Abstract:
The use of matrix metalloproteinases, mutated and not mutated, for the preparation of pharmaceutical compositions useful in the treatment of pathologies associated with an accumulation of matrix bio-polymers and/or an excess of TIMPs (Tissue Inhihitors MetalloProteinases) is described; mutated matrix metalloproteinases, in which at least an aminoacid residue in a definite position in the protein, has been mutated into a hydrophilic and/or charged aminoacidic residue, obtaining an increased stability toward autoproteolysis are also described.

Description:
FIELD OF INVENTION 
       [0001]    The invention relates to the field of proteins and in particular to the use of matrix metalloproteinases, mutated and not mutated, for the preparation of pharmaceutical compositions, as well as to mutated matrix metalloproteinases, as defined hereinafter, having an increased stability toward autoproteolysis in respect to the corresponding not mutated proteins. 
       STATE OF ART 
       [0002]    Matrix metalloproteinases constitute a family of more than 20 different Zn-dependent enzymes, which are responsible for the degradation of extracellular matrix. The extracellular matrix components carry out a fundamental role in the modulation of cellular environment during growth, morphogenesis and tissue reparation processes. For this reason, the matrix metalloproteinases activities is subtly regulated both at transcription and activation level, as well as by the endogenous inhibitors action such as TIMPs (Tissue Inhibitors MetalloProteinases) and α 2 -macroglobuline. Changes in the delicate equilibrium which regulates the matrix metalloproteinase activities are connected with arising and development of various pathologies such as scleroderma, cardiosclerosis, nephrosclerosis, hepatic fibrosis, pulmonary fibrosis and pancreatic fibrosis. Many active principles are known as inhibitors of the enzymatic activities of matrix metalloproteinases, so they are used to prepare pharmaceutical compositions useful in the treatment of pathologies which are associated with an overexpression of metalloproteinases. On the contrary, as far as the Applicant is aware of, the matrix metalloproteinases are not directly used in the pharmaceutical field as active principles in the isolated and purified form. 
         [0003]    In spite of that, there is a huge request of these proteins both in academic and industrial fields, due to their relevance in physiological and pathological fields. Indeed, the availability of purified matrix metalloproteinases is a question of primary importance in the study of biological processes in which these proteins are involved, and in the development of candidate inhibitors. 
         [0004]    However, the use of matrix metalloproteinases is usually difficult, complicated and expensive, because of their tendency towards autoproteolysis and the consequent degradation of the protein itself. This behaviour is due to the fact that one or more aminoacidic sequences exposed on the protein surface, possess good affinities towards the catalytic site of the protein which, in conclusion, hydrolyse itself. 
         [0005]    To avoid this process of autoproteolysis and degradation, the use of matrix metalloproteinases requires very narrow working conditions that often make their use in experimental conditions, very intricate and sometimes even impossible. 
         [0006]    Their transport and preservation are very complicated too, indeed manufacturing firms recommend to preserve them at a temperature of −80° C. 
         [0007]    Another important restriction is the low concentration at which the researcher is forced to work to avoid autoproteolysis. This is another limit to the use of matrix metalloproteinases in many experiments. 
         [0008]    To the present day, to reduce autoproteolysis phenomenon, researchers used synthetic inhibitors or self-inhibited protein, i.e. a protein that is provided with a prodomain. Nevertheless, both strategies present many limitations and do not represent a solution for this problem. To overcome these difficulties mutants of matrix metalloproteinases have been produced, in which aminoacid Glutamate, which is present in the active site of the enzyme and is involved in catalytic mechanism, is replaced by an Alanine (Morgunova, E. et al.,  Science  1999, 284, 1667). Indeed the Glutamate, by the carboxylic group of the side chain, coordinates the water molecule that, during hydrolysis, is responsible of the nucleophilic attack on the peptidic carbonyl (Babine, R. E. et al.,  Chem. Rev.  1197, 97, 1359-1472); following to Glutamate substitution the protein loses the water molecule and it is not able to perform its catalytic action anymore. As it can be easily understood, this approach is not valid in the case in which it would be necessary to keep the catalytic activity, because it generates inactive proteins. 
         [0009]    The need is therefore deeply felt, to develop an alternative approach able to eliminate the susceptibility of these proteins to autoproteolysis without interfering neither with their catalytic activity, nor with their affinity for substrates and natural inhibitors. 
       SUMMARY OF THE INVENTION 
       [0010]    The Applicant has surprisingly found that mutation of an aminoacid in the catalytic domain of the matrix metalloproteinases, in a specific position far from the active site, is able to increase their stability towards autoproteolysis in respect to the wild-type, not mutated protein. 
         [0011]    Moreover, the Applicant has found that both mutated and wild-type matrix metalloproteinases can be used for the preparation of pharmaceutical compositions, and in particular of compositions useful in the treatment of pathologies to which an accumulation of matrix bio-polymers and/or an excess of TIMPs is associated, such as scleroderma, cardiosclerosis, nephrosclerosis, hepatic fibrosis, pulmonary fibrosis and pancreatic fibrosis. 
         [0012]    Subject of the present invention is therefore the catalytic domain of human matrix metalloproteinases, characterised in that said catalytic domain is mutated so that the aminoacidic residue corresponding to phenylalanine 171 according to the numbering of the sequence Accession No. P39900 (SwissProt), is an hydrophilic and/or charged aminoacidic residue. 
         [0013]    Further subjects of the invention are: the matrix metalloproteinases comprising as catalytic domain the above said mutated catalytic domain; the DNA sequence codifying the above said mutated matrix metalloproteinases; the recombinant vector comprising the above said DNA sequence; and the isolated cell transfected or transformed with the above said recombinant vector. 
         [0014]    A further subject of the invention is the use of human matrix metalloproteinase and of the catalytic domains thereof, optionally mutated as said above, for the preparation of pharmaceutical composition; and the so obtained pharmaceutical compositions. 
         [0015]    Further subject of the invention is the diagnostic kit for the diagnosis of pathologies to which an accumulation of matrix bio-polymers and/or an excess of TIMPs (Tissural Inhibitors of Matrix Metalloproteinases) is associated, comprising human matrix metalloproteinases or the catalytic domains thereof, optionally mutated as said above. 
         [0016]    Further subject of the invention is the use of human matrix metalloproteinases mutated as said above, or of the mutated catalytic domain thereof, as reagents for the pharmacological characterisation of the matrix metalloproteinase as pharmaceutical target. 
         [0017]    Features and advantages of the invention will be described in detail in the following description. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0018]      FIG. 1 : SDS (Sodium Dodecyl Sulphate)/polyacrylamide gel electrophoresis of wild-type and mutated MMP3 (SEQ ID No. 3) as illustrated in Example 3. 
           [0019]      FIG. 2 : SDS/polyacrylamide gel electrophoresis of wild-type and mutated MMP10 (SEQ ID No. 10) as illustrated in Example 4. 
           [0020]      FIG. 3 : SDS/polyacrylamide gel electrophoresis of wild-type and mutated MMP13 (SEQ ID No. 10) as illustrated in Example 5. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    In the present invention, by the expression “hydrophilic and/or charged aminoacidic residue” is meant, for example, an aminoacidic residue selected from the group consisting of glutamine, asparagine, aspartic acid and glutamic acid; aspartic acid is preferably meant. 
         [0022]    In the present invention, by the expression “aminoacidic residue corresponding to the phenylalanine 171 according to the numbering of the sequence Accession No. P39900 (Swiss Prot)” is meant the aminoacidic residue in the same position of the above said phenylalanine 171 as resulting in a multiple alignment, as defined hereinafter. 
         [0023]    For the various matrix metalloproteinases (hereinafter referred to with the abbreviation MMP) not having, as such, an hydrophilic and/or charged residue in the corresponding position as defined above, i.e. for all MMPs with the exclusion of MMP-1 (SEQ ID No. 1), MMP-8 (SEQ ID No. 5), and MMP-11 (SEQ ID No. 8), the mutation is located in the following positions, identified through the multiple alignment:
       for human MMP-2 (SEQ ID No. 2) and all its isoforms determined by alternative “splicing”, the aminoacidic residue that is mutated is glycine 181 according to the numbering of the sequence Accession No. P08253 (SwissProt);   for human MMP-3 (SEQ ID No. 3) and all its isoforms determined by alternative “splicing”, the aminoacidic residue that is mutated is phenylalanine 171 according to the numbering of the sequence Accession No. P08254 (SwissProt) or Q6GRF8 (TrEMBL);   for human MMP-7 (SEQ ID No. 4) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is serine 166 according to the numbering of the sequence Accession No. P09237 (SwissProt);   for human MMP-9 (SEQ ID No. 6) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is glycine 178 according to the numbering of the sequence Accession No. P14780 (SwissProt);   for human MMP-10 (SEQ ID No. 7) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is phenylalanine 170 according to the numbering of the sequence Accession No. P09238 (SwissProt) or Q53HH9 (TrEMBL);   for human MMP-12 (SEQ ID No. 9) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is phenylalanine 171 according to the numbering of the sequence Accession No. P39900 (SwissProt);   for human MMP-13 (SEQ ID No. 10) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is phenylalanine 175 according to the numbering of the sequence Accession No. P45452 (SwissProt) or Q7Z5M0, Q7Z5M1 and Q6NWN6 (TrEMBL);   for human MMP-14 (SEQ ID No. 11) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is serine 189 according to the numbering of the sequence Accession No. P50281 (SwissProt) and Q6GSF3 (TrEMBL);   for human MMP-15 (SEQ ID No. 12) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is serine 209 according to the numbering of the sequence Accession No. P51511 (SwissProt) and Q7KZY0 (TrEMBL);   for human MMP-16 (SEQ ID No. 13 and SEQ ID No. 14) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is serine 196 according to the numbering of the sequence Accession No. P51512 (SwissProt), or Q52H48 and Q14824 (TrEMBL);   for human MMP-17 (SEQ ID No. 15) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is glycine 200 according to the numbering of the sequence Accession No. Q9ULZ9 (SwissProt) or Q5U5M0 and Q81WC3 (TrEMBL);   for human MMP-19 (SEQ ID No. 16 and SEQ ID No. 17) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is tyrosine 165 according to the numbering of the sequence Accession No. Q99542 (TrEMBL);   for human MMP-20 (SEQ ID No. 18) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is serine 179 according to the numbering of the sequence Accession No. 060882 (TrEMBL);   for human MMP-21 (SEQ ID No. 19) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is cysteine 238 according to the numbering of the sequence Accession No. Q5VZP9 and Q8N119 (TrEMBL);   for human MMP-23A (SEQ ID No. 20) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is cysteine 152 according to the numbering of the sequence Accession No. O75900 (TrEMBL);   for human MMP-23B (SEQ ID No. 21) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is cysteine 152 according to the numbering of the sequence Accession No. Q9UBR9 (TrEMBL);   for human MMP-24 (SEQ ID No. 22) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is serine 232 according to the numbering of the sequence Accession No. Q9Y5R2 and Q9H440 (TrEMBL);   for human MMP-25 (SEQ ID No. 23) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is serine 182 according to the numbering of the sequence Accession No. Q9NPA2 (TrEMBL);   for human MMP-26 (SEQ ID No. 24) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is glycine 161 according to the numbering of the sequence Accession No. Q9NRE1 (TrEMBL);   for human MMP-27 (SEQ ID No. 25) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is cysteine 168 according to the numbering of the sequence Accession No. Q9H306 and Q6UWK6 (TrEMBL);   for human MMP-28 (SEQ ID No. 26 and SEQ ID No. 27) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is glycine 193 according to the numbering of the sequence Accession No. Q9H239 and Q9BUG8 (TrEMBL);   for human MMP-like 1 (SEQ ID No. 28) and all its isoforms determined by the alternative “splicing”, the aminoacidic residue that is mutated is serine 106 according to the numbering of the sequence Accession No. O43923 (TrEMBL);       
 
         [0046]    For the alignment the program ClustalW (1.6) has been used with the following parameters: substitution matrix Gonnet 250, Gap open 10, Gap close −1, Gap extension −2, Gap distance 4. For a complete description of the alignment methodology see Andreini C. et al.,  J. of Proteome Research,  2004, 3, 21, which is herewith incorporated by reference. 
         [0047]    As showed in the following examples, the mutated proteins according to the invention presented a much more increasing stability in respect to the corresponding wild-type, not mutated protein. This makes more efficacious the use of the present mutated proteins not only as a scientific research instrument, for instance as a pharmaceutical tool to test new drugs having the matrix metalloproteinases as pharmaceutical target, but also as a pharmaceutical active principle. 
         [0048]    The following examples are reported to illustrate, and not to limit the invention. 
       Example 1 
       [0049]    According to the invention, the mutation of the aminoacidic residues has been obtained by specific site mutagenesis. A couple of nucleotides has been synthesised to this aim, wherein the nucleotides are constituted of 35 basis complementary to the wild type gene with the exclusion of the triplet codifying the aminoacid to be mutated in the protein. With these oligonucleotides a PCR reaction has been carried out on the plasmid containing the wild type gene at an annealing t m  temperature reduced by at least 15-20° C. respect to t m  of the oligonucleotides. A plasmid containing the mutated gene has been so obtained. The reaction mixture has been transformed in  E. coli  cells, and the plasmids contained in these cells have been isolated. The plasmid containing the mutated gene has been identified by gene sequencing. 
       Example 2 
       [0050]    Catalytic domains of MMPs, wild type and mutated, have been cloned, expressed and purified according to the following procedure. 
         [0051]    The cDNA of each proMMP has been cloned in a pET21 vector (Novagen). The resulting vector has been used for the transformation of BL21 cells of  Escherichia coli . A culture of the latter transformed cells have been grown in Luria-Bertani medium at a temperature of 37° C. The expression of each protein is induced during the phase of exponential growing by adding of IPTG (isopropyl-beta-D-thiogalactopyranoside) 0.5 mM. Cells have been harvested and lysated, 4 hours later the induction. After the cellular lysis, inclusion bodies have been collected and dissolved in a solution of 6 M urea and 20 mM Tris-HCl at pH 8. Then the proteins have been purified using an Hiprep 16/10 (20 ml) QFF (Pharmacia) with a buffer containing 6 M urea, 20 mM Tris-HCl at pH 8 and eluting with a linear gradient of NaCl until 0.35 M. Every purified protein has been refolded by subsequent dialysis with solutions containing 50 mM Tris-HCl (pH 7.2), 10 mM CaCl 2 , 0.1 mM ZnCl 2 , 0.3 M NaCl and 0.2 M AHA (Acetohydroxamic acid). During the refolding phases each protein is activated by prodomain cutting. In the final refolding dialysis the AHA concentration is equal to 0.5 M. For the isolation of the single catalytic domain is carried out a chromatographic column Superdex 75 16/60 (Pharmacia) eluted with the buffer of the last dialysis. 
         [0052]    The aminoacid sequences of the MMPs, prepared and purified as described above, are reported in the following. 
         [0000]    
       
         
               
             
           
               
                 SEQ ID No. 1 MMP-1 P03956 
               
               
                 &gt;gi|116852|sp|P03956|MMP1_HUMAN Interstitial 
               
               
                 collagenase precursor (Matrix metalloproteinase-1) 
               
               
                 (MMP-1) (Fibroblast collagenase) 
               
               
                 MHSFPPLLLLLFWGWSHSFPATLETQEQDVDLVQKYLEKYYNLKNDGRQV 
               
               
                   
               
               
                 EKRRNSGPVVEKLKQMQEFFGLKVTGKPDAETLKVMKQPRCGVPDVAQFV 
               
               
                   
               
               
                 LTEGNPRWEQTHLTYRIENYTPDLPRADVDHAIEKAFQLWSNVTPLTFTK 
               
               
                   
               
               
                 VSEGQADIMISFVRGDHRD N SPFDGPGGNLAHAFQPGPGIGGDAHFDEDE 
               
               
                   
               
               
                 RWTNNFREYNLHRVAAHELGHSLGLSHSTDIGALMYPSYTFSGDVQLAQD 
               
               
                   
               
               
                 DIDGIQAIYGRSQNPVQPIGPQTPKACDSKLTFDAITTIRGEVMFFKDRF 
               
               
                   
               
               
                 YMRTNPFYPEVELNFISVFWPQLPNGLEAAYEFADRDEVRFFKGNKYWAV 
               
               
                   
               
               
                 QGQNVLHGYPKDIYSSFGFPRTVKHIDMLSEENTGKTYFFVANKYWRYDE 
               
               
                   
               
               
                 YKRSMDPGYPKMIAHDFPGIGHKVDAVFMKDGFFYFFHGTRQYKFDPKTK 
               
               
                   
               
               
                 RILTLQKANSWFNCRKN 
               
               
                   
               
               
                 SEQ ID No. 2 MMP-2 P08253 
               
               
                 &gt;gi|116856|sp|P08253|MMP2_HUMAN 72 kDa type IV 
               
               
                 collagenase precursor (72 kDa gelatinase) (Matrix 
               
               
                 metalloproteinase-2) (MMP-2) (Gelatinase A) 
               
               
                 (TBE-1) 
               
               
                 MEALMARGALTGPLRALCLLGCLLSHAAAAPSPIIKFPGDVAPKTDKELA 
               
               
                   
               
               
                 VQYLNTFYGCPKESCNLFVLKDTLKKMQKFFGLPQTGDLDQNTIETMRKP 
               
               
                   
               
               
                 RCGNPDVANYNFFPRKPKWDKNQITYRIIGYTPDLDPETVDDAFARAFQV 
               
               
                   
               
               
                 WSDVTPLRFSRIHDGEADIMINFGRWEHGD G YPFDGKDGLLAHAFAPGTG 
               
               
                   
               
               
                 VGGDSHFDDDELWTLGEGQVVRVKYGNADGEYCKFPFLFNGKEYNSCTDT 
               
               
                   
               
               
                 GRSDGFLWCSTTYNFEKDGKYGFCPHEALFTMGGNAEGQPCKFPFRFQGT 
               
               
                   
               
               
                 SYDSCTTEGRTDGYRWCGTTEDYDRDKKYGFCPETAMSTVGGNSEGAPCV 
               
               
                   
               
               
                 FPFTFLGNKYESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQGYSLFLV 
               
               
                   
               
               
                 AAHEFGHAMGLEHSQDPGALMAPIYTYTKNFRLSQDDIKGIQELYGASPD 
               
               
                   
               
               
                 IDLGTGPTPTLGPVTPEICKQDIVFDGIAQIRGEIFFFKDRFIWRTVTPR 
               
               
                   
               
               
                 DKPMGPLLVATFWPELPEKIDAVYEAPQEEKAVFFAGNEYWIYSASTLER 
               
               
                   
               
               
                 GYPKPLTSLGLPPDVQRVDAAFNWSKNKKTYIFAGDKFWRYNEVKKKMDP 
               
               
                   
               
               
                 GFPKLIADAWNAIPDNLDAVVDLQGGGHSYFFKGAYYLKLENQSLKSVKF 
               
               
                   
               
               
                 GSIKSDWLGC 
               
               
                   
               
               
                 SEQ ID No. 3 MMP-3 P08254 
               
               
                 &gt;gi|116857|sp|P08254|MMP3_HUMAN Stromelysin-1 
               
               
                 precursor (Matrix metalloproteinase-3) (MMP-3) 
               
               
                 (Transin-1) (SL-1) 
               
               
                 MKSLPILLLLCVAVCSAYPLDGMRGEDTSMNLVQKYLENYYDLKKDVKQF 
               
               
                   
               
               
                 VRRKDSGPVVKKIREMQKFLGLEVTGKLDSDTLEVMRKPRCGVPDVGHFR 
               
               
                   
               
               
                 TFPGIPKWRKTHLTYRIVNYTPDLPKDAVDSAVEKALKVWEEVTPLTFSR 
               
               
                   
               
               
                 LYEGEADIMISFAVREHGD F YPFDGPGNVLAHAYAPGPGINGDAHFDDDE 
               
               
                   
               
               
                 QWTKDTTGTNLFLVAAHEIGHSLGLFHSANTEALMYPLYHSLTDLTRFRL 
               
               
                   
               
               
                 SQDDINGIQSLYGPPPDSPETPLVPTEPVPPEPGTPANCDPALSFDAVST 
               
               
                   
               
               
                 LRGEILIFKDRHFWRKSLRKLEPELHLISSFWPSLPSGVDAAYEVTSKDL 
               
               
                   
               
               
                 VFIFKGNQFWAIRGNEVRAGYPRGIHTLGFPPTVRKIDAAISDKEKNKTY 
               
               
                   
               
               
                 FFVEDKYWRFDEKRNSMEPGFPKQIAEDFPGIDSKIDAVFEEFGFFYFFT 
               
               
                   
               
               
                 GSSQLEFDPNAKKVTHTLKSNSWLNC 
               
               
                   
               
               
                 SEQ ID No. 4 MMP-7 P09237 
               
               
                 &gt;gi|116861|sp|P09237|MMP7_HUMAN Matrilysin 
               
               
                 precursor (Pump-1 protease) (Uterine 
               
               
                 metalloproteinase) (Matrix metalloproteinase-7) 
               
               
                 (MMP-7) (Matrin) 
               
               
                 MRLTVLCAVCLLPGSLALPLPQEAGGMSELQWEQAQDYLKRFYLYDSETK 
               
               
                   
               
               
                 NANSLEAKLKEMQKFFGLPITGMLNSRVIEIMQKPRCGVPDVAEYSLFPN 
               
               
                   
               
               
                 SPKWTSKVVTYRIVSYTRDLPHITVDRLVSKALNMWGKEIPLHFRKVVWG 
               
               
                   
               
               
                 TADIMIGFARGAHGD S YPFDGPGNTLAHAFAPGTGLGGDAHFDEDERWTD 
               
               
                   
               
               
                 GSSLGINFLYAATHELGHSLGMGHSSDPNAVMYPTYGNGDPQNFKLSQDD 
               
               
                   
               
               
                 IKGIQKLYGKRSNSRKK 
               
               
                   
               
               
                 SEQ ID No. 5 MMP-8 P22894 
               
               
                 &gt;gi|116862|sp|P22894|MMP8_HUMAN Neutrophil 
               
               
                 collagenase precursor (Matrix metalloproteinase-8) 
               
               
                 (MMP-8) (PMNL collagenase) (PMNL-CL) 
               
               
                 MFSLKTLPFLLLLHVQISKAFPVSSKEKNTKTVQDYLEKFYQLPSNQYQS 
               
               
                   
               
               
                 TRKNGTNVIVEKLKEMQRFFGLNVTGKPNEETLDMMKKPRCGVPDSGGFM 
               
               
                   
               
               
                 LTPGNPKWERTNLTYRIRNYTPQLSEAEVERAIKDAFELWSVASPLIFTR 
               
               
                   
               
               
                 ISQGEADINIAFYQRDHGD N SPFDGPNGILAHAFQPGQGIGGDAHFDAEE 
               
               
                   
               
               
                 TWTNTSANYNLFLVAAHEFGHSLGLAHSSDPGALMYPNYAFRETSNYSLP 
               
               
                   
               
               
                 QDDIDGIQAIYGLSSNPIQPTGPSTPKPCDPSLTFDAITTLRGEILFFKD 
               
               
                   
               
               
                 RYFWRRHPQLQRVEMNFISLFWPSLPTGIQAAYEDFDRDLIFLFKGNQYW 
               
               
                   
               
               
                 ALSGYDILQGYPKDISNYGFPSSVQAIDAAVFYRSKTYFFVNDQFWRYDN 
               
               
                   
               
               
                 QRQFMEPGYPKSISGAFPGIESKVDAVFQQEHFFHVFSGPRYYAFDLIAQ 
               
               
                   
               
               
                 RVTRVARGNKWLNCRYG 
               
               
                   
               
               
                 SEQ ID No. 6 MMP-9 P14780 
               
               
                 &gt;gi|116863|sp|P14780|MMP9_HUMAN Matrix metallo- 
               
               
                 proteinase-9 precursor (MMP-9) (92 kDa type 
               
               
                 IV collagenase) (92 kDa gelatinase) (Gelatinase B) 
               
               
                 (GELB) [Contains: 67 kDa matrix metalloproteinase- 
               
               
                 9; 82 kDa matrix metalloproteinase-9] 
               
               
                 MSLWQPLVLVLLVLGCCFAAPRQRQSTLVLFPGDLRTNLTDRQLAEEYLY 
               
               
                   
               
               
                 RYGYTRVAEMRGESKSLGPALLLLQKQLSLPETGELDSATLKAMRTPRCG 
               
               
                   
               
               
                 VPDLGRFQTFEGDLKWHHHNITYWIQNYSEDLPRAVIDDAFARAFALWSA 
               
               
                   
               
               
                 VTPLTFTRVYSRDADIVIQFGVAEHGD G YPFDGKDGLLAHAFPPGPGIQG 
               
               
                   
               
               
                 DAHFDDDELWSLGKGVVVPTRFGNADGAACHFPFIFEGRSYSACTTDGRS 
               
               
                   
               
               
                 DGLPWCSTTANYDTDDRFGFCPSERLYTRDGNADGKPCQFPFIFQGQSYS 
               
               
                   
               
               
                 ACTTDGRSDGYRWCATTANYDRDKLFGFCPTRADSTVMGGNSAGELCVFP 
               
               
                   
               
               
                 FTFLGKEYSTCTSEGRGDGRLWCATTSNFDSDKKWGFCPDQGYSLFLVAA 
               
               
                   
               
               
                 HEFGHALGLDHSSVPEALMYPMYRFTEGPPLHKDDVNGIRHLYGPRPEPE 
               
               
                   
               
               
                 PRPPTTTTPQPTAPPTVCPTGPPTVHPSERPTAGPTGPPSAGPTGPPTAG 
               
               
                   
               
               
                 PSTATTVPLSPVDDACNVNIFDAIAEIGNQLYLFKDGKYWRFSEGRGSRP 
               
               
                   
               
               
                 QGPFLIADKWPALPRKLDSVFEEPLSKKLFFFSGRQVWVYTGASVLGPRR 
               
               
                   
               
               
                 LDKLGLGADVAQVTGALRSGRGKMLLFSGRRLWRFDVKAQMVDPRSASEV 
               
               
                   
               
               
                 DRMFPGVPLDTHDVFQYREKAYFGQDRFYWRVVSSRSELNQVDQVGYVTY 
               
               
                   
               
               
                 DILQCPED 
               
               
                   
               
               
                 SEQ ID No. 7 MMP-10 P09238 
               
               
                 &gt;gi|116869|sp|P09238|MMP10_HUMAN Stromelysin-2 
               
               
                 precursor (Matrix metalloproteinase-10) (MMP-10) 
               
               
                 (Transin-2) (SL-2) 
               
               
                 MMHLAFLVLLCLPVCSAYPLSGAAKEEDSNKDLAQQYLEKYYNLEKDVKQ 
               
               
                   
               
               
                 FRRKDSNLIVKKIQGMQKFLGLEVTGKLDTDTLEVMRKPRCGVPDVGHFS 
               
               
                   
               
               
                 SFPGMPKWRKTHLTYRIVNYTPDLPRDAVDSAIEKALKVWEEVTPLTFSR 
               
               
                   
               
               
                 LYEGEADIMISFAVKEHGD F YSFDGPGHSLAHAYPPGPGLYGDIHFDDDE 
               
               
                   
               
               
                 KWTEDASGTNLFLVAAHELGHSLGLFHSANTEALMYPLYNSFTELAQFRL 
               
               
                   
               
               
                 SQDDVNGIQSLYGPPPASTEEPLVPTKSVPSGSEMPAKCDPALSFDAIST 
               
               
                   
               
               
                 LRGEYLFFKDRYFWRRSHWNPEPEFHLISAFWPSLPSYLDMYEVNSRDTV 
               
               
                   
               
               
                 FIFKGNEFWAIRGNEVQAGYPRGIHTLGFPPTIRKIDAAVSDKEKKKTYF 
               
               
                   
               
               
                 FMDKYWRFDENSQSMEQGFPRLIADDFPGVEPKVDAVLQAFGFFYFFSGS 
               
               
                   
               
               
                 SQFEFDPNARMVTHILKSNSWLHC 
               
               
                   
               
               
                 SEQ ID No. 8 MMP-11 P24347 
               
               
                 &gt;gi|116871|sp|P24347|MMP11 _HUMAN Stromelysin-3 
               
               
                 precursor (Matrix metalloproteinase-11) (MMP-11) 
               
               
                 (ST3) (SL-3) 
               
               
                 MAPMWLRSAAARALLPPMLLLLLQPPPLLARALPPDVHHLHAERRGPQPW 
               
               
                   
               
               
                 HAALPSSPAPAPATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKRFVLS 
               
               
                   
               
               
                 GGRWEKTDLTYRILRFPWQLVQEQVRQTMAEALKVWSDVTPLTFTEVHEG 
               
               
                   
               
               
                 RADIMIDFARYWDGD D LPFDGPGGILAHAFFPKTHREGDVHFDYDETWTI 
               
               
                   
               
               
                 GDDQGTDLLQVAAHEFGHVLGLQHTTAAKALMSAFYTFRYPLSLSPDDCR 
               
               
                   
               
               
                 GVQHLYGQPWPTVTSRTPALGPQAGIDTNEIAPLEPDAPPDACEASFDAV 
               
               
                   
               
               
                 STIRGELFFFKAGFVWRLRGGQLQPGYPALASRHWQGLPSPVDAAFEDAQ 
               
               
                   
               
               
                 GHIWFFQGAQYWVYDGEKPVLGPAPLTELGLVRFPVHAALVWGPEKNKIY 
               
               
                   
               
               
                 FFRGRDYVVRFHPSTRRVDSPVPRRATDWRGVPSEIDAAFQDADGYAYFL 
               
               
                   
               
               
                 RGRLYWKFDPVKVKALEGFPRLVGPDFFGCAEPANTFL 
               
               
                   
               
               
                 SEQ ID No. 9 MMP-12 P39900 
               
               
                 &gt;gi|729179|sp|P39900|MMP12_HUMAN Macrophage 
               
               
                 metalloelastase precursor (HME) (Matrix 
               
               
                 metalloproteinase-12) (MMP-12) (Macrophage 
               
               
                 elastase) (ME) 
               
               
                 MKFLLILLLQATASGALPLNSSTSLEKNNVLFGERYLEKFYGLEINKLPV 
               
               
                   
               
               
                 TKMKYSGNLMKEKIQEMQHFLGLKVTGQLDTSTLEMMHAPRCGVPDVHHF 
               
               
                   
               
               
                 REMPGGPVWRKHYITYRINNYTPDMNREDVDYAIRKAFQVWSNVTPLKFS 
               
               
                   
               
               
                 KINTGMADILVVFARGAHGD F HAFDGKGGILAHAFGPGSGIGGDAHFDED 
               
               
                   
               
               
                 EFWTTHSGGTNLFLTAVHEIGHSLGLGHSSDPKAVMFPTYKYVDINTFRL 
               
               
                   
               
               
                 SADDIRGIQSLYGDPKENQRLPNPDNSEPALCDPNLSFDAVTTVGNKIFF 
               
               
                   
               
               
                 FKDRFFWLKVSERPKTSVNLISSLWPTLPSGIEAAYEIEARNQVFLFKDD 
               
               
                   
               
               
                 KYWLISNLRPEPNYPKSIHSFGFPNFVKKIDAAVFNPRFYRTYFFVDNQY 
               
               
                   
               
               
                 WRYDERRQMMDPGYPKLITKNFQGIGPKIDAVFYSKNKYYYFFQGSNQFE 
               
               
                   
               
               
                 YDFLLQRITKTLKSNSWFGC 
               
               
                   
               
               
                 SEQ ID No. 10 MMP-13 P45452 
               
               
                 &gt;gi|1168998|sp|P45452|MMP13_HUMAN Collagenase 3 
               
               
                 precursor (Matrix metalloproteinase-13) (MMP-13) 
               
               
                 MHPGVLAAFLFLSWTHCRALPLPSGGDEDDLSEEDLQFAERYLRSYYHPT 
               
               
                   
               
               
                 NLAGILKENAASSMTERLREMQSFFGLEVTGKLDDNTLDVMKKPRCGVPD 
               
               
                   
               
               
                 VGEYNVFPRTLKWSKMNLTYRIVNYTPDMTHSEVEKAFKKAFKVWSDVTP 
               
               
                   
               
               
                 LNFTRLHDGIADIMISFGIKEHGD F YPFDGPSGLLAHAFPPGPNYGGDAH 
               
               
                   
               
               
                 FDDDETWTSSSKGYNLFLVAAHEFGHSLGLDHSKDPGALMFPIYTYTGKS 
               
               
                   
               
               
                 HFMLPDDDVQGIQSLYGPGDEDPNPKHPKTPDKCDPSLSLDAITSLRGET 
               
               
                   
               
               
                 MIFKDRFFWRLHPQQVDAELFLTKSFWPELPNRIDAAYEHPSHDLIFIFR 
               
               
                   
               
               
                 GRKFWALNGYDILEGYPKKISELGLPKEVKKISAAVHFEDTGKTLLFSGN 
               
               
                   
               
               
                 QVWRYDDTNHIMDKDYPRLIEEDFPGIGDKVDAVYEKNGYIYFFNGPIQF 
               
               
                   
               
               
                 EYSIWSNRIVRVMPANSILWC 
               
               
                   
               
               
                 SEQ ID No. 11 MMP-14 P50281 
               
               
                 &gt;gi|60392771|sp|P50281|MMP14_HUMAN Matrix metallo- 
               
               
                 proteinase-14 precursor (MMP-14) (Membrane-type 
               
               
                 matrix metalloproteinase 1) (MT-MMP 1) (MTMMP1) 
               
               
                 (Membrane-type-i matrix metalloproteinase) 
               
               
                 (MT1-MMP) (MT1MMP) (MMP-X1) 
               
               
                 MSPAPRPSRCLLLPLLTLGTALASLGSAQSSSFSPEAWLQQYGYLPPGDL 
               
               
                   
               
               
                 RTHTQRSPQSLSAAIAAMQKFYGLQVTGKADADTMKAMRRPRCGVPDKFG 
               
               
                   
               
               
                 AEIKANVRRKRYAIQGLKWQHNEITFCIQNYTPKVGEYATYEAIRKAFRV 
               
               
                   
               
               
                 WESATPLRFREVPYAYIREGHEKQADIMIFFAEGFHGD S TPFDGEGGFLA 
               
               
                   
               
               
                 HAYFPGPNIGGDTHFDSAEPWTVRNEDLNGNDIFLVAVHELGHALGLEHS 
               
               
                   
               
               
                 SDPSAIMAPFYQWMDTENFVLPDDDRRGIQQLYGGESGFPTKMPPQPRTT 
               
               
                   
               
               
                 SRPSVPDKPKNPTYGPNICDGNFDTVAMLRGEMFVFKERWFWRVRNNQVM 
               
               
                   
               
               
                 DGYPMPIGQFWRGLPASINTAYERKDGKFVFFKGDKHWVFDEASLEPGYP 
               
               
                   
               
               
                 KHIKELGRGLPTDKIDAALFWMPNGKTYFFRGNKYYRFNEELRAVDSEYP 
               
               
                   
               
               
                 KNIKVWEGIPESPRGSFMGSDEVFTYFYKGNKYWKFNNQKLKVEPGYPKS 
               
               
                   
               
               
                 ALRDWMGCPSGGRPDEGTEEETEVIIIEVDEEGGGAVSAAAVVLPVLLLL 
               
               
                   
               
               
                 LVLAVGLAVFFFRRHGTPRRLLYCQRSLLDKV 
               
               
                   
               
               
                 SEQ ID No. 12 MMP-15 P51511 
               
               
                 &gt;gi|705988|sp|P51511|MMP15_HUMAN Matrix metallo- 
               
               
                 proteinase-15 precursor (MMP-1 ) (Membrane-type 
               
               
                 matrix metalloproteinase 2) (MT-MMP 2) (MTMMP2) 
               
               
                 (Membrane-type-2 matrix metalloproteinase) 
               
               
                 (MT2-MMP) (MT2MMP) (SMCP-2) 
               
               
                 MGSDPSAPGRPGWTGSLLGDREEMRPRLLPLLLVLLGCLGLGVAAEDAEV 
               
               
                   
               
               
                 HAENWLRLYGYLPQPSRHMSTMRSAQILASALAEMQRFYGIPVTGVLDEE 
               
               
                   
               
               
                 TKEWMKRPRCGVPDQFGVRVKANLRRRRKRYALTGRKWNNHHLTFSIQNY 
               
               
                   
               
               
                 TEKLGWYHSMEAVRRAFRVWEQATPLVFQEVPYEDIRLRRQKEADIMVLF 
               
               
                   
               
               
                 ASGFHGD S SPFDGTGGFLAHAYFPGPGLGGDTHFDADEPWTFSSTDLHGN 
               
               
                   
               
               
                 NLFLVAVHELGHALGLEHSSNPNAIMAPFYQWKDVDNFKLPEDDLRGIQQ 
               
               
                   
               
               
                 LYGTPDGQPQPTQPLPTVTPRRPGRPDHRPPRPPQPPPPGGKPERPPKPG 
               
               
                   
               
               
                 PPVQPRATERPDOYGPNICDGDFDTVAMLRGEMFVFKGRWFWRVRHNRVL 
               
               
                   
               
               
                 DNYPMPIGHFWRGLPGDISAAYERQDGRFVFFKGDRYWLFREANLEPGYP 
               
               
                   
               
               
                 QPLTSYGLGIPYDRIDTAIWWEPTGHTFFFQEDRYWRFNEETQRGDPGYP 
               
               
                   
               
               
                 KPISVWQGIPASPKGAFLSNDAAYTYFYKGTKYWKFDNERLRMEPGYPKS 
               
               
                   
               
               
                 ILRDFMGCQEHVEPGPRWPDVARPPFNPHGGAEPGADSAEGDVGDGDGDF 
               
               
                   
               
               
                 GAGVNKDGGSRVVVQMEEVARTVNVVMVLVPLLLLLCVLGLTYALVQMQR 
               
               
                   
               
               
                 KGAPRVLLYCKRSLQEWV 
               
               
                   
               
               
                 SEQ ID No. 13 MMP-16 P51512 
               
               
                 Isoform 1 
               
               
                 &gt;gi|3041669|sp|P51512|MMP16_HUMAN Matrix metallo- 
               
               
                 proteinase-16 precursor (MMP-16) (Membrane-type 
               
               
                 matrix metalloproteinase 3) (MT-MMP 3) (MTMMP3) 
               
               
                 (Membrane-type-3 matrix metalloproteinase) 
               
               
                 (MT3-MMP) (MT3MMP) (MMP-X2) 
               
               
                 MILLTFSTGRRLDFVHHSGVFFLQTLLWILCATVCGTEQYFNVEVWLQKY 
               
               
                   
               
               
                 GYLPPTDPRMSVLRSAETMQSALAAMQQFYGINMTGKVDRNTIDWMKKPR 
               
               
                   
               
               
                 CGVPDOTRGSSKFHIRRKRYALTGQKWQHKHITYSIKNVTPKVGDPETRK 
               
               
                   
               
               
                 AIRRAFDVWQNVTPLTFEEVPYSELENGKRDVDITIIFASGFHGD S SPFD 
               
               
                   
               
               
                 GEGGFLAHAYFPGPGIGGDTHFDSDEPWTLGNPNHDGNDLFLVAVHELGH 
               
               
                   
               
               
                 ALGLEHSNDPTAIMAPFYQYMETDNFKLPNDDLQGIQKIYGPPDKIPPPT 
               
               
                   
               
               
                 RPLPTVPPHRSIPPADPRKNDRPKPPRPPTGRPSYPGAKPNICDGNFNTL 
               
               
                   
               
               
                 AILRREMFVFKDQWFWRVRNNRVMDGYPMQITYFWRGLPPSIDAVYENSD 
               
               
                   
               
               
                 GNFVFFKGNKYWVFKDTTLQPGYPHDLITLGSGIPPHGIDSAIWWEDVGK 
               
               
                   
               
               
                 TYFFKGDRYWRYSEEMKTMDPGYPKPITVWKGIPESPQGAFVHKENGFTY 
               
               
                   
               
               
                 FYKGKEYWKFNNQILKVEPGYPRSILKDFMGCDGPTDRVKEGHSPPDDVD 
               
               
                   
               
               
                 IVIKLDNTASTVKAIAIVIPCILALCLLVLVYTVFQFKRKGTPRHILYCK 
               
               
                   
               
               
                 RSMQEWV 
               
               
                   
               
               
                 SEQ ID No. 14 
               
               
                 Isoform 2 
               
               
                 &gt;gi|13027800|ref|NP_072086.1| matrix metallo- 
               
               
                 proteinase 16 isoform 2 [ Homo sapiens ] 
               
               
                 MILLTFSTGRRLDFVHHSGVFFLQTLLWILCATVCGTEQYFNVEVWLQKY 
               
               
                   
               
               
                 GYLPPTDPRMSVLRSAETMQSALAAMQQFYGINMTGKVDRNTIDWMKKPR 
               
               
                   
               
               
                 CGVPDQTRGSSKFHIRRKRYALTGQKWQHKHITYSIKNVTPKVGDPETRK 
               
               
                   
               
               
                 AIRRAFDVWQNVTPLTFEEVPYSELENGKRDVDITIIFASGFHGD S SPFD 
               
               
                   
               
               
                 GEGGFLAHAYFPGPGIGGDTHFDSDEPWTLGNPNHDGNDLFLVAVHELGH 
               
               
                   
               
               
                 ALGLEHSNDPTAIMAPFYQYMETDNFKLPNDDLQGIQKIYGPPDKIPPPT 
               
               
                   
               
               
                 RPLPTVPPHRSIPPADPRKNDRPKPPRPPTGRPSYPGAKPNICDGNFNTL 
               
               
                   
               
               
                 AILRREMFVFKDQWFWRVRNNRVMDGYPMQITYFWRGLPPSIDAVYENSD 
               
               
                   
               
               
                 GNFVFFKVKGDTLSVIQDGWLYKYHWKWILEQRQSVPVLSRQTEKHKTYE 
               
               
                   
               
               
                 ELSSITY 
               
               
                   
               
               
                 SEQ ID No. 15 MMP-17 Q9ULZ9 
               
               
                 &gt;gi|21264469|sp|Q9ULZ9|MMP17_HUMAN Matrix metallo- 
               
               
                 proteinase-17 precursor (MMP-17) (Membrane-type 
               
               
                 matrix metalloproteinase 4) (MT-MMP 4) (Membrane- 
               
               
                 type-4 matrix metalloproteinase) (MT4-MMP) 
               
               
                 MRRRAARGPGPPPPGPGLSRLPLLPLPLLLLLALGTRGGCAAPAPAPRAE 
               
               
                   
               
               
                 DLSLGVEWLSRFGYLPPADPTTGQLQTQEELSKAITAMQQFGGLEATGIL 
               
               
                   
               
               
                 DEATLALMKTPRCSLPDLPVLTQARRRRQAPAPTKWNKRNLSWRVRTFPR 
               
               
                   
               
               
                 DSPLGHDTVRALMYYALKVWSDIAPLNFHEVAGSTADIQIDFSKADHND G   
               
               
                   
               
               
                 YPFDGPGGTVAHAFFPGHHHTAGDTHFDDDEAWTFRSSDAHGMDLFAVAV 
               
               
                   
               
               
                 HEFGHAIGLSHVAAAHSIMRPYYQGPVGDPLRYGLPYEDKVRVWQLYGVR 
               
               
                   
               
               
                 ESVSPTAQPEEPPLLPEPPDNRSSAPPRKDVPHRCSTHFDAVAQIRGEAF 
               
               
                   
               
               
                 FFKGKYFWRLTRDRHLVSLQPAQMHRFWRGLPLHLDSVDAVYERTSDHKI 
               
               
                   
               
               
                 VFFKGDRYWVFKDNNVEEGYPRPVSDFSLPPGGIDMFSWAHNDRTYFFKD 
               
               
                   
               
               
                 QLYWRYDDHTRHMDPGYPAQSPLWRGVPSTLDDAMRWSDGASYFFRGQEY 
               
               
                   
               
               
                 WKVLDGELEVAPGYPOSTARDWLVCGDSQADGSVMGVDMEGPRAPPGQHD 
               
               
                   
               
               
                 QSRSEDGYEVCSCTSGASSPPGAPGPLVAATMLLLLPPLSPGALWTAAQA 
               
               
                   
               
               
                 LTL 
               
               
                   
               
               
                 SEQ ID No. 16 MMP-19 Q99542 
               
               
                 Isoform rasi-1 
               
               
                 &gt;gi|12643345|sp|Q99542|MMP19_HUMAN Matrix metallo- 
               
               
                 proteinase-19 precursor (MMP-19) (Matrix metallo- 
               
               
                 proteinase RASI) (MMP-18) 
               
               
                 MNCQOLWLGFLLPMTVSGRVLGLAEVAPVDYLSQYGYLQKPLEGSNNFKP 
               
               
                   
               
               
                 EDITEALRAFQEASELPVSGQLDDATRARMRQPRCGLEDPFNQKTLKYLL 
               
               
                   
               
               
                 LGRWRKKHLTFRILNLPSTLPPHTARAALRQAFQDWSNVAPLTFQEVQAG 
               
               
                   
               
               
                 MDIRLSFHGRQSS Y CSNTFDGPGRVLAHADIPELGSVHFDEDEFWTEGTY 
               
               
                   
               
               
                 RGVNLRIIAAHEVGHALGLGHSRYSQALMAPVYEGYRPHFKLHPDDVAGI 
               
               
                   
               
               
                 QALYGKKSPVIRDEEEEETELPTVPPVPTEPSPMPDPCSSELDAMMLGPR 
               
               
                   
               
               
                 GKTYAFKGDYVWTVSDSGPGPLFRVSALWEGLPGNLDMVYSPRTQWIHFF 
               
               
                   
               
               
                 KGDKVVVRYINFKMSPGFPKKLNRVEPNLDAALYWPLNQKVFLFKGSGYW 
               
               
                   
               
               
                 QWDELARTDFSSYPKPIKGLFTGVPNQPSAAMSWQDGRVYFFKGKVYWRL 
               
               
                   
               
               
                 NQQLRVEKGYPRNISHNWMHCRPRTIDTTPSGGNTTPSGTGITLDTTLSA 
               
               
                   
               
               
                 TETTFEY 
               
               
                   
               
               
                 SEQ ID No. 17 
               
               
                 Isoforma rasi-6 
               
               
                 &gt;gi|13027792|ref|NP_073628.1|matrix metallo- 
               
               
                 proteinase 19 isoform rasi-6 [ Homo sapiens ] 
               
               
                 MRQPRCGLEDPFNQKTLKYLLLGRWRKKHLTFRILNLPSTLPPHTARAAL 
               
               
                   
               
               
                 RQAFQDWSNVAPLTFQEVQAGAADIRLSFHGRQSS Y CSNTFDGPGRVLAH 
               
               
                   
               
               
                 ADIPELGSVHFDEDEFWTEGTYRGVNLRIIAAHEVGHALGLGHSRYSQAL 
               
               
                   
               
               
                 MAPVYEGYRPHFKLHPDDVAGIQALYGKKSPVIRDEEEEETELPTVPPVP 
               
               
                   
               
               
                 TEPSPMPDPCSSELDAMMLGEAPPLQAVGRRWGQPADPEAWTNGSDMGLQ 
               
               
                   
               
               
                 HEQWRAPWEDLCFQGGLCVDCIRFRTGPLVPSVCPLGGAPRKPGCCCLLA 
               
               
                   
               
               
                 SNTMDSLL 
               
               
                   
               
               
                 SEQ ID No. 18 MMP-20 O60882 
               
               
                 &gt;gi|56405303|sp|O60882|MMP20_HUMAN Matrix metallo- 
               
               
                 proteinase-20 precursor (MMP-20) (Enamel metallo- 
               
               
                 proteinase) (Enamelysin) 
               
               
                 MKVLPASGLAVFLIMALTFSTAAPSLVAASPRTWRNNYRLAQAYLDKYYT 
               
               
                   
               
               
                 NKEGHQIGEMVARGSNSMIRKIKELQAFFGLQVTGKLDQTTMNVIKKPRC 
               
               
                   
               
               
                 GVPDVANYRLFPGEPKWKKNTLTYRISKYTPSMSSVEVDKAVEMALQAWS 
               
               
                   
               
               
                 SAVPLSFVRINSGEADIMISFENGDHGD S YPFDGPRGTLAHAFAPGEGLG 
               
               
                   
               
               
                 GDTHFDNAEKWTMGTNGFNLFTVAAHEFGHALGLAHSTDPSALMYPTYKY 
               
               
                   
               
               
                 KNPYGFHLPKDDVKGIQALYGPRKVFLGKPTLPHAPHHKPSIPDLCDSSS 
               
               
                   
               
               
                 SFDAVTMLGKELLLFKDRIFWRRQVHLRTGIRPSTITSSFPQLMSNVDAA 
               
               
                   
               
               
                 YEVAERGTAYFFKGPHYWITRGFQMQGPPRTIYDFGFPRHVQQIDAAVYL 
               
               
                   
               
               
                 REPQKTLFFVGDEYYSYDERKRKMEKDYPKNTEEEFSGVNGQIDAAVELN 
               
               
                   
               
               
                 GYIYFFSGPKTYKYDTEKEDVVSVVKSSSWIGC 
               
               
                   
               
               
                 SEQ ID No. 19 MMP-21 Q8N119 
               
               
                 &gt;gi|50401068|sp|Q8N119|MMP21_HUMAN Matrix metallo- 
               
               
                 proteinase-21 precursor (MMP-21) 
               
               
                 MLAASIFRPTLLLCWLAAPWPTQPESLFHSRDRSDLEPSPLRQAKPIADL 
               
               
                   
               
               
                 HAAQRFLSRYGWSGVWAAWGPSPEGPPETPKGAALAEAVRRFQRANALPA 
               
               
                   
               
               
                 SGELDAATLAAMNRPRCGVPDMRPPPPSAPPSPPGPPPRARSRRSPRAPL 
               
               
                   
               
               
                 SLSRRGWQPRGYPDGGMQAFSKRTLSWRLLGEALSSQLSAADQRRIVALA 
               
               
                   
               
               
                 FRMWSEVTPLDFREDLAAPGAAVDIKLGFGRGRHLG C PRAFDGSGQEFAH 
               
               
                   
               
               
                 AWRLGDIHFDDDEHFTPPTSDTGISLLKVAVHEIGHVLGLPHTYRTGSIM 
               
               
                   
               
               
                 QPNYIPQEPAFELDWSDRKAIQKLYGSCEGSFDTAFDWIRKERNQYGEVM 
               
               
                   
               
               
                 VRFSTYFFRNSWYWLYENRNNRTRYGDPIQILTGWPGIPTHNIDAFVHIW 
               
               
                   
               
               
                 TWKRDERYFFQGNQYWRYDSDKDQALTEDEQGKSYPKLISEGFPGIPSPL 
               
               
                   
               
               
                 DTAFYDRRQKLIYFFKESLVFAFDVNRNRVLNSYPKRITEVFPAVIPQNH 
               
               
                   
               
               
                 PFRNIDSAYYSYAYNSIFFFKGNAYWKVVNDKDKQONSWLPANGLFPKKF 
               
               
                   
               
               
                 ISEKWFDVCDVHISTLNM 
               
               
                   
               
               
                 SEQ ID No. 20 MMP-23A O75900 
               
               
                 &gt;gi|4758730|ref|NP_004650.11 matrix metallo- 
               
               
                 proteinase 23A [ Homo sapiens ] 
               
               
                 MGRGARVPSEAPGAGVERRWLGAALVALCLLPALVLLARLGAPAVPAWSA 
               
               
                   
               
               
                 AQGDVAALGLSAVPPTRVPGPLAPRRRRYTLTPARLRWDHLNLTYRILSF 
               
               
                   
               
               
                 PRNLLSPRETRRALAAAFRMWSDVSPFSFREVAPEQPSDLRIGFYPINHT 
               
               
                   
               
               
                 D C LVSALHHCFDGPTGELAHAFFPPHGGIHFDDSEYWVLGPTRYSWKKGV 
               
               
                   
               
               
                 WLTDLVHVAAHEIGHALGLMHSQHGRALMHLNATLRGWKALSQDELWGLH 
               
               
                   
               
               
                 RLYGCLDRLFVCASWARRGFCDARRRLMKRLCPSSCDFCYEFPFPTVATT 
               
               
                   
               
               
                 PPPPRTKTRLVPEGRNVTFRCGQKILHKKGKVYWYKDQEPLEFSYPGYLA 
               
               
                   
               
               
                 LGEAHLSIIANAVNEGTYTCVVRRQQRVLTTYSWRVRVRG 
               
               
                   
               
               
                 SEQ ID No. 21 MMP-23B Q9UBR9 
               
               
                 &gt;gi|4468604|emb|CAB38176.1|MMP-23 [ Homo sapiens ] 
               
               
                 MGRGARVPSEAPGAGVERRWLGMLVALCLLPALVLLARLGAPAVPAWSMQ 
               
               
                   
               
               
                 GDVAALGLSAVPPTRVPGPLAPRRRRYTLTPARLRWDHFNLTYRILSFPR 
               
               
                   
               
               
                 NLLSPRETRRALAAAFRMWSDVSPFSFREVAPEQPSDLRIGFYPINHTD C   
               
               
                   
               
               
                 LVSALHHCFDGPTGELAHAFFPPHGGIHFDDSEYWVLGPTRYSWKKGVWL 
               
               
                   
               
               
                 TDLVHVAAHEIGHALGMHSQHGRALMHLNALRGWKALSQDELWGLHRLYG 
               
               
                   
               
               
                 CLDRLFVCASWARRGFCDARRRLMKRLCPSSCDFCYEFPFPTVATTPPPP 
               
               
                   
               
               
                 RTKTRLVPEGRNVTFRCGQKILHKKGKVYWYKDQEPLEFSYPGYLALGEA 
               
               
                   
               
               
                 HLSIIANAVNEGTYTCVVRRQQRVLTTYSWRVRVRG 
               
               
                   
               
               
                 SEQ ID No. 22 MMP-24 Q9Y5R2 
               
               
                 &gt;gi|12585280|sp|Q9Y5R2|MMP24_HUMAN Matrix metallo- 
               
               
                 proteinase-24 precursor (MMP-24) (Membrane-type 
               
               
                 matrix metalloproteinase 5) (MT-MMP 5) (Membrane- 
               
               
                 type-5 matrix metalloproteinase) (MT5-MMP) 
               
               
                 MPRSRGGRAAPGPPPPPPPPGQAPRWSRWRVPGRLLLLLLPALCCLPGAA 
               
               
                   
               
               
                 RAAAAAAGAGNRAAVAVAVARADEAEAPFAGQNWLKSYGYLLPYDSRASA 
               
               
                   
               
               
                 LHSAKALQSAVSTMQQFYGIPVTGVLDQTTIEWMKKPRCGVPDHPHLSRR 
               
               
                   
               
               
                 RRNKRYALTGQKWRQKHITYSIHNYTPKVGELDTRKIRQAFDVWQKVPLT 
               
               
                   
               
               
                 FEEVPYHEIKSDRKEADIMIFFASGFHGD S SPFDGEGGFLAHAYFPGPGI 
               
               
                   
               
               
                 GGDTHFDSDEPWTLGNANHDGNDLFLVAVHELGHALGLEHSSDPSAIMAP 
               
               
                   
               
               
                 FYQYMETHNFKLPODDLQGIQKIYGPPAEPLEPTRPLPTLPVRRIHSPSE 
               
               
                   
               
               
                 RKHERQPRPPRPPLGDRPSTPGTKPNICDGNFNTVALFRGEMFVFKDRWF 
               
               
                   
               
               
                 WRLRNNRVQEGYPMQIEQFWKGLPARIDAAYERADGRFVFFKGDKYWVFK 
               
               
                   
               
               
                 EVTVEPGYPHSLGELGSCLPREGIDTALRWEPVGKTYFFKGERYWRYSEE 
               
               
                   
               
               
                 RRATDPGYPKPITVWKGIPQAPQGAFISKEGYYTYFYKGRDYWKFDNQKL 
               
               
                   
               
               
                 SVEPGYPRNILRDWMGCNQKEVERRKERRLPQDDVDIMVTINDVPGSVNA 
               
               
                   
               
               
                 VAVVIPCILSLCILVLVYTIFQFKNKTGPQPVTYYKRPVQEWV 
               
               
                   
               
               
                 SEQ ID No. 23 MMP-25 Q9NPA2 
               
               
                 &gt;gi|2585274|sp|Q9NPA2|MMP25_HUMAN Matrix metallo- 
               
               
                 proteinase-25 precursor (MMP-25) (Membrane-type 
               
               
                 matrix metalloproteinase 6) (MT-MMP 6) (Membrane- 
               
               
                 type-6 matrix metalloproteinase) (MT6-MMP) 
               
               
                 (Leukolysin) 
               
               
                 MRLRLRLLALLLLLLAPPARAPKPSAQDVSLGVDWLTRYGYLPPPHPAQA 
               
               
                   
               
               
                 QLQSPEKLRDAIKVMQRFAGLPETGRMDPGTVATMRKPRCSLPDVLGVAG 
               
               
                   
               
               
                 LVRRRRRYALSGSVWKKRTLTWRVRSFPQSSQLSQEVRVLMSYALMAWGM 
               
               
                   
               
               
                 ESGLTFHEVDSPQGQEPDILIDFARAFHQD S YPFDGLGGTLAHAFFPGEH 
               
               
                   
               
               
                 PISGDTHFDDEETTFGSKDGEGTDLFAVAVHEFGHALGLGHSSAPNSIMR 
               
               
                   
               
               
                 PFYQGPVGDPDKYRLSQDDRDGLQQLYGKAPQTPYDKPTRKPLAPPPQPP 
               
               
                   
               
               
                 ASPTHSPSFPIPDRCEGNFDAIANIRGETFFFKGPWFWRLQPSGQLVSPR 
               
               
                   
               
               
                 PARLHRFWEGLPAQVRWQMYARHRDGRILLFSGPQFWVFQDRQLEGGARP 
               
               
                   
               
               
                 LTELGLPPGEEVDAVFSWPQNGKYLVRGROYWRYDEAAARPDPGYPRDLS 
               
               
                   
               
               
                 LWEGAPPSPDDVTVSNAGDWFYWRFPKNSIKTEPDAPQPMGPNWLDCPAP 
               
               
                   
               
               
                 SSGPRAPRPPKATPVSETCDCQCELNQMGRWPAPIPLLLLPLLVGGVASR 
               
               
                   
               
               
                 SEQ ID No. 24 MMP-26 Q9NRE1 
               
               
                 &gt;gi|136294931sp|Q9NRE1|MMP26_HUMAN Matrix metallo- 
               
               
                 proteinase-26 precursor (MMP-26) (Matrilysin-2) 
               
               
                 (Endometase) 
               
               
                 MQLVILRVTIFLPWCFAVPVPPAADHKGWDFVEGYFHQFFLTKKESPLLT 
               
               
                   
               
               
                 QETQTQLLQQFHRNGTDLLDMQMHALLHQPHCGVPDGSDTSISPGRCKWN 
               
               
                   
               
               
                 KHTLTYRIINYPHDMKPSAVKDSIYNAVSIWSNVTPLIFQQVQNGDADIK 
               
               
                   
               
               
                 VSFWQWAHED G WPFDGPGGILGHAFLPNSGNPGWHFDKNEHWSASDTGYN 
               
               
                   
               
               
                 LFLVATHEIGHSLGLQHSGNQSSIMYPTYWYHDPRTFQLSADDIQRIQHL 
               
               
                   
               
               
                 YGEKCSSDIP 
               
               
                   
               
               
                 SEQ ID No. 25 MMP-27 Q9H306 
               
               
                 &gt;gi|11066090|gb|AAG28453.1|AF195192_1 matrix 
               
               
                 metalloprotease MMP-27 [ Homo sapiens ] 
               
               
                 MKRLLLLFLFFITFSSAFPLVRMMENEENVQLAQAYLNQFYSLEIEGNHL 
               
               
                   
               
               
                 VQSKNRSLIDDKIREMQAFFGLTVTGKLDSNTLEIMKTPRCGVPDVGQYG 
               
               
                   
               
               
                 YTLPGWRKYNLTYRIINYTPDMARAAVDEAIQEGLEVWSKVTPLKFTKIS 
               
               
                   
               
               
                 KGIADIMIAFRTRVHGR C PRYFDGPLGVLGHAFPPGPGLGGDTHFDEDEN 
               
               
                   
               
               
                 WTKDGAGFNLFLVAAHEFGHALGLSHSNDQTALMFPNYVSLDPRKYPLSQ 
               
               
                   
               
               
                 DDINGIQSIYGGLPKEPAKPKEPTIPHACDPDLTFDAITTFRREVMFFKG 
               
               
                   
               
               
                 RHLWRIYYDITDVEFELIASFWPSLPADLQAAYENPRDKILVFKDENFWM 
               
               
                   
               
               
                 IRGYAVLPDYPKSIHTLGFPGRVKKIDAAVCDKTTRKTYFFVGIWCWRFD 
               
               
                   
               
               
                 EMTQTMDKGFPQRWKHFPGSIRVDAAFQYKGFFFFSRGSKQFEYDIKTKN 
               
               
                   
               
               
                 ITRIMRTNTWFQCKEPKNSSFGFDINKEKAHSGGIKILYHKSLSLFIFGI 
               
               
                   
               
               
                 VHLLKNTSIYQ 
               
               
                   
               
               
                 SEQ ID No. 26 MMP-28 Q9H239 
               
               
                 Isoform 1 
               
               
                 &gt;gi|37538314|sp|Q9H239|MMP28_HUMAN Matrix metallo- 
               
               
                 proteinase-28 precursor (MMP-28) (Epilysin) 
               
               
                 (UNQ1893/PR04339) 
               
               
                 MVARVGLLLRALQLLLWGHLDAQPAERGGQELRKEAEAFLEKYGYLNEQV 
               
               
                   
               
               
                 PKAPTSTRFSDAIRAFQWVSQLPVSGVLDRATLRQMTRPRCGVTDTNSYM 
               
               
                   
               
               
                 WAERISDLFARHRTKMRRKKRFAKQGNKWYKQHLSYRLVNWPEHLPEPAV 
               
               
                   
               
               
                 RGAVRAAFQLWSNVSALEFWEAPATGPADIRLTFFQGDHND G LGNAFDGP 
               
               
                   
               
               
                 GGALAHAFLPRRGEAHFDQDERWSLSRRRGRNLFVVLAHEIGHTLGLTHS 
               
               
                   
               
               
                 PAPRALMAPYYKRLGRDALLSWDDVLAVQSLYGKPLGGSVAVQLPGKLFT 
               
               
                   
               
               
                 DFETWDSYSPQGRRPETQGPKYCHSSFDAITVDRQQQLYIFKGSHFWEVA 
               
               
                   
               
               
                 ADGNVSEPRPLQERWVGLPPNIEAAAVSLNDGDFYFFKGGRCWRFRGPKP 
               
               
                   
               
               
                 VWGLPQLCRAGGLPRHPDAALFFPPLRRLILFKGARYYVLARGGLQVEPY 
               
               
                   
               
               
                 YPRSLQDWGGIPEEVSGALPRPDGSIIFFRDDRYWRLDQAKLQATTSGRW 
               
               
                   
               
               
                 ATELPWMGCWHANSGSALF 
               
               
                   
               
               
                 SEQ ID No. 27 
               
               
                 Isoform 2 
               
               
                 &gt;gi|14589912|ref|NP_116568.1|matrix metallo- 
               
               
                 proteinase 28 preproprotein isoform 2 [ Homo   
               
               
                   sapiens ] 
               
               
                 MVARVGLLLRALQLLLWGHLDAQPAERGGQELRKEAEAFLEKYGYLNEQV 
               
               
                   
               
               
                 PKAPTSTRFSDAIRAFQWVSQLPVSGVLDRATLRQMTRPRCGVTDTNSYA 
               
               
                   
               
               
                 AWAERISDLFARHRTKMRRKKRFAKQGNKWYKQHLSYRLVNWPEHLPEPA 
               
               
                   
               
               
                 VRGAVRMFQLWSNVSALEFWEAPATGPADIRLTFFQGDHND G LGNAFDGP 
               
               
                   
               
               
                 GGLALAHAFLPRRGEAHFDQDERWSLSRRRGRNLFVVLAHEIGHTHGLTH 
               
               
                   
               
               
                 SPAPRALMAPYYKRLGRDALLSWDDVLAVQSLYGKPLGGSVAVQLPGKLF 
               
               
                   
               
               
                 TDFETWDSYSPOGRRPETQGPKYCHSSFDAITVDRQQQLYIFKGSHFWEV 
               
               
                   
               
               
                 AADGNVSEPRPLQERWVGLPPNIEMAVSLNDGDFYFFKVQSV 
               
               
                   
               
               
                 SEQ ID No. 28 MMP-like1 O43923 
               
               
                 &gt;gi|4758728|ref|NP_004133.1| matrix metallo- 
               
               
                 proteinase-like 1 [ Homo sapiens ] 
               
               
                 MDPGTVATMRKPRCSLPDVLGVAGLVRRRRRYALSGSVWKKRTLTWRVRS 
               
               
                   
               
               
                 FPQSSQLSQETVRVLMSYALMAWGMESGLTFHEVDSPQGQEPDILIDFAR 
               
               
                   
               
               
                 AFHQD S YPFDGLGGTLAHAFFPGEHPISGDTHFDDEETWTFGSKASQQLE 
               
               
                   
               
               
                 QELAGGSPVDEELGFSRGWRVNPLGPGSPERLS 
               
             
          
         
       
     
       Example 3 
     Evaluation of the Mutated MMP-3 Stability (SEQ ID No. 3) 
       [0053]    Studies have been carried out to evaluate stability of the wild type human metalloproteinase 3 (MMP-3) (SEQ ID No. 3) catalytic domain and of the mutated human metalloproteinase 3 (MMP-3) (SEQ ID No. 3) catalytic domain, wherein Phenylalanine has been mutated in Aspartate. The analysis has been carried out comparing stability towards autoproteolysis of the two proteins in the same conditions. 
         [0054]    The two proteins have been prepared following the procedure reported above in Example 1, and the stability has been analysed by SDS/polyacrylamide gel electrophoresis according to the following procedure. 
         [0055]    A sample of the protein is used, dissolved in a buffer containing Tris hydroxymethylaminoethane (Tris) 50 mM pH=7.2; CaCl 2  5 mM; ZnCl 2  0.1 mM; NaCl 0.3 M; and acetohydroxamic acid (AHA) 0.5 M, and having a concentration of 0.45 mM. The sample is maintained at room temperature. 
         [0056]    At intervals corresponding to 0, 4, 7, 11 and 14 days 5 μL aliquots of the two samples maintained at room temperature are collected and immediately frozen at −80°; all the collected samples having the above said protein concentration are analysed by electrophoresis on SDS/polyacrylamide gel (17%), thus obtaining the results showed in  FIG. 1  and illustrated in the following. 
         [0057]    The analysis shows a higher stability of the mutated protein in respect of the wild-type protein. In fact, both wild-type and mutated proteins show a degradation band since time=0, but during the next 14 days the intensity of the band corresponding to the non degraded wild-type protein shows a significant decrease. On the contrary, the intensity of the band corresponding to the non degraded mutated protein is substantially constant during 14 days. Moreover, the fragmentation of wild-type protein involving the aminoacidic residue 171 corresponding to the mutation site in the mutated protein yields to a fragment with lower molecular weight if compared with the fragment obtained for the mutated protein. This indicates different cutting sites for wild-type and mutated proteins, so that the mutation site of the mutated protein is less preferred as cutting site than that in the same position of the wild-type protein, this being a suggestion that the non degraded mutated protein is more stable. 
         [0058]    Finally, as showed in  FIG. 1 , the band corresponding to the fragment coming from degradation of the mutated protein has an intensity which is substantially maintained, and this indicates that the mutated protein is not only less susceptible to fragmentation but the fragment coming from its fragmentation is also more stable than that forming from fragmentation of the wild-type protein. 
       Example 4 
     Evaluation of Stability of the Mutated MMP-10 (SEQ ID No. 7) 
       [0059]    Studies have been carried out on stability of the wild type human metalloproteinase 10 (MMP-10) (SEQ ID No. 7) catalytic domain and of the mutated human metalloproteinase 10 (MMP-10) (SEQ ID No. 7) catalytic domain, wherein Phenylalanine has been mutated in Aspartate. The analysis has been carried out comparing stability towards autoproteolysis of the two proteins in the same conditions. 
         [0060]    The two proteins have been prepared following the procedure reported above in Example 1, and the stability has been analysed by SDS/polyacrylamide gel electrophoresis according to the following procedure. 
         [0061]    A sample of the protein is used, dissolved in a buffer containing Tris 10 mM pH=7.2; CaCl 2  10 mM; ZnCl 2  0.1 mM; NaCl 0.3 M; and AHA 0.5 M, and having a concentration of 0.3 mM for the wild-type protein and of 0.26 mM for the mutated protein. The samples are maintained at 4° C. 
         [0062]    At intervals corresponding to 0, 1, 2, and 3 days for the wild-type protein and 0, 6 and 30 days for the mutated protein, 5 μL aliquots of the two samples maintained at 4° C. are collected and immediately frozen at −80° C.; all the collected samples having the above said protein concentrations, are analysed by electrophoresis SDS/polyacrylamide gel (17%), thus obtaining the results showed in  FIG. 2 . Besides the observations already made above for the results concerning MMP-3 (SEQ ID No. 3), that are substantially the same results as those obtained for MMP-10 (SEQ ID No. 10), it is significant to notice the high stability showed by the mutated protein even after 30 days. 
       Example 5 
     Evaluation of Stability of the Mutated MMP-13 (SEQ ID No. 10) 
       [0063]    Studies have been carried out on the stability of the wild type human metalloproteinase 13 (MMP-13) (SEQ ID No. 10) catalytic domain and of the mutated human metalloproteinase 13 (MMP-13) (SEQ ID No. 10) catalytic domain where phenylalanine has been mutated in aspartate. The analysis has been carried out comparing stability towards autoproteolysis of the two proteins in the same conditions. 
         [0064]    The two proteins have been prepared following the procedure reported above in Example 1, and the stability has been analysed by SDS/polyacrylamide gel electrophoresis according to the following procedure. 
         [0065]    A sample of the protein is used, dissolved in a buffer containing Tris 50 mM pH=7.2; CaCl 2  5 mM; ZnCl 2  0.1 mM; NaCl 0.3 M; but without AHA, and having a concentration of 80 μM. The sample is maintained at room temperature. 
         [0066]    At intervals corresponding to 0, 2, 7, 14 and 21 days 5 μL aliquots of the two samples maintained at room temperature, are collected and immediately frozen at −80° C.; all the collected samples having the above said concentration are analysed by electrophoresis on SDS/polyacrylamide gel (17%), thus obtaining the results showed in  FIG. 3  and illustrated in the following. 
         [0067]    The analysis shows a significantly higher stability of the mutated protein in respect of the wild-type protein. In fact, it is evident from  FIG. 3  a decrease of intensity of the band corresponding to the non degraded wild-type protein, which is almost disappeared after 21 days; on the contrary, the intensity of the same band for the protein mutated according to the invention, is almost unchanged after 21 days. 
         [0068]    Moreover,  FIG. 3  shows bands corresponding to low molecular weight fragments only for the wild-type protein, while no such bands are visible for the protein mutated according to the invention. 
       Example 6 
     Evaluation of the Activity 
       [0069]    The activity measurements have been carried out with the following methodology. The colorimetric substrate is a thiopeptide (Ac-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gly-OC 2 H 5 , a reagent from Biomol catalogue. The hydrolysis of the substrate due to matrix metalloproteinases produces a sulphydrilic group that reacts with DTNB (5,5′-dithiobis(2-nitrobenzoic acid), called Ellman reagent) to produce 2-nitro-5-thiobenzoic acid, detectable using its absorbance at 412 nm. The reaction conditions are reported hereinafter: 
         [0000]    Reaction cell=500 μl
 
[thiopeptolidel]*=100 μM
 
[protein]*=50 nM
 
*final concentration in the cell
 
         [0070]    Buffer for the activity measurement: 
       50 mM HEPES pH 7 
     5 mM CaCl 2    
     0.1 mM ZnCl 2    
     0.05% Brj-35 
       [0071]    1 mM DTNB [5,5′-dithiobis(2-nitrobenzoic acid)] 
         [0072]    The results at 25° are: 
         [0000]    MMP3 (SEQ ID No. 3) (F171)—specific activity at 25° C.: 43 U/μg. An U=100 pmol/min
 
MMP7 (SEQ ID No. 4) (S166D)—specific activity at 25° C.: 66 U/μg. An U=100 pmol/min
 
MMP10 (SEQ ID No. 7) (F171)—specific activity at 25° C.: 41 U/μg. An U=100 pmol/min
 
MMP12 (SEQ ID No. 9) (F171)—specific activity at 25° C.: 91 U/μg. An U=100 pmol/min
 
         [0073]    The activity values of the MMP refer by Biomol are measured at 37° C. on the same substrate used by us, and they are as follows: 
         [0000]    MMP3 (SEQ ID No. 3)—specific activity at 37° C.: 50 U/μg. An U=100 pmol/min
 
MMP7 (SEQ ID No. 4)—specific activity at 37° C.: 34.5 U/μg. An U=100 pmol/min
 
MMP10 (SEQ ID No. 7)—specific activity at 37° C.: 42.5 U/μg. An U=100 pmol/min
 
MMP12 (SEQ ID No. 9)—specific activity at 37° C.: 86 U/μg. An U=100 pmol/min
 
       Example 7 
       [0074]    The cDNA of the catalytic domain for each MMP has been cloned in a pET21 vector (Novagen); the resulting vector has been then used for transformation of BL21-DE3 cells of  Escherichia coli . The transformed cells have been grown in Luria-Bertani medium at a temperature of 37° C. The expression of the protein has been induced during the exponential growth phase by addition of IPTG 0.5 mM. 5 hours after induction, cells have been harvested and lysated, and inclusion bodies have been dissolved in a buffer containing Tris 20 mM pH 8 and urea 6 M. The proteins are then purified by means of an anionic exchange column (HiTrap QHP-Pharmacia) using a buffer containing Tris 20 mM pH 8 and urea 6 M, and eluting with a linear gradient of NaCl until 0.5 M. 
         [0075]    The purified proteins have been then refolded by subsequent dialysis with solutions containing Tris 50 mM pH 7.2; CaCl 2  10 mM; ZnCl 2  0.1 M; NaCl 0.3 M and AHA 0.2 M. In the final refolding dialysis the concentration of AHA is brought to 0.5 M. For the isolation of the catalytic domains a chromatographic column Superdex 75 16/60 (Pharmacia), eluted with the buffer of the last dialysis, has been used. 
         [0076]    Following the procedure described above, the catalytic domain of MMP-1 (SEQ ID No. 1), MMP-3 (SEQ ID No. 3), MMP-7 (SEQ ID No. 4), MMP-8 (SEQ ID No. 5), MMP-10 (SEQ ID No. 7) and MMP-12 (SEQ ID No. 9) have been prepared and purified.