinity column (HisTrap ™ FF) coupled to a medium pressure system, preferably AKIA 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 oolagenase 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. 4). 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 Aerc / scnas collagenase on proliferation and differentiation of cultured mammalian cells. For this we use the EC3T3-E1 (ATCC) cell line, a mouse osteoblast cell line. These cells are located on the bone surface and are directly responsible for bone formation. They were particularly selected because during the differentiation process osteoblast precursor cells alter their morphology and the expression of functional markers, starting by secreting bone matrix proteins such as type 1 collagen, which represents about 90% of the organic matrix and provides bone mineralization.

Thus, the established chord culture Notwithstanding, the cells in humid atmosphere, 5% backward structure of the MC3T3 - E.1 cell line was described (P12010). were maintained at 37 C ^S essential medium with balanced .Minimum sup1ementado% (v / v) of a s t p imo three Ci in the sub -conf1uen TES 1: 5 to 0.25% w ith

CQ:;, was containing. 2 ml glntamine in a salt solution (Eagle's Balanced Balt Bolution),% (v / v) fetal bovine serum (FSS), 1 penicillin solution (160 D / ral) and (100 mg / ml) and 3.7 g / 1% Na ECO; .. Cultures (80 - 90% confluence] are divided: trinsine / EDTA solution at 5 ° C / 37 ° C.

[00651 To determine the dose - dependent cellular viability and evaluation of the effect of the proliferation Colah - ^1x10:; Cells are seeded into 35 mm wells in 6 - well culture plates (at a density of 1x10 cells / cm ^@;) and cultivated in the above conditions. The environment is renewed: every 2 days of culture. After 18 days in culture, which corresponds to 15 days after confluence, the culture medium is replaced with medium supplemented with 1.00, 150 and 306 nM pure collagenase, and 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 resinsrin solution to determine cell viability. After 4 h incubation at 37 C in humidified atmosphere with 5% CO:, 1 ml melo is collected and the optical density (OD) at 570 and 600 nm is measured for all samples, the final OD (DO- ?) results subtraction between reason DQrô / DGetm each amosbra and the ratio of the m / DOscm cesium negative control (medium containing 10% of a solution 0, _f 1 mg / ml resazurin) As a positive control, DCn of. Cells incubated in 0 nM reeombinant collagenase are considered to be 100% and cell viability is calculated as a relative percentage of this control value. As depicted in the graph of Fig. 8, the reombinant GolAh concentrations tested and obtained by the method of the present invention do not affect the viability of MC3T3 cells in order to determine the effect of GolAh on cell proliferation of cultured osteoblasts. the expression profile and segregation of collagen type I using immunodetection based methodologies.

Thus, after GolAh exposure assay, the culture medium (1 ml) of each condition is collected into micro tubes containing 10% sodium dodecylate (SDS). The respective cells are carefully washed with phosphate buffered saline (P8SG and collected with a 1% SDS (w / v) solution ...). The samples are boiled for 10 minutes in a water bath (100 ^{3 * S} C) and the Total protein concentration (BCA Protein Assay lleageut, Pierce) <Next, the same amount of total protein (60 pg) for both types of cell fractions obtained (cells and culture medium) was resolved on gel 6.5 % SDS - FAGE in triscglycine buffer Proteins separated by electrophoresis were electrotransferred to a nitrocellulose membrane for subsequent immunodetection with anti-collagen type .1 primary antibody after membrane blockage with 5% solution (w / v skim milk in Tf6S). -T (10 µM Tris - HC.1, pK 8.0, 150 mM NaCl, 0.5% Ween) (DU .... 2005). The detection of protein-antibody binding is performed by guiolumumescence. , with ³ DBS (ix) anti-XgGs secondary antibody; Dissolve 8g NaCl, 0 , 2g PCI, 1, g: Ot> 10 _4z 0.24, and KftPO. _; 800 ml of distilled water; set the pa to 4; perfater 1 autoclave you (latsi, 121 * 0, 20tninS.

rabbit, conjugated with. horseradish peroxidase and using the ECL Kit according to the manufacturer's instructions and revealed in a darkroom on autoradiographic plates.

The results clearly show the reduction of collagen in both fractions, cells and extracellular medium, demonstrating the effect of collagenase on the expression of the extracellular matrix by osteoblasts (Fig. 9), the same effect. is observed at non-toxic collagenase concentrations.

purified collagenase The cellular distribution of collagen was analyzed by immunocytochemistry according to PI_2010. As in the previous description the cultures were set up in 35 mm diameter plates containing coverslips and were treated under the same exposure conditions as the end of the test. The coverslips were% (v / v) formaldehyde in PBS and permeabilized with methanol. . Non-specific sites were blocked by incubation (1 h) with 3% (w / v) bovine serum albumin (BSA) in PBS and immunocytogenesis was performed with type I anti-collagen antibody diluted in 3% BSA-PBS. After .3 washes in PBS, the cells with a fluorescein isothiocyanate fluorochrome antibody (FICT) solution. The coverslips were mounted® on microscopy slides in mounting medium of Î ± 6'.amid.ino-2'-phenylindoi dihydrochloride (DAPI) with vectashild reagent.

Fixed wells were incubated secondary to (to reduce fluorescence photodecoration).

At? These images were acquired on a Li3M S10 META confocal microscope with an Argon laser (488nm) to excite the EITC conjugate and a Diode 4005-4000 laser to assess DAPI fluorescence therein.

The acquired images show that: treatment of collagen-secreting cells with recombinant collagenase promotes a dose-dependent response, reflecting increased proliferation of asteoplasts for the highest concentration of coagenesis used in the assay {Fig. 10), The reduction of the deposition of: type X collagen in the extra-cellular matrix of the cultured cells is also evident. The results obtained show that ColAh re-ant and induces phosphite properties. They also raise osteoblasts, even at sub-toxic concentrations to cells. In fact, the first phase of bone formation is characterized by massive segregation of coiagens, 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 deposition of coiagenes.

In another possible embodiment of the present invention recombinant collagenase, ColAh, as known for other microbial collagenases, may have different applications, with the advantage of having high storage stability, e.g. , its potential application to enzymatic tissue debridement, due to its degradative capacity of coiagenes, 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, the North American, and the Australian (SY _ .. 2012), For treatment, the surgical phase is often used, which does not limit the recurrence of the pathology and, as an invasive method, leads to an increase in the patient's morbidity. A new noninvasive therapeutic strategy. has recently been implemented and is based on the use of Ciostridia disroiyeicum ^: (GCH) xiaflex® Collagenase (with constant Xyl ratio for ColG and ColH collagenases, respectively).

100751 Recent publication: Results on the molecular and cellular effect of xiaflex® CCB (S ¥ __2012) underlie and reinforce the potential application of recombinant CoiAh collagenase in the treatment of Dupuytren's disease in one of the preferred embodiments of the present invention. .

In a further possible embodiment of the present invention the use of recombinant collagenase is applied in methodologies of transforming tumor cell transfection into processes related to gene or electro-gene therapy.

In another possible embodiment of the present invention the use of recombinant CoiAh for the treatment of sciatica, noraeadamence in herniated intervertebral discs is applied.

In another embodiment: possible of the present invention the use of GolAh recombinant collagenase is applied to techniques involving cultures of animal cells and tissues (isolation of endothelial cells, ueuronal cells, cardaomyocytes, hepatocytes, stem cells, among others). ), In another possible embodiment of the present invention the use of the recombinant ColAh collagenase in the food industry is applied, namely in the processing of meat to increase its properties and textures, In another possible embodiment of the The present invention applies the use of ColAh recombinant glue in the tanning industry, in particular to aid in the leather dyeing process.

[0081] In another possible embodiment of the present invention the use of recombinant ColAh collagenase for the extraction and purification of collagens is applied.

Nucleotide Sequence List (SSQ. Nx No. 1-4). Nucleotide sequences are described by the single letter nucleotide code. The nucleotide label is as follows (single letter code): Adenine, T - thymine, G - guanidine, C - oitosin.

SEQ ID, No. 1 - Nucleotide sequence of stewardesses collagenase ge. (. 05 .... 0517) hpdrophíia ATCC 7966 '·

ArGAAGAATCTGGGTACGAGGTTGGTGGTGCTGGCAGCAGCGGGCCTGTTTGCGACTTC

GGCGCTGGCCAACTACTGCeCCTCCGACTGGCAACAGTATCAACTCAAAGGTGAATCCA

GCCGTCAGCTGGGAGATGGTeTCACCGAAGTGACCTACGAGCTGAGCGCCCGCAGCGGe

GGCGCCCCCTATe & GCAGCTGAGGGTGTATCGGCGeTTCGÁCTGGAGCGATGCGA.ACGT

GGCTGeGCTGGCAGAACAGCAGTGCGGCG.AGCCGCÃGCTCAAGATCGAGCTGGGGTGGC

AGATCCGCTAGGTCAGeTGTGAAGAGGTGGTGGCGGCTGGGAAGGCCGTTCGGGGCAGe agctatgactatggctacggcatgaagcagggccgçtgggagccactggcaggcaccgg

GACCGCGCGGGGTGAGGATGGTCTGGGGGTGGGGGAGCGGGTGATGGTGGGTCAGAGGG

AGGAGGAAGTGGATGGGTGGGAGCTGAAGGGCGAAGGCGGCTGCGGGGAGGAGGGATGG gagtagcaggcggaaaagtggcagcagttgaaggtggtggaagagaggggcaatgaagg ggatggcgggggggggggggggggggggggggggg

GGAAGeAGGTGAGGGAGCTGGACGTCTGGGGCCAGTACACCTGGTTGCTGGAAeAAGGe

AAGAGGGAGCAAGAGTGGGATGAAGGCCAGAGGCGTGAGGAAGGGGACAAGACGATGAG

CTACGGGGTGTGGGGGC ^ AGACCGTGGCGGGCGGGAGCGAAGTGGAGGTGGTGTTGAAGG

ACACCGGCTATCAATACCCGGTGGGGGGGAGGGAATGGGAGAGCGTGCGGGxAAAGAAGC

GAGTGGCAGGAGAGGGGGGTGCTCAACCACGCCATCGTGCTGGCCAGCAAGGAAGAGCA

GGTGGACTGTGGGCGTGCCGAGGGGCGCGGCTGTTGCGAGGGAGATGTGCGGGGCACGG

AGGTGCTGGAGGCGGAAGGGGGCAAGATAGTGCAGGATGGGAGTGGCCAGGGTGGGCCG

GTCTGGCAGGAGAACTATGGTCACGATGACAGGAAGCTGCTGGCCGTGTCGGGGGGGAT

GGAGAGGGTGCTGGGGGCGATATAGAGGGGGGGGATGCAGGGATGAAGGTGGTGeTGGAAT

AGGTGGGTGCCCAGAAGTAGGAGAACTAGGGCAAGGACAIGGAAGAGGGGGGGGGGGGG

GGCGAGGGGCTGGGGGAGGGGTTGAGGGGGCTGGGGGCGGATGCGCTGCTCTTCGCGGA

GCAGGGCAGCGACGAGGTTGGTGCAGTGATGGGTGGGTGGAGCATeGCCGTGCACGGTC

ÁGTTGAAGAGGGCAGCAGTGCAGAGCCGCTTTGGCAGGeTGCTGGGCGAGTTCAACCAG

ATGCTGGeCTACAGCACCCGGCATGCCAGCGAGATCAACGGTGAGCAGGGCTGGGGAAG

CGGCCTGTTGGATCTGGTCAAGTTGGTGGACTTCGGCAGCGAGTACAGCGAGeCGTTGG

CCAACGAÇTTCCGCCAAGAGGACGGTGAGCTGCGGAAGCAGGTGeACGCCGTCGGCATG

AtGCGAACTCGCGTTGTGGAAGGGAGGGGATGGGGGGGATGTGTTeCTGeTGAAGAACGT gçtggatgggtaçaggggcctctagggggtggggcggtataçcggccgggaggagctgg

ACGGCTATGG ^ AAAGTGTTGGATGAGTeCGTGATCGGACTGGirTGGCGACCAGAAGGTG:

atgcgcggtgggcagcagagtcaggatctggtggaagagatgtcagtgãgcgtgtgc

GTACTACGTGACGTACxAÇGGAGÚGCAGCAGGGAGGÇGTGCATCAGGGGTTGGGCG

GGGTGTGGACGCCTGTeGGGGTGGAGGAeGTGCTGqGGTTGGÃGGAGÃCGTGGTeGGCG agcgtgcgggtgggggçgcaggaggtgaggatggatgaggccgãagggatgtggggtga agtgggtgccgaagagcaggagttggatcaagagatggagagcgggtgggãggçggtgg gggacgatcagaacgaggcgctggaactggtggtgttgaagtgctgcgcggactggaaa cgctatggcagtgctctgttcggcggcgtgtggagggagaagggcgggatgtagctgga

AGGGGATGCGGGTCGGeGCGGGAAÇGAGGGGGGGTTGTTCGGGTAGGAGGCGGAGTGGA agcggggagcgttccaggtgtggaacgtgggggaggagtatgtgcactaggttggagggg

GGGTTGAACCAGTACGGCAGCTTGGGGCAGTACGGGGTGAACCGGACeAGCTGGTGGTG ggaaggggtgggggagttcatggçggaggggcagtggttggggcggqgtctggâgaagg

TGGGGGGCCGTCGXOCCAGTGATGGmGGGGGGTTGGGeGACATCCTGCACCTGGATTAG

GAGAAGGGTGGCGAGATGGTGTAGTGCTGGTCGTACAGGGTGGAGCGCTTCGTGAAGGA

AACGGGTCGGGGGGCGAGGTGGGTGGGCADGGCCGAGGCGCTGGGCAAGCeGGATCAGC

AGCAGGGCATGAGCACGTTCGAAGCCGAGGTGGACCãGGTGATTGCCA & TGACAGCGAG

GCGTACCAGCAGTGGCTCGGCGGCGAGCTGCTGGCGTGGTGGGAAGCCAAGAAGGACTC cgacgagtggaaggccaaggactcctcccactga

SKQ, XD. No. 2 - Sequence: primer nueleotide

AHCF ... ScpHI 5'- GGGATAATCATGAACAATGTGGGTACGAG-1 '

SEQ XD. No. 3 ~ Nucleotide Primer Sequence

Hindi AHCR 5'-TTAAGGTTGTGGGAGGAGDGGTTGGG -3 '

SEQ ID No. 4 - Nucleotide sequence obtained by sequencing with the universal primers T7 promoter primer and T7 termineter primer

TAATAGGAGTCACTAXAGGGGAATTGTGAGGGGATAACÃ † TCGGCTeTAGAAATAATT

TTGTTTAAGTTiAGAGAGAGTATATAGATATGAAATAeGTGCTGGGGAGCGGTGCTGGT

GGTCTGCXGCTCCTGGCTGCGGAGGCGGGGATGGCGADGAACAATCTGGGTACGAGGTT

GGTGGTGCTGGCAGCACCGSGCCTGTTTGCÇAGTTCGGCGGTGGGCAAGTAGGGCGGGT

CCGACTGGGAACAGTAIC ^ AAGTCAAAGGTGAATCGAGGCGTGAGCTGGGAGADGGTGTC

AGGGAAGTGAGGTAGGAGGTGAGCGGCCGGAGGGGGGGGGGCGGCTATGAGCAGGTGAG ggtgtatcgcggçttcgactggagcgatgcgaacctggctgcgçtggcagaacagcacít qgggcqagccgcaggtcaagatcgagctgggctggcagatgggctaçctgagçtGtgaa gaggtggtggcgggtgggaagggcgttggggcgagcagçtatgagtatgggtacggcat

GAAGCAGGGCCGGTGGGAGGCAGTGGCAGGCAGGCGGACGGGGCGGCGTGAGGATeGmG tggcgctggcggagggggtgatggdgggtcacagcgaggaggaactggatcgctgcag

GTCAAGGGCGAAGGGGGCTGGGGGGAGCAGGCA-PGGGAGTAGGAGGGGGAAAACTGGGA

GGAGTTGAAGGTGCTGGAAGAGAGGGGGAATGAAGGGGATGGGGGGGTGGAGGAGATCT

TGTTGGGGCTGGAGGGGATGGXOGGGAGCCAGGGGGCGAAGCAGGTGAGGGAGGXGGAC

GTGTGGCGGeAGTAGACGTGGTTGCIGGAAGAAGCGAAGAGCCAGGAAGAGTGGGATGA agggcagacgggtcaggaaggggacaagacgatgaggtagggggtgtgcggcgagaccc

TGGGCGGCGGCAGCGAAGTGCAGGTGGTGTTGAAGGAeAGCGGGrATGAATAGGCGGTG

GGGGGGAGCGAATGGGAGACGCTGCCGGAAACAACCGAGTGGGAGGAGAGGGGGGTGGm

GAAGCACCGGATCGTGGTGGGGAGCAAGGAAGAGGAGGTCGAGTGTCGCGGTGCCGACG

GCGGGGGGTGTTCeGAGGeAGATCTGCGGGGCACGGAGGTGCTGGAGGCCGAAGCGGGC

AAGATAGTGCAGGATGCCAGTGGCCAGCCTGCGCCGGTCTGGCAGGAGAMCTATGGTCA

CGATGACAÇÁÁGeTGCTGfâCGGTG ^ CGCGCGGCATCCAGAGCCTGCTGGCGGCCAATC agccggccgatccagccatgaaggtgctgctggaatacgtgcgtgggacacaagtacgac

AACTGGCAAGCAGAAGGAAGAeGGCGCGGCCGCCGCCGAGGCGCTGGCCGAGGCGTT

GAGGGCGGTGGGGGGCCAlGCGGTGCTGT ^ CCeGGAGCAGGCCAGCGACGAGGTTGGTG

CAGTCATGGGTGCCTGGAGCATCGCCGTGGACGGGGAGirTCAAGAGCCGAGCAGTGCAG

AGGCGGTTGGGGAceGTGCTGGGCGAGTTCAAGCAGATGGTGGCGTACAGCAGGCGCGA

TGCCAGGGAGAXCAAeGGT / GAGGAeGCCTGGGCAA.CCGGGG.TGTTGGATGTGCTCAACT tgctcgacttggcçagcgactagaggaggggttcgcgaacgacttcggggaagaggag

GGTGAGeTGGGGAAGGAGÇTGCACGCGÇTeGGÇATGAGGGAAGTGGCGTTGTGGAAGGG

AGGGGAIGGGGÇCGAXGTGTTGGTGGXGAAGAAGGTGGTGGATGCeTACAGCGGCCTGT

AGCGGGT.GGGCCGGTATACCGGCGGGGACGAGGTGGAGGGCTA ^ CGCAAAGTGTTGGAT

GAGTGCGTGAXGGCAGTGGTTCGGGACGACAãGGTGATGGGGGGTGGCGAGGAGAGGA

GGATCTGGTGGAAGACAT @ rGAGTGACGGTCTGCAGGTACTAGCTGACÇTAGAGGGe

GGACGAGCGAGGGGXGGAreAGGGGTGACTTGGGeGGGGTGTGCAGCGCTGTGeGGGTG

GAGGAGGXGG ^ GCGGTTGGAGCAGAGCTGGTGGGGGÁGGCTGGGCGTGCGGGGCGAGGã

GGTGAGGAXGGAXGAGGCGGAAGGGAXCXGGGGTGAACXGGGTGCCGAAGAGCAGeAGT

GÇGATÇAACAGAGGGAGACGGGCGGGGAGGCGGTGGGGGAGGATÇAC: AAGGAGGCGGTG

GAACTGGGGG: TGTTGAACTGGTGGGCÇGAGTGGAAÇGÇTATGGGAGTGCTGTÇ4Tsr‘CGG

GGGCGTGTCCAGCGAGAACGGGGGCATCTAGGTGGAAGGGGATGGGGCTGGGGGGGGGA

AGGAGGGGGGGTTGTTGGÇGTaAGGAGGGGGAGTGGAAGGGCGAGGGTTCGAGGTGTGG aaggtgggccaggagtatgtggagtaggtggacggggggttgaagcagtagggc: aggtt

GGGGCAÇGAÇGGGGTCAAGGGGAGGAaCGTGGXGGTGGGAAGGGGTGGCGGAGTTGAIGG

CGGACGGCGAGTGGTTGGGGCGGGGTCTGGAGAÂGGTGGGGGGGCGTGGTGGGAGTGAT

GGTGGGGCGTTGGCGGAGATGGIGGACGTGGATTAGGAGAAGGGTGGGGAGATGGTCTA

GTGCTGGGGGXAGAGGGIGGAGGGGTTGGTGAAeGAAAGGGGTGGCGGGGCGAGCTGGG

TGGGGATGGGGCAGGGCCTGCGGAAGGGGGATGAGGAGGAGGCGATGAGGAGGTTGGAA

GGGGAGGTGGACGAGGTGATTGGGAAXGAGAGCGAGGGGTACGAGGAGTGGCTGGGGGG eGAGÇTGGTGGGGTGGGGGGGAAGGGAACAAGGAGiTGCGAeGAGTGGAAGggggggggggggtgggggtgg

TAÃCAAAGCGGGAAAGGAAGGTGAGTTGGeTGGTGCÇACCGGTGAGCAATAACTAGG

Τ7 Promoter Prírfter # 69348-3

SEQ ID No, 6 ~ 5 '- A.ATACGACTCACTATAGGG-3';

T7 Terminator Prirner «69337-3

SEQ ID No. 7 - 5'-GCTAGTTATTGCTCAGCGG-3 '.

Direct T7: SEQ _x T7 Reverse: SEQ.

: d. No. 8-5 '

ID No. 7 TAATACGACTCACTATAGGG - GGTAGTTATTGCTCAGGGG

Leading sequence: underlined; Coding region: bold Protein sequence _: active recombinant collagenase (Açromonaa Agdrophia ATCC 7966 '· colAhl (SEG. ID. No. 5) is expressed 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 - L Phenylanyanine - F, Leucine - L, Tryptophan M ..

Pi

P, 7a read on

W, Alanine - A, Cisternal Metiopine - · C, Asparagine - N, Giicine - G. Serina. - S, Glutamisa - Q, Tyrosine - Threonine - T, Aspartic acid - D, Glutamic acid - E.

The magnet

V,

SEQ ID Node, 5 ΜΙ4ΝΕ0ΤΡ.ΕΕΕΕΑΑΡ & ΕΕΑΤΕΑΕΑΝΥΕΡ3ΝΝβθΥ0ΕΚ0Ξ38Ε9Ι, ΟΟΚΙ.ΤΕ7ΤΥΕΙ, ΕΑΙ <ΕΤτ

GAPYQQLRWRREDWSDANLAALíAEQQCGEPQDKIELGWQIEYDSGEEíWPAGKAVPAG δΎΡΥΟΥύΜΝΟαΕνίΕΡΕΑΟΤΡΤΑΡΕΟΕΕΕΡΕΑΕΕνίΕάΗΕΕάΕΕηΕΟΕΕΝΑΕβΕσΑΕδΑΚ

QyQPQWQQLKVLEETPNEmiGRLEQIEFRDQPiWSQAAKQVSELHVWRQYTIftJLDEQA

KTQQECDEPQTRQEGDKTISYRVCRQTLPA.GSEVQYVDKDTGYQYPVGGSÊWQTLPETT

ΕΧ7αΕΕΚνΕΝΗΡΐνΕΑΕ: ΚΕΕ.ζ5 _: ΕΠ € ΕΝΑΟ6ΝΑ €: .9ΕΝϋΕΡ ^ ΤΕΙ..Ι.Ι} ΑΒΑΑΚΤν0ϋΑΕΟ0ΡΑΡ

WQSNYGHDDTKLLAVSSGIQSLLAANQPAHPAMELLLEYVRANNYHNYGíqíKEDGPAA

ΑΕΑΕΑΕΑΕΤΑΕβΑΗΡΝΕΕΡΕ £ Α3ΕΕνθΑ ^ 0ΑΝ8ΙΑΕΗΟ’ζ5ΒΚΕΡΑνθδΕΡ0ΤΙίΤ, ιΟΕΕΝζ) .KÍLAYSTRHASEINGQHAWATGLFnLLNFLDFASDYSDPFANDFR ^ QD ^ SIJS.KOPHALÇlí

ΕΕΕΑΚ4ΚσΡηθΑΕΕΕΕΕΡΝνΕΠΑΥΤΝΕΥΗνΑΕΥΤΚΡΡΕΕΠ6ΥΕΚΙ, ΝΏηΕνΐΑΙΑ ^ ΗΗΝ.Τ .. 'iPGGQQ.SQDLLEDMEt.TLSTYYLTYTDWEAGrSGDFAGLCTpVRVEDVLPFÈHTQSP

ΤίΗΕΗΑΟϋΝΤΝΡΟΑΕβίΕΕΕΕαΑΕΕΟαΕΗΟάΜΕΤ ^ ΝαΡνΑΒηΕΝΕΑΕΕΙ, ννΕΝΕΕΑβΐ ^ Κ

RYGSALFGGVSTDNGG1 ¥ LEGDPARPGNQARESAYEAEWKRPAFQWNLRHEYVHYLDG RFNQyGSFGMY ^ ElJRTTWSEGLASFIAHGGCEARGhDWAGEPASDHPALAUXLHLDY DKGJI íJJ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^

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Pina S, Vieira SI, Rego P, PM Torres, Cruz e Silva OA, Cruz e Silva SR, Ferreira JM {2010}. Biological Responses © f brushite-forming Zn- and ZnSr-sufestituted beta-ficalcium phosphate bone cements. Bur Cell I killed. 20: 162-77,

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VA Z, Wei I, X, Li J. Wang Y, Ni D, Yang P, Shang Y (2009). Percutaneous treatmeni of non-confined daytime disc herniation by ingestion of oxygen-ozone combined with collagenase. Sur 0 Radiol. 72 (3): 499-504.

The preferred embodiments described above are combinable with each other. The following claims further define preferred embodiments of the present invention.

(I) Q8S) The present invention is not, of course, in any way restricted to the embodiments described herein. A person of ordinary skill in the art may foresee many possibilities for modification thereof and substitution of technical characteristics for other equivalents, depending upon the particulars of the invention. requirements of each situation as defined in the appended claims.

Claims (2)

Isolated collagenase characterized by the sequence SEQ, ID. 5, Method for recombinant obtaining and active purification. Romanian A s, of collagenase described in claim 1, comprising the following steps: isolate Genomic DNA from a culture of Aeromonas amplify a sequence with a degree of homology. at least 95% with SEQ ID. 1 N ^s? integrating into an inducible expression vector the coding sequence for a fusion protein, wherein said coding sequence codes for a set of consecutive histidines; To produce and select the desired recombinant plasmid, select Cu. said cell; recombinant and induce expression of çolagenase: recombinant with appropriate inducer; collect and purify recombinant coiagenesis. A method for recombinant obtaining and purifying active descriptive collagenase from Aeromonar, claim 1 according to claim comprising the following steps; Progenous genomic DNA in the a) isolate flight attendants; b) amplifying the putative and collagenase gene of said culture with a sequence with a degree of homology of at least 95% with SEQ, ID. t. ^;? 1 - AHA ... 0517 using primers which include the restriction enzyme recognition site BopH.I and Aindllx; c) digesting a c: lonon vector comprising the loci for the enzymes Νησί and Hlndlll; d) cloning said βolagenase gene into said cloning vector; e) producing and selecting: the desired reeombinant plasmid; inserting said reeombinant plasmid into suitable host cells, preferably E. coli, namely: M. coli TuP10F 'to, transform and select reeorobinophil vectors, · g) transforming said: recombinant plasmid into reeombinant expression cells: preferably E, ccli, namely E, coli BL21 (DE3); h) selecting and cultivating said recombinant expression cells and inducing expression of reeombinant collagenase; i) collect and purify collagenase: reeombinant. A method for obtaining and purifying the active re -ombinant Aeromonas collagenase described in claim 1 according to claims 2-3, wherein said: Aercmónas are microorganisms of the species aeraonas hydrapa. ATCC V966 ^r . The method for obtaining and purifying Aeromonas active recombinant collagenase described in claim 1 according to claims 2-4, wherein said primers comprise the recognition site of the restriction enzymes SEQ, ΪΡ No, 2 or SEQ. . 1D No. 3 and further based on the SEQ coding nucleotide sequence. ID No. 1 of the pre-ColÂh polypeptide, including the signal peptide, A method for obtaining and purifying recombinant Aeromonas active eolagenase as described in claim 1 according to claims 2-5, wherein said primers comprise the following homologous sequences; SEQ ID No. 2, SEQ. T.D. No. 3. A method for obtaining and activating eolagenase purification The method described in claim 1 according to claims 2-6, wherein said amplification occurs by polymerase chain reactions. . δ. A method for obtaining and purifying recombinant active Aertwonas eolagenase as described in claim 1 according to the preceding claim, wherein said polymerase chain reactions are carried out in 50 µl volumes containing 3 mM MgClz, 250 pM dNIPs. 0.5 μΜ of said primers - SEQ. ID No. 2, SEQ. ID No. 3 -5% DMSO (v / v)> .1.5 D lag3NA polymerase in appropriate buffer and 50-100 ng SNA. Method: for obtaining and purifying Aeromonás active recombinant cobngenase, described in claim 1 according to claims 2-8, wherein said amplification is performed in a thermocycler. comprising the initial denaturation of the Aeromenas ερρ dna genómied · the temperature 91 - 96 ^ô C for 2 - 5 min; followed by 40 cycles of 30 s at 94-96 b, 30 sec 58 ° C and 3 min at 70-75 ° C, preferably 72 ° C; final extension 20-30 min at 68-75 ° C, preferably 72 ° C. A method for obtaining and purifying recombinant active Î ± -romenal collagenase as described in claim 1 according to claims 2-9, wherein the cloning vector is pET26b (i). A method for obtaining and purifying recombinant active eromonan collagenase, described in claim 1, according to claims 2-10, wherein the host cells for amplification of the recombinant vector are E. coli, preferably coli TOPIOF cells. · ' The method for obtaining and purifying eromonu collagenase as described in claim 1 according to claims 2 -1,1, wherein the host cells for overexpressing the active recombinant and in the recombinant protein are β cells. preferably oli BL21 (DE3). in A method for obtaining and purifying recombinant atheromatic recombinant collagenase described in claim 1 according to claims 2-12, wherein said host cells are cultured at 25 ° C - 37 '-. -c, and 100-150 rpm during J. 6-13 h, preferably 26 ° C, 130 rpm. A method for obtaining and purifying recombinant recombinant Î ± -ememone collagenase according to claim 1 according to claims 2-13, wherein the selection of a thione from step d) is by culturing in selective medium, wherein the positive elones are selected on kanamycin supplemented medium. The method for obtaining and purifying the active stewarding collagenase of Aeromeças described in claim 1 according to claim 2-14 was that the collection of recombinant collagenase is by centrifugation supernatant post er i or £ i1í chilled then dialysis connecting and at 1 ^G C raction. against tampon Method for. obtaining and purification from collagenase r e c omb i na nte active of Λ e r cm ona s, described in claim 1 according to claims 2-13, wherein said purification of recombinant collagenase is by affinity eromatography, preferably with immobilized metal ion. Isolated collagenase described in claim I for use as a medicament in medicine. The isolated collagenase described in claim 1 for _: the use in the treatment of traumatized tissues according to claim 17, namely in the treatment of traumatic wounds, varicose ulcers and burns or also in the treatment of other conditions associated with the trauma. excessive deposition Dupuytren's disease. of collagen like for example The 19 Descriptive collagenase in claim 1 for use in gene therapy or claim 17, electro-gene a deal with The 2 Q. Collagenase described in claim 1. for use Π ©: sciatica treatment according to claim 17, namely in herniated intervertebral discs. Composition characterized in that it comprises the collagenase described in claim 1. A composition according to the preceding claim wherein the collagenase is lyophilized. The composition of claims 21-22 further comprising a biomaterial, in particular chitosan. The composition of claims 21-23 wherein the composition is a topical formulation, uses oral or parental administration formulation or an injectable formulation, Use of the recombinant collagenase described in claim 1 in techniques for culturing animal cells and tissues such as isolation of endothelial cells, neuronal cells, cardiomyocytes, hepatoates and stem cells, Use of the recombinant collagenase described in claim I in the food industry, particularly in meat processing to improve its properties and textures. Use of the recombinant collagenase described in claim 1 in the tanning industry, in particular to aid in the leather dyeing process.