EP1623232A2 - Enrichment of enzymatic cleavage products - Google Patents
Enrichment of enzymatic cleavage productsInfo
- Publication number
- EP1623232A2 EP1623232A2 EP04729627A EP04729627A EP1623232A2 EP 1623232 A2 EP1623232 A2 EP 1623232A2 EP 04729627 A EP04729627 A EP 04729627A EP 04729627 A EP04729627 A EP 04729627A EP 1623232 A2 EP1623232 A2 EP 1623232A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- mutant
- protease
- enzyme
- enzymatically inactive
- cleavage products
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006882 induction of apoptosis Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000004987 nonapoptotic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000007030 peptide scission Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 238000000575 proteomic method Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 150000003355 serines Chemical class 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- AFVLVVWMAFSXCK-UHFFFAOYSA-N α-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(C#N)=CC1=CC=C(O)C=C1 AFVLVVWMAFSXCK-UHFFFAOYSA-N 0.000 description 1
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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
- C12N9/6408—Serine endopeptidases (3.4.21)
-
- 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
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the invention relates to a method . for the enrichment, isolation and / or identification of enzymatic cleavage products as well as certain mutants and their use.
- proteases as enzymes that catalyze cleavage play a major role in the breakdown of proteins.
- proteases are the enzymes that catalyze the hydrolytic cleavage (proteolysis) of the peptide bond in proteins and peptides.
- the proteases can be divided into the so-called proteinases (formerly: endopeptidases) and peptidases (formerly exopeptidases).
- the former cleave petid bonds inside a protein and thereby produce peptides as cleavage products.
- the latter cleave proteins at the amino or carboxy end.
- proteases which are preferably proteinases.
- proteinases and their cleavage products are of particular interest for degradomics research.
- the caspases play an important role in the controlled degradation of various cellular substrates, in particular they are involved in apoptotic processes, that is to say in degradation processes which are associated with controlled cell death.
- the caspases are a highly conserved protease family with at least 12 human members. Because of their role in inflammatory processes and in cell apoptosis, caspases are of enormous scientific interest.
- the caspases are cysteine proteases, which means that these proteases carry cysteine at a critical point in the active center, which is crucial for proteolytic activity.
- Caspases are very specific proteases that cleave in their substrate after an aspartic acid residue. All cleavage products of the caspases therefore carry an aspartic acid residue at the C-terminal end of the peptide cleavage product (position P1). At position P3 there is often glutamic acid.
- the object of the invention is to provide a method with which cleavage products of a specific enzyme or a group of enzymes can be investigated with a few process steps.
- Claim 16 relates to a specific mutant of a protease and claim 25 relates to a corresponding nucleotide sequence.
- Claims 27 and 29 deal with the use of the mutant or with a corresponding affinity matrix. Preferred embodiments can be found in the subclaims. By reference, the wording of all claims is made the content of the description.
- cleavage products of at least one enzyme from a sample can be enriched, isolated and / or identified.
- This is done using an enzymatically inactive mutant of a protease as affinity material, it being decisive for the method according to the invention that the enzymatically inactive mutant of the protease continues to have its substrate specificity. It is also important that the enzyme or enzymes to be analyzed have at least a structural similarity to the hydrolytic products of the protease, the mutant of which is used.
- the enzymatic inactivity is advantageous since otherwise the protease used as the affinity material could itself produce fission products which would possibly falsify the results of the method according to the invention.
- the sample containing the cleavage products to be detected is first incubated with the enzymatically inactive mutant, so that interactions between the cleavage products to be detected in the sample and the mutant can develop. These interactions are based on the fact that the mutant has a high binding affinity for substrates with certain structural features. The cleavage products to be detected have these structural features, so that they are specifically bound by this mutant. In a further step of the method, material that does not interact with the mutant can be removed. The cleavage products that had been bound by the mutant can then be analyzed. Whether a separation of the interacting cleavage products from the mutant before the actual analysis of the cleavage products is useful and possibly necessary depends on the specific design of the method and in particular on the analysis method.
- the method according to the invention is based on the one hand that the protease, the mutant of which is used as an affinity material, has a high binding affinity for its own substrates and also for the products which result from the proteolytic cleavage of the substrates. Furthermore, it is necessary that this binding activity of the protease can be separated from its catalytic activity. Such a separation of the catalytic activity from the binding activity is already known for the proteases trypsin and chymotrypsin. A so-called anhydro modification in the catalytic center of these proteases can destroy the catalytic activity, that is to say the catalysis of hydrolytic cleavages, while the binding affinity for the cleavage products is retained.
- anhydrotrypsin or anhydrochymotrypsin
- the cleavage products are peptides that have arginine and lysine at the C-terminal end.
- anhydrochymotrypsin these are peptides with hydrophobic amino acids at the C-terminal end.
- the anhydro mutants of trypsin and chymotrypsin can be achieved by chemical modification or treatment, with serine in the active center of the enzymes being replaced by alanine, ie the anhydro form of serine.
- the enzyme whose cleavage products are to be enriched, isolated and / or identified is a protease, this protease preferably differing from the protease whose enzymatically inactive mutant is used as affinity material.
- This embodiment of the invention has the great advantage that the enzymatically inactive mutant of a protease can be used as a universal tool for examining the cleavage products of any enzyme, as long as the cleavage products to be examined have the corresponding structural features as they do for the binding activity of the enzymatically inactive mutant used for certain substrates are required. This makes it a widely applicable process for proteome research, which is based on the use of functional features. With the aid of the method according to the invention, results can be achieved, among other things, which allow conclusions to be drawn about the identity of different substrates or products of certain enzymes. This also enables the activities of enzymes or entire enzyme families to be quantified.
- the structural similarity between the cleavage products to be investigated and the products which are bound by the protease, the enzymatically inactive mutant of which is used is one or more identical terminal amino acid residues, in particular C-terminal residues.
- the mutants of which can be used according to the invention the binding affinity to their substrates is based on one or more specific C-terminal amino acids.
- V8 proteinase from Staphylococcus aureus shows a specific binding affinity for peptides which have glutamic acid (Glu) or aspartic acid (Asp) at the C-terminal end.
- Glu glutamic acid
- Asp aspartic acid
- An enzymatically inactive mutant of this V8 proteinase, which still has its substrate specificity, is therefore suitable according to the method according to the invention for the analysis of cleavage products of other enzymes which have corresponding C-terminal amino acids or corresponding residues.
- the enzymatically inactive mutant of the protease carries a change in the active center. This destroys the catalytic activity according to the invention, but the binding activity is retained.
- the protease whose mutant is used is a serine protease.
- Serine proteases are characterized in that they carry serine at a critical point in the active center. Removing or replacing this serine destroys the enzymatic activity, whereas the substrate specificity is retained. These proteases are therefore particularly suitable according to the invention since a single change which brings about a corresponding amino acid exchange can provide a mutant which can be used according to the invention.
- These particularly suitable serine proteinases include, for example, the V8 proteinase already mentioned.
- the enzymatically inactive mutant is advantageously an anhydro mutant.
- this is an exchange of serine for alanine. Since in the serine proteases mentioned a serine in the catalytic center of the protease is responsible for the hydrolytic activity, such an anhydro mutation can destroy the hydrolytic activity while maintaining the substrate specificity.
- other mutants of proteases can also be used according to the invention as long as they are enzymatically inactive, that is to say can no longer catalyze hydrolytic cleavage, and continue to have their substrate specificity.
- the enzymatically inactive mutant is used in immobilized form.
- the method can be carried out in the form of column chromatography, in which case the enzymatically inactive mutant can be immobilized on a conventional chromatography material, such as, for example, Sepharose, Agarose or Fractogel. Immobilization can be carried out using customary methods.
- the mutant can be coupled to immobilized nickel ions (eg Ni-NTA agarose) via a sequence of histidines.
- the enriched fission products can be analyzed using customary methods. Analysis using one- and / or two-dimensional polyacrylamide gel electrophoresis is particularly preferred. Furthermore, the analysis can be carried out using conventional mass spectrometric methods. Mass spectrometry can also be combined with polyacrylamide gel electrophoresis or other common methods.
- Chromatography in particular column chromatography, for example conventional affinity chromatography, can be carried out to carry out the actual method, that is to say incubating the sample and removing non-interacting material.
- the analysis can comprise one or more chromatography steps, in particular column chromatography steps.
- a further separation of different enriched cleavage products can be achieved by one or more chromatography steps.
- the fission products to be analyzed are modified in the course of the method. In particular, this can involve further cleavage of the cleavage products, which is achieved, for example, by treatment with suitable enzymes. Tryptic digestion or the like is particularly suitable for this. Such a modification takes place above all with regard to an analysis of the fission products, the fission products being further fragmented, for example, for a mass spectrometric analysis.
- the protease, the enzymatically inactive mutant of which is used is a V8 proteinase, for example a V8 proteinase from Staphylococcus aureus.
- a corresponding mutant, in particular an anhydro mutant of this enzyme is particularly suitable according to the invention. It is to be used for the enrichment, isolation and / or identification of cleavage products which carry a C-terminal residue of glutamic acid or aspartic acid. It is particularly preferred here to enrich or isolate and / or identify peptides with a C-terminal aspartic acid residue.
- the method according to the invention can advantageously be used to examine cleavage products of cysteine proteases.
- the method is very particularly suitable for the investigation and characterization of fission products of one or more caspases.
- Caspases are very specific proteases whose cleavage products have aspartic acid at the C-terminal end.
- the use of an enzymatically inactive mutant of V8 proteinase is particularly suitable for the investigation of cleavage products of the caspases, since a corresponding mutant has a high affinity for cleavage products with a C-terminal aspartic acid residue.
- the invention further comprises an enzymatically inactive mutant of a protease, the substrate specificity being retained in this mutant.
- a corresponding mutant of a serine protease, in particular a V8 proteinase, for example a V8 proteinase from Staphylococcus aureus, is particularly preferred.
- Such a mutant can be used with great advantage in the method according to the invention described.
- this mutant it is the V8 proteinase mentioned, for example from Staphylococcus aureus, in which serine is modified, exchanged or removed at position 237.
- the serine at this position is preferably replaced by another amino acid, in particular by alanine.
- the exchange at position 237 from serine to alanine is preferably carried out by a single base exchange at position 712 from thymine to guanine.
- the serine at position 237 is the critical serine in the active center of the proteinase. A change at this point destroys the catalytic, ie the hydrolytic, activity, while maintaining the substrate specificity.
- the mutant according to the invention can be produced by chemical modification of the protease. However, it is particularly preferred if the mutant is produced by molecular biological methods.
- the enzymatically inactive mutant according to the invention can be characterized in that it comprises at least part of the amino acid sequence according to SEQ ID No. 1 has. Furthermore, the invention comprises a corresponding mutant which is at least 70%, in particular at least 90%, and preferably at least 99% identical to the amino acid sequence according to SEQ ID No. 1 or one or more parts thereof. This mainly includes mutants that the part or parts of the amino acid sequence according to SEQ ID No. 1, which are responsible for the substrate specificity. In addition, the invention also encompasses mutants which are so similar to such sequences that they still bring about a corresponding substrate specificity in the sense of the invention.
- the enzymatically inactive mutant is further characterized in that it is present in immobilized form.
- the enzymatically inactive mutant is further characterized in that it is present in immobilized form.
- the invention furthermore comprises a nucleotide sequence which codes for an enzymatically inactive mutant of a protease, the substrate specificity of which is retained.
- a nucleotide sequence which codes for an enzymatically inactive mutant of a protease, the substrate specificity of which is retained.
- the nucleotide sequence according to the invention is characterized in that it comprises at least part of the nucleotide sequence according to SEQ ID No. 2 includes.
- the parts of the nucleotide sequence which code for the region of the protease and which are decisive for the substrate specificity are preferred.
- the invention comprises the use of an enzymatically inactive mutant of a protease, the substrate specificity of which is retained, as an affinity material in a process for the enrichment, isolation and / or identification of cleavage products of at least one enzyme, at least one cleavage product of the protease and at least one cleavage product of the enzyme at least have a structural similarity.
- the invention comprises an affinity matrix for the enrichment, isolation and / or identification of enzymatic cleavage products.
- This affinity matrix comprises an immobilized, enzymatically inactive mutant of a protease while maintaining its substrate specificity.
- This affinity matrix according to the invention can be used as a universal tool for investigations in the proteome area or in degradomics research. For example, changes in the activity of entire enzyme families, such as the caspases, can be examined under different conditions.
- the affinity matrix can be used, for example, as a simple column chromatography matrix, comparable to a conventional affinity chromatography.
- the cleavage products to be examined can be eluted, for example, by changing the buffer conditions and then analyzed.
- the analysis can be carried out, for example, with a conventional two-dimensional polyacrylamide gel electrophoresis. With such a method, results can be achieved in a few steps, which provide information about enzymatic activities.
- substrates and products of enzymes, in particular proteinases can also be characterized and / or identified.
- the method according to the invention provides an effective tool for selectively enriching or purifying specific classes of proteins or peptides which are of great interest in particular for proteome research.
- Such a group-specific affinity tool opens up the possibility of creating profiles of proteins based on their activity. As a result, changes in the functional status of enzymes can be examined even if the quantitative level of the enzyme remains constant. Furthermore, the invention could be used to carry out tests on the effectiveness of inhibitors which influence the various members of an enzyme family, for example the caspases. For example, the intact cell or a cell extract with a potential inhibitor are treated, then to analyze the activity of the enzyme family according to the invention in the manner described above.
- Fig. 1 shows a schematic representation of the construction of Ser237Ala-V8 ⁇ 48.
- Fig. 2 Expression of the anhydro mutant of the V8 proteinase.
- the protein samples were separated on a 12% SDS-polyacrylamide gel (SDS-PAGE) with a discontinuous buffer. The coloring was done with Coomassie Brillantblau R-250. 1: total cell lysate, 2: run, 3: elution, 4: marker proteins.
- Fig. 3 Enzymatic activities of the wild type and the anhydro mutant of the V8 proteinase.
- the proteinase activity was measured at 30 ° C. in 0.1 M Tris-HCl pH 7.8, 0.01 M CaCl 2 with 0.4% universal protease substrate (Röche) in a volume of 0.2 ml. The activity was measured by absorption
- the concentration of the proteinase was 1 ⁇ g / ⁇ l in each case.
- the Ni-NTA agarose beads were treated with Ser237Ala-V8 ⁇ 48 in 50 mM Tris-HCl, 300 mM Load NaCI, 1% CHAPS at pH 7.4.
- the neurotropic factor for retinal cholinergic neurons and [Cys (Bz) 84 , Glu (OBz) 85 ] - CD 4 (81-92) were incubated with the loaded agarose.
- the agarose was washed and eluted with 200 mM acetic acid.
- the samples were lyophilized in a Speed Vac and for the
- Fig. 5 Two-dimensional polyacrylamide gel electrophoresis of the eluates of the anhydro V8 agarose. Readystrip IPG strips (pH 5-8) were loaded with 40 ⁇ g cell extract. Isoelectric focusing (IEF) was carried out with up to 70 kVh. The SDS-PAGE was carried out with 11% polyacrylamide gels (70x70x1.0 mm) at 11 ° C. with constant current (40 mA). The second
- the gels were stained with silver nitrate.
- the left gel shows the control, the right gel shows the separated cell extract of the cells with induced apoptosis.
- FIG. 6 Two-dimensional polyacrylamide gel of the separated cell extract of the cells with induced apoptosis, the different protein spots compared to the control gel (see FIG. 5) being marked with numbers.
- FIG. 7 database comparison of mass spectrometric results of different spots from a two-dimensional polyacrylamide gel from cell extract from cells with induced apoptosis (see FIG. 6).
- FIG. 8 Amino acid (A) and nucleotide sequences (B) and (C) of the 6xHis-Ser237Ala-V8 ⁇ 48 mutant.
- (A) is the amino acid sequence the mutant including the 6xHis linker is shown (SEQ ID No. 1).
- the coding nucleotide sequence including the 6xHis linker and the stop codon from the vector pQE9 is shown under (B) (SEQ ID No. 2).
- the nucleotide sequence which was used as an insert for the cloning into the vector pQE9 is listed under (C) (SEQ ID No. 3).
- 6xHis is italic and the alanine mutation is shown in bold and underlined.
- (B) and (C) the restriction sites (BamHI and HindIII) are in italics and the point mutation (t to g) is shown in bold and underlined.
- Staphylococcus aureus was cultivated in LB medium at 37 ° C. overnight. Genomic DNA was purified using the RNA / DNA QIAGEN mini kit using 1 ml culture. The polymerase chain reaction (PCR) was carried out with the primers PF1 (SEQ ID No. 4) and PR1 (SEQ ID No. 5) (Table 1), a dNTP mix and the PfuTurbo ® DNA polymerase. By means of these primers according to Yabuta et al. (Appl. Microbiol. Biotechnol.
- V8 ⁇ 48 protease V8 ⁇ 48 protease
- the duplication products were separated by electrophoresis on a 1.2% agarose gel and stained with ethidium bromide.
- the resulting PCR products were purified, digested twice with BamHI and HindIII and ligated into the vector pUC18, so that the plasmid pUC18-V8 ⁇ 48 was formed.
- the ligation mixture was used to transfect Escherichia coli DH5 (competent cells). All clones produced were checked by DNA sequencing.
- Table 1 Sequences of the primers used for the cloning and mutagenesis of the anhydro V8 proteinase.
- the site-directed mutagenesis of V8 ⁇ 48 was performed using QuikChange ® XL site-directed mutagenesis kit and the plasmid pUC 18-V8 ⁇ 48 performed.
- the mutation of serine (Ser) 237 to alanine (Ala) was achieved with the primers PF2 (SEQ ID No. 6) and PR2 (SEQ ID No. 7) (Table 1).
- the vectors were isolated and sequenced. Positive clones were digested twice with BamHI and HindIII and ligated into the expression vector pQE9, so that the plasmid pQE9-Ser237Ala-V8 ⁇ 48 was formed.
- the competent E. co // ' strain BL21 (DE3) was transfected with the plasmid pQE9-Ser237Ala-V8 ⁇ 48.
- Freshly transfected cells with the plasmid were cultured in 100 ml LB (Luria Bertani) medium with 1 ⁇ g / ml ampicillin at 37 ° C.
- LB Lia Bertani
- protein expression was induced by adding isopropyl thiogalactoside to a final concentration of 1 mM.
- the cells were harvested by centrifugation and suspended in 2 ml of lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 1% CHAPS, pH 7.4).
- lysis buffer 50 mM Tris-HCl, 300 mM NaCl, 1% CHAPS, pH 7.4
- 0.1 ml of a solution with lysozyme (20 mg / ml) was added. The mixtures were for 30 min. Incubated at 37 ° C and sonicated for complete cell destruction. After the solutions had been incubated for 20 min. Centrifuged at 15,000 xg. The supernatant was used further and the pellet was discarded. A 8 ⁇ l sample was saved from each supernatant for SDS gel analysis.
- Ni-NTA agarose beads were packed in 0.5 ml columns and loaded with the supernatant. The columns were washed with 5 column volumes of lysis buffer and 5 column volumes of lysis buffer with 20 mM imidazole. The bound protein was eluted with elution buffer (50 mM Tris-HCl, 300 M NaCl, 1% CHAPS, 400 mM imidazole, pH 7.4). The protein content was determined using the BCA method with bovine serum albumin as the calibration standard. The eluted fraction were analyzed on a 12% SDS-PAGE with discontinuous buffer according to Laemmli. The coloring was done with Coomassie Brillantblau R-250.
- elution buffer 50 mM Tris-HCl, 300 M NaCl, 1% CHAPS, 400 mM imidazole, pH 7.4
- the purified enzyme fractions were combined and desalted using NAP-5 gel filtration columns.
- the NAP-5 columns were equilibrated with 50mM Tris-HCl, 1% CHAPS, 10% glycerin, pH 7.4 before use.
- the samples were stored at -20 ° C until further use.
- V8 protease activity was measured at 30 ° C. in 0.1 M Tris-HCl, pH 7.8, 0.01 M CaCl 2 with 0.4% universal protease substrate (Röche) in a volume of 0.2 ml.
- the activity of the enzyme was determined by observing the absorption at 574 nm over a period of 10 min. certainly.
- Ni-NTA agarose beads (20 ⁇ l) were loaded with 20 ⁇ g Ser237Ala-V8 ⁇ 48 in 50 mM Tris-HCl, 300 mM NaCl, 1% CHAPS, pH 7.4.
- the agarose beads were washed with 2 x 200 ⁇ l 0.1 M acetic acid and then 3 x 1 ml 50 mM Na phosphate, 300 mM NaCl, 1% CHAPS, pH 7.2.
- [Cys (Bz) 84 , Glu (OBz) 85 ] -CD 4 (81 -92) is the short amino acid sequence AS81 -92 of the CD4 protein, which is at position 84 (Cys) with Bz and at position 85 (Glu) is derivatized with OBz.
- the beads were washed three times with 500 ul ice-cold buffer and washed 500 ⁇ l of 20 mM NH4HCO3, pH 8.4 and eluted with 20 ⁇ l of 200 mM acetic acid.
- the samples were lyophilized in a Speed Vac and used for mass spectrometric analysis (MALDI-TOF).
- the fibroplast cell line from rat kidney NRK-49F was in Dulbecco's Modified Eagle's Medium (nutrient mixture F12 urine, with 10% fetal calf serum (FCS), 1% antibiotic antifungal (100 X solution with 10,000 U / ml penicillin-G, 10 mg / ml streptomycin and 25 ⁇ l / ml amphotericin-B) cultured in a humidified atmosphere at 5% CO 2 at 37 ° C.
- the cells were cultivated in 10 cm culture dishes to subconfluence and using trypsin EDTA in a ratio of 1 : 5.
- 80% confluent NRK-49F cells were immobilized for 24-48 h by incubation in an appropriate medium with 0.5% FCS, and the cells were stored by freezing in 90% FCS, 10% DMSO in liquid nitrogen.
- Apoptosis in 80% confluent cells was induced by adding 1 M hydrogen peroxide to a final concentration of 1 mM. Control cells were incubated without hydrogen peroxide for a corresponding period of time. The cells were lysed in hypotonic buffer (10 mM Na phosphate, pH 7.2, 1% Triton X-100, 1X complete protease inhibitors) and the insoluble cell debris at 10,000 xg for 20 min. centrifuged. 5 M NaCl was added to the supernatant to reach a final concentration of 300 mM. The samples thus obtained were placed on 100 ⁇ l packed Ni-NTA agarose columns, which were loaded with Ser237Ala-V8 ⁇ 48 protease as described above.
- the beads were washed 3 ⁇ with 2 ml ice-cold buffer with 300 mM NaCl and with 100 ⁇ l 1% SDS in 10 mM Tris-HCl, pH 7.4, 300 mM NaCl at 80 ° C. for 5 min. eluted.
- the salts were removed with Millipore BIOMAX 5K ultrafiltration membrane devices and the samples diluted in electrophoresis sample buffer.
- Ready-to-use Readystrip IPG strips (pH 5-8) from Bio-Rad were rehydrated overnight with 50 ⁇ l cell extract. The isoelectric focusing was carried out up to a total of 70 kVh.
- the IPG strips were in a solution of 20 mg / ml dithiothreitol (DTT) in equilibration buffer for 20 min. incubated and then in a solution of 45 mg / ml iodoacetamide in the same buffer for 20 min. given.
- SDS-PAGE was carried out with an 11% polyacrylamide gel (70x70x1, 0 mm) at 11 ° C. with a constant current of 40 mA. The second dimension was carried out until the bromophenol blue front reached the end of the gel.
- the gels were coated with silver according to Shevchenko et al. (Anal. Chem. 68, 850-858 (1996)).
- the samples were analyzed in an Autoflex MALDI-TOF mass spectrometer (Bruker, Germany). All spectra were recorded in a positive ion reflector mode. Typically, the first 10 shots from a new spot were discarded and the next 200 shots were taken.
- the coding regions of the V8 ⁇ 48 protease were determined by a polymerase chain reaction with the genomic DNA of S. aureus according to Yabuta et al. amplified. The product obtained was ligated into the plasmid pUC18 via the BamHI / HindIII restriction sites. It was shown by double-stranded sequencing that the coding region is identical to the sequence described in previous work.
- Site-directed mutagenesis was used to produce a mutation from Ser-237 to Ala (T712> G) in the pUC18 plasmid with the .V ⁇ protease gene. This step was checked by DNA sequencing. The identified colony carrying this mutation was cultivated in LB medium and the corresponding plasmid was isolated and included BamHI and Hindill digested. The gene Ser237Ala-V8 ⁇ 48 obtained was subcloned into the expression vector pQE9 using the BamHI / Hindlll restriction sites and used for transfection of E. coli BL21 (DE3).
- the mutant Ser237Ala-V8 ⁇ 48 with an N-terminal histidine tag (FIG. 1) was successfully expressed in E. coli BL21 (DE3) using isopropyl ⁇ -D-thiogalactopyranoside at 37 ° C. for 3 h. Ni-NTA agarose columns were used to purify the mutant.
- the protein fractions with Ser237Ala-V8 ⁇ 48 were desalted as soon as possible after purification by NAP5 columns, which were equilibrated with 25 mM Tris-HCl, pH 7.4, 1% Triton X-100 and 100 mM NaCI.
- the purity of the enzyme was examined by SDS-PAGE, a single band of 26 kDA with a purity of over 95% being observed (FIG. 2). Almost all of the V8 ⁇ 48 proteinase mutant was expressed as soluble protein, which could be purified in a single step using Ni-NTA agarose affinity columns.
- the expressed purified protein showed no proteolytic activity.
- the wild type of V8 proteinase was used as a positive control in the same approach (Fig. 3).
- the neurotropic factor for retinal cholinergic neurons and [Cys (Bz) 84 , Glu (OBz) 85 ] -CD 4 (81-92) were used to test the binding properties of Ser237Ala-V8 ⁇ 48 against peptides at the C-terminal end Have aspartic acid or glutamic acid.
- the mutant immobilized on Ni-NTA agarose beads was incubated with each of the peptides and after washing the beads, the bound peptides were eluted with 200 mM acetic acid. Both peptides were detected in the eluates by MALDI-TOF (Fig. 4). As a control for this experiment, calibration peptides were used used for MALDI-TOF mass spectrometry.
- spots were marked on the two-dimensional gel (FIG. 6) and cut out of the gel.
- the proteins or peptides were obtained from the gel by tryptic digestion.
- the fragments obtained were subjected to MALDI-TOF mass spectrometry (Vogt et al., 2003. Rapid Communications in mass spectrometry 17: 1273-1282).
- the masses found were compared with data from a database (FIG. 7) and the proteins from the two-dimensional gel were identified in this way.
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