WO2004033679A1 - Abbau und modifizierung von silicaten und siliconen durch silicase und verwendung des reversiblen enzyms - Google Patents
Abbau und modifizierung von silicaten und siliconen durch silicase und verwendung des reversiblen enzyms Download PDFInfo
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- WO2004033679A1 WO2004033679A1 PCT/EP2003/010983 EP0310983W WO2004033679A1 WO 2004033679 A1 WO2004033679 A1 WO 2004033679A1 EP 0310983 W EP0310983 W EP 0310983W WO 2004033679 A1 WO2004033679 A1 WO 2004033679A1
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- polypeptide
- silicase
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
Definitions
- Silicon is the second most common element in the earth's crust and occurs in a wide variety of compounds. Silicon compounds are not only the most species-rich class of minerals, but are also economically significant. Technically used materials made of silicates are, for example, glass, porcelain, enamel, pottery, cement and water glass. Some silicates have catalytic properties. By partially replacing the lattice sites occupied by silicon with other elements, especially aluminum, the variety in the possible structures and technical applications is expanded. The aluminosilicates, to which the feldspars and the zeolites belong, are of importance u. a. due to their molecular sieve and ion exchange properties. Other silicon compounds such as silicones (siloxanes) have u. a. also medical significance, such as for the manufacture of implants.
- Silicon dioxide occurs in both crystalline and amorphous form.
- the various forms of crystalline SiO 2 include quartz, tridymite and cristobalite.
- Agate, opal and flint are amorphous silicon dioxide minerals. In all these forms, silicon has the coordination number 4 and is tetrahedrally surrounded by four oxygen atoms.
- the shells of diatoms (diatoms) and the needles (spiculae) of sponges are made of amorphous SiO 2 .
- the acid molecules formed by proton absorption condense with one another to form polysilicic acids, whereby the solution becomes gel-like.
- three-dimensional structures, which correspond to the composition SiO 2 result from the initially obtained chains or networks.
- the silicates can be divided into .1.) Silicates with discrete anions, namely 1a.) Island silicates (ortho-silicates with the anion [SiO 4 ] 2 " ; example: phenakite, olivine, zirconium), 1b.) Group silicates (linking the SiO 4 tetrahedra to short-chain units; example: disilicates and tri-silicates) and 1c.) Ring silicates (ring linking the SiO 4 tetrahedra; example: benitoid with a 3-membered ring, Axinite with 4-membered ring and beryl with 6-membered ring), 2.) chain silicates and band silicates (chain-like interconnected SiO 4 tetrahedra, which are polymers of the anion [SiO 3 ] 2 " , and band-shaped Molecules that are created by linking several SiO 4 chains; examples: hornblende, asbestos), 3.)
- silicones are formed by partially replacing the OH groups of the silica with monovalent organanyl residues that do not participate in the condensation process. They are divided into: 1.) linear polysiloxanes (type: R 3 SiO [R 2 SiO] nSiR 3 ), 2.) branched polysiloxanes (with trifunctional or tetrafunctional siloxane units at the branching points), 3.) cyclic polysiloxanes (from difunctional siloxane units) and 4.) cross-linked polymers (linking of chain or ring-shaped molecules to two- or three-dimensional networks).
- linear polysiloxanes type: R 3 SiO [R 2 SiO] nSiR 3
- branched polysiloxanes with trifunctional or tetrafunctional siloxane units at the branching points
- cyclic polysiloxanes from difunctional siloxane units
- cross-linked polymers linking of chain or ring-shaped molecules to two- or three-dimensional networks.
- Silicones are important technical materials.
- the viscosity of the chain-shaped silicones increases with increasing chain length.
- Silicones that are cross-linked to a small extent show rubber elasticity (silicone rubber), highly cross-linked chains are resin-like (silicone resins).
- silicates are only expensive to produce.
- the process of chemical synthesis of the silicates often requires drastic conditions such as high pressure and high temperature.
- Silica sponges like diatoms, are capable of using special enzymes to form silicate structures under mild conditions, ie at a relatively low temperature and pressure.
- the SiO 2 synthesis in these organisms is characterized by high specificity, controllability and the possibility of the synthesis of defined microstructures (nanostructures).
- the main elements of the skeleton of the pebble sponges are the acicular spicules, which in the group of demospongia (horn sponges) and hexactinellida (glass sponges) consist of amorphous, non-crystalline silicon dioxide.
- demospongia and Hexactinellidae are the only metazoa that have silica instead of calcium in their skeleton.
- the opal silicon dioxide in the spicules of the silica sponges contains 6-13% water, which gives the approximate formula (SiO 2 ) 2 - 5 'H 2 O (Schwab DW, Shore RE (1971) Mechanism of internal stratification of siliceous spicules. Nature 232 : 501-502).
- silicatein isolated from natural sources is able to synthesize amorphous silicon dioxide (polysilicic acids and polysilicates) from organic silicon compounds (alkoxysilanes) (Cha JN, Shimizu K, Zhou Y, Christiansen SC, Chmelka BF, Stucky GD, Morse DE (1999) Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro. Proc Natl Acad Sei USA 96: 361-365).
- sicase an enzyme that is able to dissolve both amorphous and crystalline silicon dioxide.
- Silicase is able to perform two functions: first, it has the ability (i) to dissolve lime material in analogy to carbonic anhydrase and (ii) - and this was surprising - also to dissolve silicon dioxide with the formation of silica. Therefore, the silicase - first found in S. domuncula - is able to participate both in the catabolism of calcareous material and in the catabolism of the siliceous spicules.
- the silicase gene can be induced by increasing the silicon concentrations in the medium (to usually 60 ⁇ M) (see Figure 7).
- a process for the in vitro or in vivo degradation of amorphous or crystalline silicon dioxide (condensation products of silicic acid, silicates), silicones and other silicon (IV) or metal (IV) compounds and of Copolymers of these compounds wherein a polypeptide or a metal complex of a polypeptide is used for degradation, characterized in that the polypeptide comprises an animal, bacterial, vegetable or fungal carbonic anhydrase domain which has at least 25% sequence similarity (see Figure 3 ) to the sequence shown in SEQ ID No. 1. It was not previously known that enzymes containing such carbonic anhydrase domains are able to degrade such silicates or silicones.
- a further aspect of the present invention relates to a process for the synthesis of amorphous silicon dioxide (condensation products of silicic acid, silicates), silicones and other silicon (IV) or metal (IV) compounds and of mixed polymers of these compounds , wherein a polypeptide or a metal complex of a polypeptide for synthesis is used, characterized in that the polypeptide comprises an animal, bacterial, vegetable or fungal carbonic anhydrase domain which has at least 25% sequence similarity to the sequence shown in SEQ ID No. 1.
- a process according to the invention is preferred which is characterized in that compounds such as silicas, monoalkoxysilane triols, dialkoxysilane diols, trialkoxysilanols, tetraalkoxysilanes, alkyl or aryl silane triols, alkyl or aryl monoalkoxysilane diols, alkyl or aryl dialkoxysilanols are used for the synthesis - Or aryl trialkoxysilanes or other metal (IV) compounds are used as reactants (substrates).
- metal (IV) compounds are used as reactants (substrates).
- the formation can be defined by the polypeptide or a metal complex of the polypeptide or the binding of the polypeptide or a metal complex of the polypeptide to other molecules or the surfaces of glass, metals, metal oxides, plastics, biopolymers or other materials two- and three-dimensional structures.
- a method for modifying a structure or surface containing silica or silicon (IV) or metal (IV) compound, a polypeptide or a metal complex of a polypeptide being used for the modification characterized in that that the polypeptide comprises an animal, bacterial, vegetable or fungal carbonic anhydrase domain which has at least 25% sequence similarity to the sequence shown in SEQ ID No. 1.
- the structure or surface containing silicic acid is preferably in the form of a precious stone or semi-precious stone.
- a method according to the invention is preferred, the modification comprising smoothing, etching or producing bores in the structure or surface containing silica or silicon (IV) or metal (IV) compound by means of the polypeptide or a metal complex of the polypeptide.
- Another aspect of the present invention relates to a chemical compound or silica-containing structure or surface, which was obtained by a method according to the invention, in particular in the form of a gemstone or semi-precious stone.
- a further aspect of the present invention also relates to a polypeptide of a silicase from Suberites domuncula according to SEQ ID No. 1 or a polypeptide homologous thereto which has at least 25% sequence similarity in the amino acid sequence of the carbonic anhydrase domain to the sequence shown in SEQ ID No. 1 , a metal complex of the polypeptide, or parts thereof.
- nucleic acid in particular according to SEQ ID No. 2, characterized in that it essentially codes for a polypeptide of the invention.
- the nucleic acid according to the invention can be in the form of a DNA, cDNA, RNA or mixture thereof and can be characterized in that the sequence of the nucleic acid has at least one intron and / or a polyA sequence.
- Another aspect of the present invention relates to the nucleic acid according to the invention in the form of its complementary “antisense” sequence.
- a still further aspect of the present invention also relates to a nucleic acid according to the invention in the form of a (a) fusion protein (chimeric protein) construct, (b) construct with separate protein expression (protease cleavage site) or (c) construct with separate protein expression (cassette expression).
- the nucleic acid according to the invention can have been produced synthetically. Methods of doing this are well known in the art.
- Another aspect of the invention relates to a vector, preferably in the form of a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, adenoviral vector or particle, nanoparticle or liposome, the vector containing a nucleic acid according to the invention.
- vectors can be provided for the transfer of proteins, preferably in the form of a nanoparticle or liposome, which comprise a polypeptide of the invention.
- a host cell transfected with a vector or infected or transduced with a particle according to the invention.
- This host cell can be characterized in that it expresses a polypeptide according to claim 1, a metal complex of the polypeptide or parts thereof.
- All known host cell organisms are suitable as host cells, such as yeasts, fungi, sponges, bacteria, CHO cells or insect cells.
- the polypeptide according to the invention can be characterized in that it has been produced synthetically or in that the polypeptide or the metal complex of the polypeptide is present in a prokaryotic or eukaryotic cell extract or lysate.
- the cell extract or lysate can be obtained from a cell ex vivo or ex vitro, for example a recombinant bacterial cell or a sea sponge.
- polypeptide according to the invention can be purified by conventional methods known in the art and can therefore be essentially free of other proteins.
- a further aspect of the present invention then relates to a method for finding inhibitors or activators of a polypeptide of a silicase from Subites domuncula according to SEQ ID No. 1 or a polypeptide homologous thereto which has at least 25% sequence similarity in the amino acid sequence of the carbonic anhydrase domain of the sequence shown in SEQ ID No. 1, wherein a) a polypeptide of a silicase from Suberites domuncula according to SEQ ID No. 1 or a homologous polypeptide which has at least 25% sequence similarity in the amino acid sequence of the carbonic anhydrase domain to that in SEQ ID No.
- step b) the polypeptide from step a) is brought into contact with an optional inhibitor or activator, and c) the ability of the polypeptide is measured to degrade or synthesize silicates or silicones.
- This procedure can be used to detect valuable substances that may be suitable as therapeutic agents (see below for more information).
- Methods for finding such substances are known to the person skilled in the art and include, for example, the use of radioactively labeled or enzymatically labeled candidate Connections. Methods for measuring the activity of the silicase are described below and can easily be adapted to a test format by those skilled in the art.
- An inhibitor substantially completely reduces the activity of the enzyme, an activator induces an activity or increases it above the initial level.
- the polypeptide of a Silicites from Suberites domuncula according to SEQ ID No. 1 or a polypeptide homologous thereto which has at least 25% sequence similarity to the sequence shown in SEQ ID No. 1 in the amino acid sequence of the carbonic anhydrase domain is provided in vivo, in a prokaryotic or eukaryotic cell extract or lysate or in a purified form for the test.
- a still further aspect of the invention relates to a method for producing a pharmaceutical composition, comprising a) finding an inhibitor or activator according to claim 25 or 26 and b) mixing the inhibitor or activator found as stated above with a pharmaceutically suitable carrier or auxiliary.
- This composition provides valuable pharmaceuticals which, like the polypeptide or a nucleic acid of the invention, can be used for the prevention or therapy of silicosis. Use is preferred, the prevention and therapy of silicosis being carried out by dissolving quartz crystals.
- the use of a polypeptide or a nucleic acid or pharmaceutical composition according to the invention for resorption or for modulating the resorbability of silicones and silicone implants can take place.
- the present invention can also be used for transfecting cells with nucleic acids according to the invention for resorption or for modulating the resorbability of silicones and silicone implants.
- the above-mentioned uses and the methods for this are known to the person skilled in the art and can easily be adapted to the needs and requirements present here.
- the cDNA (called: SDSIA) coding for the silicase from the sea sponge S. domuncula and the polypeptide derived from the nucleotide sequence (called: SIA_SUBDO) have the following properties. Length of the cDNA: 1395 nucleotides (nt); open reading grid: from nt ⁇ 22 - nt ⁇ 24 to nti25_ - nti26i (stop codon); Length of the polypeptide: 379 amino acids; molecular weight (M r ) of the polypeptide: 43131; isoelectric point (pl): 6.5 (calculated with: PC / GENE (1995) Data Banks CD-ROM; Release 14.0. IntelliGenetics, Inc. Mountain View, CA).
- Figure 2 shows the nucleotide sequence of the sponge silicase cDNA identified using the differential display technique
- Figure 2 top and bottom
- Figure 3A show the polypeptide of the sponge silicase (SIA_SUBDO) derived from the nucleotide sequence.
- the deduced amino acid sequence of the sponge silicase is very similar to the amino acid sequences of the carbonic anhydrase family. So far, more than seven isoenzymes of carbonic anhydrases have been identified in humans (Sun MK, Alkon DL (2002) Carbonic anhydrase gating of attention: memory therapy and enhancement. Trends Pharmac Sei 23: 83-89).
- the "Expect value” [£] (Coligan JE, Dünn BM, Ploegh HL, Speicher DW, Wingfield PT (2000) Current protocols in protein science.
- the carbonic anhydrases form a family of zinc metal enzymes that are involved in the reversible hydration of CO 2 (Sly WS, Hu PY (1995) Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu. Rev. Biochem. 64: 375-401).
- the three conserved histidine residues are found in the silicase at the amino acids aa ⁇ s ⁇ , aai83 and aa 2 _6 ( Figure 3A).
- Figure 3B shows the position of the sponge silicase among various selected representatives of the carbonic anhydrase family (phylogenetic tree; "rooted tree” with the bacterial carbonic anhydrase sequence from Neisseria gonorrhoeae).
- the sponge silicase together with the carbonic anhydrase from Caenorhabditis elegans, forms the basis for the carbon anhydrases of the other metazoa.
- the metazoan enzymes are separated from the plant enzymes and also from the bacterial enzymes.
- the silicase can be purified from tissues or cells or produced recombinantly.
- the homogenized tissue (or, for example, in a Tris-SO / sodium sulfate buffer (pH 8.7)) is (which the cells homogenized in this buffer) are centrifuged and an affinity chromatography matrix such as CM-Bio-Gel A, coupled with p-aminomethylbenzenesulfonamide, is added to the supernatant obtained.
- an affinity chromatography matrix such as CM-Bio-Gel A, coupled with p-aminomethylbenzenesulfonamide
- the affinity gel is then collected by suction through a glass filter and washed with a buffer (for example 0.1 M Tris-SO 4 , pH 8.7, containing 0.2 M Na 2 SO 4 , 1 mM benzamidine and 20% glycerol). It is then expedient to connect a second washing step with the same buffer at a lower pH (for example pH 7.0) in order to remove non-specifically bound proteins.
- the gel is then transferred to a column and washed with the same buffer (pH 7.0).
- a 0.1 M Tris-SO 4 buffer, pH 7.0, containing 0.4 M NaN 3 , 1 mM benzamidine and 20% glycerol can be used to elute the enzyme.
- the eluted enzyme protein is then dialyzed, for example, against a 10 mM Tris-SO 4 buffer, pH 7.5, containing 1 mM benzamidine and then placed on an ion exchange column (for example DEAE-Sephacel), which is used, for example, with 10 mM Tris SO, pH 7.5, has been equilibrated.
- an ion exchange column for example DEAE-Sephacel
- the enzyme is eluted by applying a linear salt gradient (for example 0 to 0.1 M Na 2 SO 4 ) and collected. This procedure can be used to purify the silicase from the S. domuncula sponge, among others.
- RNA is isolated from control cultures (maintained at a low silicon concentration of 5 ⁇ M) and from cultures treated in the presence of 60 ⁇ M silicon using TRIzol reagent (GibcoBRL).
- the synthesis of the first strand of cDNA is carried out using "anchored" oligo (dT) primers and AMV reverse transcriptase according to the manufacturer's instructions (Promega).
- the resulting cDNA is diluted ten-fold with H 2 O and an aliquot part of it (2 ul) subjected to the polymerase chain reaction (PCR).
- the reaction is carried out in a volume of 20 ⁇ l after addition of the “arbitrary” primer 1 (5'-GTGATCGCAG-3 ') or 2 (5'-CTTGATTGCC-3') and 2 ⁇ M dNTP, TuGC, 5 units of BioThem polymerase ( Genecraft) and [ ⁇ - 32 P] dATP.
- reaction conditions have proven to be suitable for the PCR: initial denaturation at 95 ° C. for 5 minutes, then 40 amplification cycles at 95 ° C. each for 20 seconds, 42 ° C. for 120 seconds, 72 ° C. for 30 seconds, followed by one Final incubation at 72 ° C for 10 minutes.
- the samples are then separated in a 5% polyacrylamide gel (in 1 x TBE). After the run, the gel is dried and exposed to an X-ray film for 4 days.
- the bands of interest, detected in the autoradiogram are cut out, boiled in 200 ⁇ l H 2 O for 15 minutes, cooled on ice and centrifuged for 10 minutes at 14,000 ⁇ g.
- the resulting supernatants are mixed with the same volume of 10 M ammonium acetate, 20 ⁇ g / ml tRNA and precipitated with 2.5 volumes of ethanol at -80 ° C. overnight.
- the cDNA pellets are washed three times in 75% ethanol and dissolved in 20 ⁇ l H 2 O.
- transcripts are selected which are differentially expressed, ie which are additionally obtained in the gels with the RNAs of cells which have been treated with 60 ⁇ M silica ( Figure 1).
- the identified cDNAs / transcripts are compared with the sequences contained in the BLAST database.
- CaM kinase II gamma (XM_044349; Expect value [£]: 1e -16 ); hypothetical protein (XP 01359, E 1, 6); MUC3B mucin (AJ291390, E 0.20); hypothetical protein (XP_067115, E 5.9); hypothetical protein (XP_090138, E 2.9); ATP-binding cassette, subfamily A member 4 (XM_001290, E 1.6); Polypeptide similar to zinc finger protein 91 (XM_091947, E 3.1); hypothetical protein (XP_104250, E 0.48), hypothetical protein (XP_169372, E 8.6); hypothetical protein (XP_104250, £ 4.1), hypothetical protein (XP_098020, E 3.3) and hypothetical protein (XP_169372, E 8.6).
- the silicase was identified as another transcript and analyzed in greater
- the silicase gene can also be obtained from cDNA libraries, e.g. B. in ZapExpress and in Ash chia coli XL1-Blue MRF ', with suitable degenerate primers using the PCR technique; Appropriate vector-specific primers are used for this.
- the synthesis products obtained are used for screening in the relevant cDNA libraries.
- the identified clones are then subcloned into a vector (for example pGem-T) and then sequenced.
- the recombinant silicase (called: rSIA_SUBDO) is preferably produced in E. coli. However, production in yeast and mammalian cells is also possible and has been carried out successfully.
- the expression of the SDSIA gene from S. domuncula in E. coli using the "GST (Glutathione-S-Transferase) Fusion" system (Amersham) is described below as an example.
- GST Glutathione-S-Transferase
- two inserts are used to eliminate potential effects of signal peptides during expression; one insert comprises the entire derived protein (long form; from amino acid aai to amino acid aa 3 _) and the other insert only amino acids aa_ 6 to aa 3 g (short form) ( Figure 3A).
- SDSIA-I and SDSIA-s are cloned into an appropriate vector, e.g. B. in the plasmid pGEX-4T-2, which contains the glutathione S-transferase (GST) gene from Schistosoma japonicum.
- GST glutathione S-transferase
- Other expression vectors have also been found to be suitable.
- expression of the silicase is usually induced by IPTG (isopropyl- ⁇ -D-thiogalactopyranoside) and carried out in the presence of 1 mM ZnSO 4 for 4 or 6 hours at 37 ° C.
- the GST fusion proteins obtained with the designation rSIA_SUBDO-l (long form; Mr 69 kDa) or rSIA_SUBDO-s (short form; M r 58 kDa) are z.
- the proteins are then subjected to gel electrophoresis in the presence subjected to 2-mercaptoethanol.
- Gel electrophoresis can be carried out in 10% polyacrylamide gels with 0.1% NaDodSO (PAGE). The gels are stained with Coomassie Brilliant Blue.
- the isolation, cloning and expression of the silicase cDNA can also be carried out from other organisms, for example from (silicon dioxide-producing) diatoms (for example Cylindrotheca fusiformis).
- diatoms for example Cylindrotheca fusiformis.
- the extraction of diatoms in axenic cultures is state of the art (Kroger N, Bergsdorf C, Sumper M (1996) Europ J Biochem 239: 259-264).
- the silicase is purified on an antibody affinity matrix.
- the affinity matrix is prepared by immobilizing a silicase-specific antibody on a solid phase (CNBr-activated Sepharose or other suitable carrier). Monoclonal or polyclonal antibodies against the silicase are used as antibodies, which are produced by standard methods (Osterman LA (1984) Method of Protein and Nucleic Acid Research, Vol 2, Springer-Verlag, Berlin). The antibody is coupled to the column matrix according to the manufacturer's instructions (Pharmacia). The pure silicase is eluted by changing the pH or changing the ionic strength.
- an assay can be used which is based on the hydrolysis of p-nitrophenylacetate (Armstrong JM, Myers DV, Verpoorte JA, Edsall JT (1966) Purification and properties of human erythrocyte carbonic anhydrase. J Biol Chem 241: 5137-5149).
- 0.5 ml of a 3 mM p-nitrophenylacetate solution (Sigma) is mixed with 0.05 ml of a 0.3 mM Tris-HCl buffer (pH 7.6). After preincubation at 25 ° C for 5 minutes, 50 ul of the recombinant silicase (rSIA_SUBDO) are added and the increase in absorbance at 348 nm is determined over a period of 5 minutes.
- Figure 5 shows that the activity of the recombinant silicase depends on the concentration of the enzyme in the assay. Enzyme activity is expressed in optical density (OD) units per minute. The addition of 1 ug per assay Silicase (0.56 ul) gave an activity of 0.005 OD 348n m, which increased with increasing protein concentration up to 0.04 OD 3 8nm.
- Spicule from S. domuncula can serve as the substrate (amorphous silicon dioxide) for the silicase.
- PBS phosphate buffer salt solution, consisting of 1.15 mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 , 137 mM NaCI and 2.7 mM KCI
- Silicase activity can be determined as follows. Usually 100 ⁇ g of the dried spicules (powder) are added to a suitable buffer such as 50 mM Tris-HCl buffer (pH 7.2; 10 mM DL-dithiothreitol, 100 mM NaCI) and 0.5 mM ZnSO 4 in 2 ml Eppendorf- Added vessels. Then usually 50 ul of the recombinant silicase are added and incubated at 25 ° C (the incubation is also possible at other temperatures between 5 ° C and about 65 ° C). The average incu bation time is 60 minutes.
- a suitable buffer such as 50 mM Tris-HCl buffer (pH 7.2; 10 mM DL-dithiothreitol, 100 mM NaCI) and 0.5 mM ZnSO 4 in 2 ml Eppendorf- Added vessels. Then usually 50 ul of the recombinant silicase are added and in
- the undissolved spicules are centrifuged off (14000 ⁇ g; 15 minutes; 4 ° C.).
- the released, soluble silica can e.g. B. with the aid of a molybdate-based detection method, such as. B. the colorimetric "Silicon Test"(Merck; 1.14794) can be determined quantitatively.
- the amount of silica is calculated using a calibration curve with a silicon standard (Merck 1.09947) from the absorbance values at 810 nm.
- Figure 5 shows that the recombinant silicase catalyzes the breakdown (dissolution) of amorphous silicon dioxide.
- 3 ng of silica / assay are released at an enzyme concentration of 1 ⁇ g of recombinant silicase per assay.
- the release of silica is 20 or 43 ng / assay.
- sponge spicules needleles; 1 mg
- lysate 1.5 ml
- ZnCl 2 and 0.1 M NaCl 1 mM ZnCl 2 and 0.1 M NaCl were added
- the mixture was then centrifuged (5 min, 14000 rpm) and the molybdate assay (kit from Merck; see above) was carried out to determine the silicate released. It was found that at 4 ° C only a very small amount of silicate was released, but at room temperature (22 ° C) and 56 ° C up to 3.4 and 4.1 ng / ml (24 h).
- silicate-degrading activity was also present in bacterial cell extracts.
- Silicase activity is not only measurable with the sponge enzyme, but surprisingly also with commercial carbonic anhydrases.
- Table 2 shows the release of silicate from diatom skeletons (silicate frameworks of diatoms) and from sand by a commercial carbonic anhydrase preparation (from bovine erythrocytes; Calbiochem company).
- the silicase reaction is reversible. Therefore, the reaction can also be used to synthesize amorphous silica or silicones.
- a suitable expression vector for example pQE-30 vector; Qiagen
- the stop codon in the silicase cDNA is removed.
- the PCR technique is used and primers which have the relevant restriction sites are used for the amplification.
- the cDNA for the second protein is obtained accordingly, the same interface at the 5'-terminus as at the 3'-terminus of the silicase cDNA (in the example Sall) and at the 3'- Terminus is different from the others (e.g. an H / ll position). If there are internal restriction sites in the cDNAs in question, alternative restriction enzymes can be used. Linkers can also be inserted between the two cDNAs.
- the two cDNAs are ligated, purified and ligated into the pQE-30 vector by the usual methods.
- the ligation follows the histidine tag (about 6 histidine codons).
- the expression and purification of the fusion protein via e.g. B. the histidine tag, which is present on the recombinant protein, can on appropriate affinity columns, e.g. B. a Ni-NTA matrix can be carried out (Skorokhod A, Schcke H, Diehl-Seifert B, Steffen R, Hofmeister A, Müller WEG (1997) Cell Mol Biol 43: 509-519).
- a protease cleavage site (such as an enterokinase site) can be cloned between the cDNA for the silicase and the cDNA for another protein.
- a codon for a new start methionine can be inserted in front of the coding region of the gene for the further protein.
- the fusion protein is cleaved proteolytically. Now both proteins are available separately.
- both proteins can be expressed separately on one construct.
- the silicase gene is connected downstream of the His tag in an expression vector.
- a stop codon is inserted at the end of the silicase cDNA.
- a ribosome binding site with a codon for a start methionine is cloned between the cDNA for the silicase and the cDNA for the further protein. Again, a His day precedes the cDNA for the further protein. This gene also receives a stop codon.
- the His tags can be deleted if the proteins are used for functional analysis in the host cells concerned. 4.4. extensions
- Both bacterial and eukaryotic cells can be used for the expression described under 4.1 to 4.3.
- the expression described under 4.1 to 4.3 can also be used for three or more open reading frames.
- a patent has been filed for the primmorph system (DE 19824384. Production of primmorphs from dissociated cells of sponges, corals and other invertebrates: Process for culturing cells of sponges and other invertebrates for the production and detection of bioactive substances, for the detection of environmental toxins and for Cultivation of these animals in aquariums and outdoors, inventor and applicant: Müller WEG, Brummer F).
- Primmorphs are aggregates consisting of proliferating and differentiating cells (Müller WEG, Wien M, Batel R, Steffen R, Borojevic R, Custodio MR (1999) Establishment of a primary cell culture from a sponge: Primmorphs from Suberites domuncula. Marine Ecol Progr Ser 178: 205-219). Primmorphs are formed from sponge single cells, which are obtained from sponge tissue after dissociation in Ca 2+ and Mg 2+ -free, EDTA-containing artificial sea water. After transfer into sea water containing Ca 2+ and Mg 2+ , aggregates form from the single sponge cells, which after 3 days reach a size of 1 mm, and after 5 days, primmorphs with a diameter of about 5 mm.
- the primmorphs are surrounded by epithelial cells, the pinacocytes.
- the cells within the primmorphs are predominantly spherical cells, along with amoebocytes and archaeocytes. 5.2. Effect of silicon on the formation of spicules
- the primmorph system of sponges e.g. B. S. domuncula
- the primmorph system of sponges can be used to study spicula formation or dissolution.
- primmorphs are cultivated for 8 days in sea water, which is supplemented with 30 ⁇ M Fe (+++) (added as citrate) and 10% RPMI1640 medium.
- the silicon concentration in the seawater / medium is 5 ⁇ M.
- the primmorphs are either further incubated in this medium or transferred to a medium which contains 60 ⁇ M silicon (the optimum silicon concentration for the formation of spicules; added as Na hexafluorosilicate) and cultured for 1 or 3 days.
- Primmorphs which are cultivated without the addition of silicon, show predominantly a round, spherical shape.
- Figure 6A shows that most of the primmorphs, however, become oval in shape after additional 3 days of cultivation in the presence of 60 ⁇ M silicon. In the presence of silicon, the primmorphs begin to form spicules. The synthesis of long (> 100 ⁇ m) spicules can be observed in some cases ( Figure 6B), but smaller spicules (30 ⁇ m) are usually found ( Figure 6D). In the absence of silicon, no spicules are present (Figure 6C).
- the expression of the silicase gene is upregulated in primmorphs of S. domuncula in the presence of silicon.
- the expression of the following genes is increased: silicatein, collagen, myotrophin and isocitrate dehydrogenase.
- the expression of the silicase gene can be determined by Northem blotting using methods which are state of the art and have been used, for example, to determine the expression of silicatein and collagen (Krasko A, Batel R, Schröder HC, Müller IM, Müller WEG (2000) Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin. Europ J Biochem 267: 4878-4887).
- RNA was then extracted. A quantity of 5 ⁇ g total RNA was separated electrophoretically on a 1% formaldehyde / agarose gel and blotted onto a Hybond-N + nylon membrane in accordance with the manufacturer's instructions (Amersham). Hybridization was performed with 400 to 600 bp segments of the following probes: SDSIA (encoded for silicase), SDSILICA (encoded silicatein) and SDIDH (encoded for the ⁇ -subunit of isocitrate dehydrogenase).
- SDSIA encoded for silicase
- SDSILICA encoded silicatein
- SDIDH encoded for the ⁇ -subunit of isocitrate dehydrogenase
- the probes were labeled with the PCR-DIG-Probe-Synthesis Kit according to the manufacturer's instructions (Röche). After washing, the DIG-labeled nucleic acid with anti-DIG Fab fragments (conjugated with alkaline phosphatase; dilution: 1: 10,000) was detected and visualized using the chemiluminescence technique using CDP (Röche), the chemiluminescent substrate of alkaline phosphatase.
- Figure 7 shows the Northern blots obtained. It can be seen that the genes for the silicase and silicatein are highly upregulated in response to higher silicon concentrations. Furthermore, the isocitrate dehydrogenase gene (encoded for an enzyme involved in the citrate cycle) is upregulated, which indicates that the formation of amorphous silicon dioxide requires an increased metabolic rate of the cells.
- the silicase also has carbon anhydrase activity, such as with a colorimetric assay (Armstrong JM, Myers DV, Verpoorte JA, Edsall JT (1966) Purification and properties of human erythrocyte carbonic anhydrase. J Biol Chem 241: 5137-5149) can be demonstrated.
- a colorimetric assay Armstrong JM, Myers DV, Verpoorte JA, Edsall JT (1966) Purification and properties of human erythrocyte carbonic anhydrase. J Biol Chem 241: 5137-5149
- the silicase causes a change in pH due to the conversion of CO 2 to HCO 3 " ( Figure 8 [1]). This enables etching of lime substrates, but not of silica materials, whose solubility increases with increasing, but does not increase with falling pH.
- a method which is "mild” and gentle on biomaterials compared to the physical / chemical methods used represents a modification of the surfaces which is based solely on biochemical / enzymatic reactions, which is possible with the aid of the method according to the invention (silicase-mediated enzymatic degradation and - as a reversible reaction - enzymatic synthesis of S1O 2 - or siloxane-containing surfaces with the help of recombinant / purified silicase).
- the recombinant or purified from natural sources silicase in the production of surface modifications (in the coating) of silicone materials, such as silicone breast implants, endoprostheses or metal implants (improving the connection between bone and metal -Implant, biologization of the metal implants) and contact / plastic lenses.
- silicone materials such as silicone breast implants, endoprostheses or metal implants (improving the connection between bone and metal -Implant, biologization of the metal implants) and contact / plastic lenses.
- Other uses relate to the coating of collagen, which serves as a bone replacement material, and of collagen nonwovens, which, for. B. can be used for "tissue engineering". The aim here is to increase the stability and porosity and to improve the resorbability.
- Crystalline silica (silicon dioxide) in the form of quartz, tridymite or cristobalite is probably one of the most important workplace hazardous substances. The severity of the Health impairments and the variety of possible sources of exposure have long been known. Because of the widespread occurrence of crystalline silica in the earth's crust and the frequent use of materials containing it, workers in a variety of different industries are particularly exposed to crystalline silica. It can be assumed that in agriculture, mining, in the glass and fiber glass industry, in cement production, in the production of ceramics, in foundries, in the production of paints, soaps and cosmetics or in the dental manufactory / repair, millions of employees are regularly exposed to crystalline silicon dioxide. According to the American Thoracic Society, silicon dioxide is one of the main causes of lung diseases worldwide. There is therefore a great need to develop strategies for prevention and therapy.
- silica Inhaled crystalline silica is known to cause lung fibrosis (silicosis) and lung cancer.
- Silicosis is a malignant pneumoconiosis, which is caused by an accumulation of silicon dioxide particles in the lung tissue and is characterized by the appearance of silicotic nodules. There is no rational therapy for this disease, which leads to severe disabilities.
- Silicosis generally develops very slowly over decades. It is a progressive disease that is not curable. It first manifests itself through shortness of breath, irritable cough and stinging in the chest. Overworking the heart and restricting breathing and circulation ultimately lead to death. The average period between exposure to dust and the appearance of silicosis is around 20 years. A dangerous complication of silicosis is the culosis. The mechanism that leads to the development of lung cancer by crystalline silicon dioxide is still poorly understood.
- Silicosis is the most common dust lung disease among occupational diseases.
- the average total cost of a silicosis patient is around 130,000 euros.
- the silicase which is involved in the dissolution of biogenic silicon dioxide, can be used as a therapeutic / protective agent for the treatment of silicosis.
- Silicase is not only able to dissolve amorphous, but also crystalline silicon dioxide (quartz crystals).
- the silicase thus has the properties necessary to eliminate silicon dioxide from the lungs and / or to modulate the course of this lung disease.
- Microspheres as a carrier system for the recombinant silicase for the treatment of silicosis can, for. B. from sponge collagen analogous to calf callagen microspheres (Rössler et al., Pharmazie 49 (1994) 175-179).
- the sponge collagen microparticles are loaded by adsorption of the recombinant protein (silicase) as described (Roessler et al., J. Microencapsulation 12 (1995) 49-57; Berthold et al., Eur. J. Pharm. Biopharm 45 (1998) 23-29).
- the advantages of collagen are its biodegradability as well as its low toxicity and immunogenicity.
- Other delivery systems include liposomes with the enclosed recombinant enzyme and lipid nanoparticles (Jenning et al., Eur. J. Pharm. Biopharm. 49 (2000) 211-218).
- SEQ ID No. 1 the amino acid sequence of the silicase according to the invention from S. domuncula (SIA_SUBDO),
- SEQ ID No. 2 The nucleic acid sequence of the cDNA of the silicase according to the invention from S. domuncula.
- Illustration 1
- FIG. 3 (A) Alignment of S. domuncula silicase (SIA_SUBDO) with human carbonic anhydrase II (carbonate dehydratase II) (CAH2_HUMAN; P00918).
- the carbonic anhydrase domain is boxed (
- the characteristic amino acids which form the eukaryote-type carbonic anhydrase signature are marked (A: found in both sequences; ⁇ : only present in the carbonic anhydrase, but not in the silicase).
- the additional signs (+) show those residues that form the hydrogen network of the active center.
- the three zinc-binding histidine residues are marked (Z).
- PCC 7120 (CAH_ANASP; P94170).
- the latter sequence served as an outgroup.
- the measurement bars indicate an evolutionary distance of 0.1 amino acid substitutions per position in the sequence.
- the phylogenetic tree was constructed using "neighbor joining"("Neighbor” program: Felsenstein, J. (1993). PHYLIP, ver. 3.5. University of Washington, Seattle).
- the recombinant S. domuncula silicase (rSIA_SUBDO) was produced as a GST fusion protein. Both the long and the short SDSIA were cloned into a pGEX-4T-2 plasmid containing the glutathione S-transferase (GST) gene. The fusion proteins were either outgoing induction with IPTG (-IPTG) or after incubation with IPTG (+ IPTG) isolated for 4 or 6 hours, then split, cleaned and then subjected to Na-DodSO 4 -PAGE. The gel was stained with Coomassie Brilliant Blue. The purified long form rSIA_SUBDO-l with a size of 43 kDa and the short form (M r 32 kDa) of the silicase were obtained.
- RNA was extracted from primmorphs, which in the absence of additional silicon (- Si) or in the presence of 60 ⁇ M silicon (+ Si) had been incubated for 1 to 3 days. 5 ⁇ g of the total RNA was separated electrophoretically, blotted on nylon membranes and hybridized with the following probes: SDSIA (silicase), SDSILICA (silicatein) and SDIDH ( ⁇ -subunit of isocitrate dehydrogenase). The sizes of the transcripts are given.
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DE50308707T DE50308707D1 (de) | 2002-10-03 | 2003-10-02 | Abbau und modifizierung von silicaten und siliconen durch silicase und verwendung des reversiblen enzyms |
AU2003276044A AU2003276044A1 (en) | 2002-10-03 | 2003-10-02 | Decomposition and modification of silicate and silicone by silase and use of the reversible enzyme |
EP03807840A EP1546319B1 (de) | 2002-10-03 | 2003-10-02 | Abbau und modifizierung von silicaten und siliconen durch silicase und verwendung des reversiblen enzyms |
CA002501208A CA2501208A1 (en) | 2002-10-03 | 2003-10-02 | Decomposition and modification of silicate and silicone by silase and use of the reversible enzyme |
JP2004542423A JP2006501832A (ja) | 2002-10-03 | 2003-10-02 | シリカーゼによるケイ酸塩及びシリコンの分解及び修飾並びに可逆酵素の使用 |
DK03807840T DK1546319T3 (da) | 2002-10-03 | 2003-10-02 | Nedbrydning og modificering af silicater og siliconer med silicase og anvendelse af det reversible enzym |
US10/530,240 US7229807B2 (en) | 2002-10-03 | 2003-10-02 | Decomposition and modification of silicate and silicone by silase and use of the reversible enzyme |
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WO2000035993A1 (en) * | 1998-12-18 | 2000-06-22 | The Regents Of The University Of California | Methods, compositions, and biomimetic catalysts for in vitro synthesis of silica, polysilsequioxane, polysiloxane, and polymetallo-oxanes |
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US5962270A (en) * | 1996-02-06 | 1999-10-05 | Bionebraska, Inc. | Recombinant preparation of calcitonin fragments and use thereof in the preparation of calcitonin and related analogs |
DE10037270B4 (de) * | 2000-07-28 | 2007-09-13 | Müller, Werner E. G., Prof. Dr. | Silicatein-vermittelte Synthese von amorphen Silikaten und Siloxanen und ihre Verwendung |
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2002
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WO2000035993A1 (en) * | 1998-12-18 | 2000-06-22 | The Regents Of The University Of California | Methods, compositions, and biomimetic catalysts for in vitro synthesis of silica, polysilsequioxane, polysiloxane, and polymetallo-oxanes |
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CHA J N ET AL: "SILICATEIN FILAMENTS AND SUBUNITS FROM A MARINE SPONGE DIRECT THE POLYMERIZATION OF SILICA AND SILICONES IN VITRO", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 96, January 1999 (1999-01-01), pages 361 - 365, XP001037314, ISSN: 0027-8424 * |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2005106003A1 (de) * | 2004-04-30 | 2005-11-10 | Heiko Schwertner | Enzym- und template-gesteuerte synthese von silica aus nicht-organischen siliciumverbindungen sowie aminosilanen und silazanen und verwendung |
WO2005106004A1 (de) * | 2004-04-30 | 2005-11-10 | Heiko Schwertner | Enzymatisches verfahren zur herstellung bioaktiver, osteoblasten-stimulierender oberflächen und verwendung derselben |
JP2007535319A (ja) * | 2004-04-30 | 2007-12-06 | シュヴェルトナー,ハイコ | 酵素とテンプレートで制御された、非有機シリコン化合物、アミノシラン、シラザンからのシリカの合成、並びにその使用 |
JP2007535320A (ja) * | 2004-04-30 | 2007-12-06 | シュヴェルトナー,ハイコ | 生体活性を有する骨芽細胞刺激表面を製造するための酵素的方法およびその利用 |
CN102257148A (zh) * | 2008-12-19 | 2011-11-23 | 诺维信公司 | 具有二氧化硅酶活性的酶的用途 |
DE102016004090A1 (de) | 2015-04-16 | 2016-10-20 | Florian Draenert | ZUSAMMENSETZUNG ElNES BlOMATERlALS |
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AU2003276044A1 (en) | 2004-05-04 |
CA2501208A1 (en) | 2004-04-22 |
US20060029939A1 (en) | 2006-02-09 |
JP2006501832A (ja) | 2006-01-19 |
EP1546319A1 (de) | 2005-06-29 |
DE10246186A1 (de) | 2004-04-15 |
US7229807B2 (en) | 2007-06-12 |
DE10246186B4 (de) | 2005-07-07 |
US20070218044A1 (en) | 2007-09-20 |
DE50308707D1 (de) | 2008-01-10 |
DK1546319T3 (da) | 2008-03-31 |
EP1546319B1 (de) | 2007-11-28 |
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