DE10041238A1 - Process for the identification of specifically cleavable peptides and use of such peptide sequences - Google Patents

Abstract

The invention relates to methods for detecting and identifying peptides that can be specifically cleaved, using a defined amino acid sequence based on a library of nucleic acid peptide fusion molecules. According to said methods, the variable part of the peptides is coded by the respective nucleic acid fused thereto, using proteolytically active solutions. The invention also relates to the use of said peptides for the targeted release of chemical active ingredients and for diagnosis.

Description

The invention specifically relates to methods of finding and identifying cleavable peptides with a defined amino acid sequence using proteolytic solutions and the use of these peptides for targeted Release of chemical agents and for diagnostics.

A method for the detection of specifically cleaved peptides under Use of a specific protein construct that is used in proteolytic cleavage fluorescent of the construct is described in US 5981200. The method is based on fluorescence resonance energy technology (FRET). An advantage of this method is that they are also used for in vivo detection of protein cleavage events can be. In contrast, it is disadvantageous that only specific peptides are labeled can be marked or in the case of an examination of different marked Proteins an exact assignment of the cleavage event to the respective Sequence would involve a lot of effort. Another disadvantage of this The method is that relatively large amounts of labeled substrates are used must be in order to obtain a detectable signal.

Another method for the selective identification of enzyme substrates can be used is the phage display (e.g. laid down in WO 97/47314). A disadvantage of this method is that the phage display peptide libraries less than other surface-represented peptide libraries Show number of possible different sequences and that the subsequent Processing of the phage particles and determination of the identified substrates is relatively expensive.  

The object of the present invention is a method for finding and Identify specific proteolytically cleavable peptides to develop as well Provide substances that target chemical release Active substances allowed.

The object is achieved by a method for the identification of specifically proteolytically cleavable peptides, which comprises the following method steps:

• a) Incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with a proteolytically active sample,
• b) isolation of the proteolytically cleaved components of the fusion molecules,
• c) Determination of the sequence of the nucleic acid component of the isolated Fusion molecules.

The method according to the invention is based on the use of Fusion molecules that have a phenotypic peptide part and a genotypic Contain nucleic acid part, which has a sequence coding the peptide part. The The peptide part is linked to the nucleic acid part via a suitable linker. A protein acceptor, e.g. B. Puromycin used the is covalently bound to the nucleic acid part. The linker can include other components such as z. B. a non-coding nucleic acid sequence, preferably a poly-A sequence, include. In a preferred embodiment, the nucleic acid part contains other regions that are constant in sequence and that are located on one or both sides of the coding region. These constant nucleic acid regions can e.g. B. as Primer binding sites for carrying out a PCR or as Restriction enzyme interfaces. The coding region of the The nucleic acid part of the fusion molecules contains a variable part which is for at least two amino acids, preferably for at least four, particularly preferred encoded for seven to twelve amino acids. This can be further constant coding nucleotide sequences occur. The constant nucleic acid sequences can encode peptide segments that e.g. B. a simple fixation of the allow peptide part of the fusion molecule on a solid surface or that create interesting structural elements. Such structural elements are e.g. B. Epitopes for antibodies, tags for cleaning or for the detection of the constructs or Elements that determine certain tertiary structures.  

The preparation of such fusion molecules can, for. B. according to WO 98/31700. A system is described there in which the nucleic acid, preferably RNA, a protein acceptor, for example a puromycin, via a suitable linker, is bound. This ensures that shortly before the in vitro translation ends RNA into the corresponding protein, the protein synthesized covalently to its coding RNA can be bound and characterized in more detail. comparable Systems that can be used for the present invention are for example in DE 196 46 372 C1, WO 98/16636, US 5843701 or WO 94/13623 described.

In a preferred embodiment of the method according to the invention, the Fusion molecules fixed to a surface (support) via their peptide part. The term "sluggish" is understood in the sense of the present invention Material that is in solid or gel-like form. As carrier materials are suitable, for example, ceramics, metals, in particular semiconductors, precious metals, Glasses, plastics, crystalline materials or thin layers of the support, in particular the materials mentioned, or (bio) molecular filaments such as Cellulose, framework proteins. But not only flat materials come as carriers but also particles in question, such as B. with protein-loaded materials Column chromatography or beads made from organic polymers. The carrier is generally covalent, quasi-covalent, supramolecular or physical.

The peptide part of the fusion molecules can e.g. B. via biotin streptavidin binding the carrier can be fixed, but also specific domains of the peptide part of the Fusion molecules such as e.g. B. metal-binding domains (z. B. His-Tag), terminals Cysteine residues or domains, the epitopes recognizable by specific antibodies contain such a fixation.

Libraries whose fusion molecules contain all possible permutations with respect to the variable part of the peptide sequence are preferred in the method according to the invention. Libraries of such fusion proteins that can still be handled practically have a size of approximately 10 ¹³ different sequences and are thus 10 ³ -10 ⁴ times (e.g. phage display) or 10 ⁶ -10 ⁷ times larger than other known peptide libraries (classic, chemically synthesized libraries) larger. In order to cover all permutations, a variable peptide part of less than 11 amino acids is preferred; variable peptide parts of seven or eight amino acids are particularly preferred. Of course, libraries can also be created which contain longer variable peptide parts in their fusion molecules, but then a complete coverage of the sequence diversity is no longer possible. Since the previously known, specific peptide bond-cleaving proteases require a recognition sequence of at least four amino acids, libraries with a variable peptide part of at least four amino acids are preferred.

The library of fusion molecules is either fixed in solution or preferably exposed to a support of a proteolytically active sample. As proteolytic active sample, pre-sample or comparative sample come mainly tissue-specific Extracts of animal, human or vegetable origin, extracts of certain Cell compartments, such as B. cytosolic extracts, subcellular extracts, extracts of membrane components, extracellular extracts or mixtures of such Extracts in question. Extracts from pathological substances are also of particular interest Tissues such as B. from carcinomas. But also extracts that all or part the proteolytic activity of an organism, especially viruses, Representing microorganisms such as bacteria or protists can be used become. As a proteolytically active sample, preliminary sample or comparative sample can also solutions containing known proteases are used.

The library is preferably incubated with the proteolytically active sample under physiological conditions, preferably at temperatures between 0 ° C and 45 ° C. This can be done using the extracts to be examined for their proteolytic activity be used directly or the extracts are in a suitable solvent, such as B. a physiological saline solution and for incubation used.

During the incubation, the variable peptide components of the Fusion molecules cleaved by the proteases contained in the sample. It the result is a specific, characteristic for the sample extract used, proteolytic cleavage pattern. The split parts are the Fusion molecules encoded by the nucleic acid part.  

After incubation of the library with the proteolytically active to be examined The proteolytically cleaved fusion molecule parts are isolated and the sample Sequence of the coding nucleic acid part determined. So that’s through the Sample cleavable peptide sequences identified.

Isolation of the cut fusion proteins after incubation of library and Proteolytically active extract in solution can be carried out as follows.

To isolate the nucleic acid areas of the cut from the uncut Fusion proteins can e.g. B. a constant structural feature, e.g. B. a known one Epitope for an antibody in which the peptide part which is used due to the proteolytic cleavage no longer with the rest of the fusion molecule, including the nucleic acid part. The approach is a Affinity chromatography, taking advantage of e.g. B. said antibody. The Nucleic acid part of the cut fusion proteins cannot bind, is from the Nucleic acids of the uncut fusion proteins separated, and is therefore in the Eluate included. Other structural features such as e.g. B. His day or strep tag can be used for separation. The one on the affinity matrix remaining fusion molecules can be eluted and in solution or advantageously directly continue to be used in the fixed state.

Isolation of the cut fusion proteins after incubation of fixed Library with extract can be done with different methods.

In a preferred embodiment of the method according to the invention after incubation of the fixed library with the extract of the nucleic acid part of the cut fusion molecules obtained directly in the eluate.

The simplest case is to identify the sequences of the cleaved peptides directly by PCR amplification of the nucleic acid parts contained in the eluate cleaved fusion proteins with subsequent cloning and sequencing respectively. But it is also possible to start with another one Affinity chromatography using specific structures in the peptide or Nucleic acid part of the cleaved fusion proteins first cleaning the cleaved fusion proteins compared to other proteins present in the eluate  Components. After elution of the cleaved fusion proteins from chromatographic material, e.g. B. by incubation with a KOH solution the sequence of the cleaved peptides again by means of PCR, cloning and Sequencing done.

In addition, there are a number of other chromatographic or electrophoretic ones Methods for isolating the proteolytically cleaved fusion molecule parts possible. For this purpose, it is advantageous if the fusion molecules are lighter Identification is marked or modified. So z. B. the nucleic acid part of the Fusion molecules e.g. B. fluorescently labeled or radioactively labeled. A magnetic labeling of the nucleic acid part of the fusion molecules is special preferred, it can also be used in conjunction with a fluorescent label or radioactive labeling can be used. The magnetic insulation of the cleaved fusion molecule parts can be carried out very easily and selectively become. The proteolytic pattern is evaluated e.g. B. via a Hybridization of the isolated nucleic acid sequences on a biochip as z. B. is available from Affymetrix or Nanogen. So the isolated mixture can be made directly cleaved fusion molecule parts containing different Nucleic acid sequences are analyzed analytically.

When preparing the extracts to be examined, care must be taken that the proteases are isolated in their active form. At the same time, however DNA-degrading nucleases are inactivated as far as possible.

The proteolytically active sample can be processed mechanically as follows: If necessary, the entire tissue is cleaned by external washing in cold buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 150 mM NaCl, 0.5 mM DTT ; Dignam, JD, Preparation of Extracts from Higher Eukaryotes. From: Deutscher, MP (ed.) Guide to Protein Purification. Methods in Enzymology, Vol 182, Academic Press, (1990). Page 194-202). The tissue is crushed in the cold buffer and, if necessary, components such as. B. connective tissue, skin, blood vessels removed. The tissue pieces can be homogenized with Kobayashi et al. (Kobayashi, H., Fujishiro, S., Terao, T., Cancer Res. (1994) 54, 6539-6548): 0.01 M HEPES buffer, pH 7.2, 0.25 M sucrose, 0.5% Triton X. -100 in a volume ratio of 1: 2 to 1: 5 (w / w) and processed in a suitable tissue homogenizer (see below) with ice cooling to the homogenate. The method must be adapted to the desired tissue type. Suitable tissue homogenizers are e.g. B .:

• - Mixer: e.g. liver can be easily processed into porridge in a mixer, then continue rub through a close-meshed nylon net. It creates a fine one Tissue homogenate.
• - Homogenizer "Potter": rotating glass rod that fits tightly in a fitting Vessel is inserted so that the tissue between the vessel wall and the Glass rod is ground. Constant cooling must be ensured.
• - Dispersing machines: homogenizers with rotating knives in one Shaft.

The homogenized tissue is mixed with further homogenization buffer and shaken at 4-8 ° C for up to 45 min. Then that will Tissue homogenate centrifuged at 4 ° C with 16,000 g and the supernatant directly or, e.g. B. via column chromatography or dialysis, further purified and Analysis used according to the inventive method.

If frozen material is used instead of fresh tissue, the method should be modified. Do not simply let frozen tissue thaw, otherwise it will proteases released from lysosomes inactivate the enzymes. The tissue will preferably with constant cooling with liquid nitrogen in a mortar pulverized and the powder freeze-dried in a lyophilizer overnight. The dry powder can then continue to be used.

In the following, a suitable processing of brain tissue and its separation into core fraction, cytosolic fraction, membrane fraction and Cytoskeleton fraction given. The extracts obtained can be directly or below Addition of other auxiliary substances as a proteolytically active sample, preliminary sample or Comparative sample can be used.

Brains, e.g. B. from mice, from several preparations (about 50 pieces) are in about 25 ml of solution buffer 1 (LP1) added (20 mM Tris-HCl pH 7.5; 150 mM NaCl, 1 mM  EDTA, 1 mM EGTA) and with the Ultraturrax 1 min at maximum Homogenized speed. Alternatively, the glass potter is used with less material used.

To isolate a core fraction, the homogenate can be kept at 1000 × g for 10 min at 4 ° C be centrifuged. The supernatant is collected for further preparations. The Centrifugate (nuclear pellet) is resuspended in 12 ml LP1 and as before centrifuged. The nuclear pellet is in LP2 (20 mM Tris-HCl pH 7.5; 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40) resuspended.

The collected supernatants can be used to isolate and clean a cytosolic fraction can be used. To do this, they are at 100,000 × g, 1 h, 4 ° C centrifuged. The supernatant forms the extract containing cytosolic proteins.

To isolate the membrane fraction, the pellet is made from the cytosolic Separation washed three times in LP1 and centrifuged at 100,000 × g, 1 h, 4 ° C. After the third washing step, the pellet is resuspended in 15 ml LP2 and over Night solubilized. The mixture is then centrifuged at 100,000 × g, 1 h, 4 ° C. The The membrane fraction forms a supernatant.

To obtain a cytoskeleton fraction, the pellet is removed from the Membrane fraction separation washed three times in LP2 and at 100,000 × g, 1 h, 4 ° C centrifuged. After the third washing step, the pellet is poured into LP3 (12.5 mM Tris HCl pH 7.5; 0.4% SDS) resuspended and solubilized at 95 ° C. concluding Centrifugation at 20,800 × g, 15 min RT. The supernatant contains the Zytoskelettfraktion.

For the extraction of proteolytically active extracts from cells and tissues contain secreted proteases, z. B. human cells or cell lines in the Cell culture cultivated as a monolayer. The cells are washed three times with cell culture medium washed. The medium is then replaced by fresh medium. there the secretory proteases are secreted into the medium at 37 ° C. The A suitable secretion period is 1 to 3 hours. In the same way, with Tissue pieces or tissue sections are moved.  

To obtain tissue fluid from tissue associations, for. B. organs or tissue is carefully shredded with scissors and a scalpel. The Tissue pieces are washed several times with tissue buffer. The tissue pieces or even small organs are in physiological buffer or medium at 37 ° C. incubated for 1-3 hours. After the incubation, the tissue pieces are left in one Sediment 15 ml pointed vessel, the medium is removed and can continue to be examined.

Alternatively, the tissues can be transferred to centrifugation vessels and gently be centrifuged. The supernatant contains the proteases of the interstitial Fluid from the spaces between the tissues.

Alternatively, protein extracts can also be obtained using the following method: Comminution of the tissue in ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40, 0.25% sodium deoxycholate, 150mM NaCl, 1mM EGTA, 1mM PMSF, 1mM Na3V04; 1 mM NaF and 1 µg / ml aprotonin, leupeptin and pepstatin), freeze of the shredded tissue and then crush it. For homogenization of the tissue, RIPA buffer is added in a volume ratio of 1 to 2 and the Approach is then thawed.

After 15 min the lysate is centrifuged at 13,000 x g, 4 ° C for 10 min. The lysate can be aliquoted, snap frozen and stored in liquid nitrogen.

In a further modified method, the proteolytic activity of an unknown sample compared to a comparison sample can be determined. The method for the differential determination of proteolytic activity comprises the following method steps:

• a) Incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide, with a proteolytically active sample,
• b) Incubation of the same library of fusion molecules in at least one parallel approach, with at least one proteolytically active comparative sample,
• c) isolation of the proteolytically cleaved components of the fusion molecules,
• d) Creating a differential nucleic acid bank
• e) determination of the sequence of the nucleic acids determined.

The differential determination of the proteolytically cleaved nucleic acid Fusion molecules can e.g. B. by applying an excess  single-stranded nucleic acid parts isolated from the comparison sample a suitable carrier, saturation of the free binding sites of the carrier and subsequent hybridization with single-stranded nucleic acid parts resulting from the Sample was isolated. The detachable, not hybridized Nucleic acid parts represent specifically cut only in the sample Peptide sequences.

In a further method according to the invention, the proteolytic activity of a sample against at least one pre-sample is determined. The differential proteolytic activity can be determined by successively incubating the fusion molecule library with the preliminary sample and sample. The process comprises the following process steps:

• a) Incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with at least one proteolytic active pre-trial,
• b) removing the split-off components of the fusion molecules,
• c) incubation of the library prepared in this way with a proteolytically active sample,
• d) isolation of the proteolytically split components of the fusion molecules,
• e) determination of the sequence of the nucleic acid component of the separated Fusion molecules.

In preferred embodiments of all three according to the invention Process variants are the nucleic acid parts of the proteolytically cleaved and isolated parts of the fusion molecule reproduced by PCR. To do this Fusion molecules used, the nucleic acid part on both sides of the coding Nucleic acid sequence constant nucleic acid sequences as primer binding sites have.

To both the amount of fusion molecule that is in the investigated extract is cut, as well as the specificity of the selection step (cleavage in the proteolytic active extract) is preferred to increase greatly The inventive method the isolated nucleic acid of the cleaved Fusion proteins amplified by PCR and transcribed in vitro. Starting from the RNA thus produced is a again by methods already described  new library of fusion proteins made and with the same extract containing incubated proteolytic activities. This cycle can be repeated be repeated.

In a further variant of the method according to the invention, PCR can be carried out in vitro Transcription and / or in vitro translation in one or more of the described cycles with a higher than normal error rate be performed. This will increase the available ones to be tested Peptide sequences achieved (in vitro protein evolution).

It is also fast thanks to the variant of directed in vitro protein evolution and easily possible to change known cleavage sequences so that others result in kinetic properties with respect to the cutting proteases.

The proteolytically active sample can contain other additives, such as. B. specifically acting protease inhibitors or nuclease inhibitors or non-specific RNA and / or DNA to saturate possible nucleases.

Especially when the sample extracts used are non-specific cutting lysosomal or endosomal proteases (acidic peptidases, such as aspartate peptidases) it contains the addition of one which acts specifically against these proteases Inhibitors such as B. the addition of pepstatin A, recommended.

The specific inhibition of exoproteases can also be of benefit to the Degradation of the proteins contained in the sample, comparative sample or preliminary sample, especially those of other proteases and their peptide cofactors prevent. The fixed fusion molecules themselves do not have to be used against exoproteases be protected since there are no free N- or C-terminal peptides are. The breakdown of the peptide portion of the proteolytically cleaved fusion molecules is irrelevant for the further course of the procedure.

Removal of nuclease activities from tissue extracts to protect the Nucleic acid parts of the fusion molecules can be freshly prepared Tissue homogenate already during tissue processing and subsequent steps by treatment with RNAse or DNAse inhibitors can be achieved. In a further embodiment, the nuclease inhibitors  be covalently coupled to particles or suitable column materials. nucleases that bind to surface-coupled nuclease inhibitors are thus from the remaining tissue homogenate separated. An alternative to inhibiting the Nucleases, is the protection of the nucleic acid portion of the fusion molecules by Excipients. The auxiliary substances surround the DNA and RNA, form a complex and thus protect them from nucleases (Katayose, S. (1998) J. Pharm. Sci., 87: 160-163). These auxiliaries are in particular polyethyleneimine or PEG-PLL, but can also be proteins or chaparones. Protection against nucleases is also conceivable by using artificial nucleic acids in the fusion molecule, such as. B. PNA. The nucleic acids can also chemically increase stability be modified, e.g. B. by methylation.

The incubation times of library and extract depend on the proteolytic Activity of the sample used. In an expanded embodiment the methods according to the invention are repeated with variation of the incubation times carried out. This has the advantage of making kinetic statements regarding the sample and their peptide substrates that make up the library of fusion molecules include, can be hit. Peptidic substrates used in short Incubation times proteolytically cleaved have a high Rate constant compared to proteolytically active components of the sample on.

Another easy way to make kinetic statements about the proteolytic To be able to meet the activity of the sample is either the variation of the Concentration of the fusion molecules of the library that the peptide substrates form, or preferably in the variation of the concentration of the incubation provided sample.

A great advantage of the method according to the invention is that the proteolytic Activity of the sample examined phenomenologically, that is, without complete Knowledge of the proteolytic active components of the sample can be. When comparing different samples, it is irrelevant whether one different proteolytic activity on the presence of different proteases, or based on a direct mutation of a protease, which is rarely the case,  or for a malregulation of a protease gene or a protease inhibitor or activator goes back. Also changes in proteolytic activity that over Extracellular signals can be induced using the present method be recorded. It is known that e.g. B. by binding ligands, such as. B. Hormones on transmembrane receptors which have kinase and / or phosphatase activity in the A cell's cytosol can be altered. The degree of phosphorylation plays a role in Regulation of enzyme activities in the cells has a crucial role thus also a decisive influence on the protease activity. A deregulated one intracellular phosphatase and, coupled to it, the protease activity plays e.g. B. play a crucial role in the development of cancer.

The differential determination of specifically cleavable ones is particularly advantageous Peptides with regard to extracts from healthy and diseased tissue, as well from healthy tissue infected with pathogens. Also from Of particular interest is the finding of specifically cleavable peptides in comparison individual organisms.

It can also be done quickly and easily with the method according to the invention Sequences of peptides are determined by the sample under investigation cut, especially cut specifically. On the other hand sequences can also be found which do not result in any proteolytic cleavage by the examined sample are subject.

In addition to the identification of specific proteolytically cleavable peptide sequences, The methods described above can also be used to find specific acting inhibitors or activators are used without the to be known to inhibit proteases. This is simply the Sample solution a potential inhibitor or activator is added and the differential proteolytic activity between sample and potential inhibitors or Samples containing activators determined. The number of split is reduced Sequences when it is an inhibitor, the number of cleaved increases Sequences is an activator.  

In a preferred embodiment, a is used to identify inhibitors preselected library of fusion molecules used after several Selection cycles with the proteolytically active sample was obtained. alternative can also be identified specifically with the method according to the invention cleavable peptides directly for the screening of inhibitors, such as. B. in a FRET Assay.

This provides a quick and easy method to screen for Protease inhibitors are available.

The quick and easy screening of protease inhibitors also exists the possibility of combining many effective inhibitors in a mixture of active ingredients, to prevent the occurrence of resistance.

Another object of the present invention are proteolytically cleavable Substances that are available with one of the described methods Contain peptide sequence.

The specific proteolytically cleavable peptide sequences can be used to synthesize specifically acting inhibitors can be used. For this, z. B. chemical Modifications to the peptide sequence are made or one or several α-amino acids of the peptide against β-amino acids or L-amino acids exchanged for D-amino acids to prevent proteolysis (see e.g. Werder M. & Hauser H. (1999) Helvetica Chimica Acta, 82, 1774-1783).

The specific proteolytic identifiable with the present methods fissile peptides can be used to design drug delivery systems can be used to achieve selective drug release at the target site. It can be at the destination z. B. a specific organ, tissue, a certain cell type, a subcellular compartment or to face sick healthy tissue or around microbially infected versus non-infected tissues or act cells.

In the case of an active ingredient which can be activated by protease cleavage, the specifically proteolytically cleavable substances as covalently bound inhibitors be used. The specific proteolytically cleavable peptides can also be used as  Linkers can be used to target a drug with a ligand, such as. B. a specific antibody or receptor ligand connect. But also the construction of drug-based systems that use specifically proteolytically fissile substances can be closed (Minko T. et al., (2000) Int. J. Cancer, 86: 108-117).

The variant of directed in vitro protein evolution makes it possible to use known ones to change peptide cleavage sequences so that other cleavage kinetics result. The time profile of the active ingredient release can thus be controlled.

With the help of the specifically proteolytically cleavable peptides, consequently, via a Targeted drug release, diseases are combated more efficiently those who have changed proteolytic activities compared to the healthy state desired site of action occur or which is due to a malregulation or mutation of specific proteases or their activators or inhibitors. Examples of diseases with disturbed protease activity are e.g. B. asthma, Osteoporosis, cancers such as leukemia, breast cancer, colon cancer, Stroke, neuronal diseases such as Alzheimer's, arthritis, pancreatitis, High blood pressure, thrombosis, colds or schistosmiasis.

The targeted release of active ingredients can reduce the side effects minimized these active ingredients and the amount of active ingredient to be used can be due to the changed distribution profiles in the organism can be reduced. Are interesting especially active pharmaceutical ingredients such. B. against bacteria, fungi, viruses or against diseased tissue cells, but also herbicides, fungicides or insecticides be used.

Another application of the method according to the invention is in the use as a diagnostic assay. The assay can e.g. B. on a comparison of the profile of proteolytic activity of extracts against one in their composition defined library of fusion molecules between pathological tissue, e.g. B. cancer cells, and a sample to be examined. But are preferred Assays that are compared to a comparison or pre-test on only from pathological tissue proteolytically cleavable peptides are based. With such a selective assay can be carried out after separation of the split off parts of the  Fusion molecules provided sequencing step are omitted. The identification the presence of a specific disease-specific protease activity can be done in this case using a hybridization probe. On such procedure can be highly integrated by many as disease-specific markers identified cleavable peptides in such an assay be used.

Alternatively, those identified with the method according to the invention can also be used specifically cleavable peptides directly for the screening of inhibitors, such as. B. in a FRET assay. The specific cleavable sequences can also be used in vivo as a substrate for FRET assays.

Another use of the specific from a proteolytically active sample cleavable peptides is used to isolate the protease cleaving this peptide sequence (see, e.g., Li Y.M. (2000) Nature, 405: 689-694).

An applicable method for isolating serine proteases is e.g. B. in US 5843701. Serine proteases are protein enzymes that prevent the hydrolysis of Catalyze peptide bonds within proteins. Often the target protein, due to the specific peptide linkage within the substrate, selective cut. The serine proteases include, among others Tissue plasminogen activator, trypsin, elastase, chymotrypsin, thrombin and Plasmin. Many stages of the disease can be treated with serine protease inhibitors such as blood clotting disorders. Elastase inhibitors can reduce the clinical progression of emphysema.

The isolation of proteases can be carried out according to the methods described above determined specifically proteolytically cleavable peptides z. B. with standard methods coupled to column material and an affinity chromatography of the proteolytic active samples can be performed on it. The buffer of the during the Binding cycles used must not be denaturing for the protease is not deactivated. The proteases interacting with the peptides can Required for the fixed peptides by common crosslinking methods be bound. This can also include cleavage sequences containing β-amino acids are used, which are specifically recognized by proteases but are not cleaved can.

Claims (34)

1. A method for identifying specific proteolytically cleavable peptides comprising the method steps:

• a) incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with a proteolytically active sample,
• b) isolation of the proteolytically cleaved components of the fusion molecules,
• c) Determination of the sequence of the nucleic acid component of the separated fusion molecules.

2. Method for the identification of specifically proteolytically cleavable peptides comprising the method steps:

• a) incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with a proteolytically active sample,
• b) incubation of the same library of fusion molecules in a parallel approach, with at least one proteolytically active comparison sample,
• c) isolation of the proteolytically cleaved components of the fusion molecules,
• d) Creating a differential nucleic acid bank
• e) determination of the sequence of the nucleic acids determined.

3. A method for identifying specific proteolytically cleavable peptides comprising the method steps:

• a) incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with at least one proteolytically active preliminary sample,
• b) separation of the split-off components of the fusion molecules,
• c) incubation of the library prepared in this way with a proteolytically active sample,
• d) isolation of the proteolytically split components of the fusion molecules,
• e) determination of the sequence of the nucleic acid component of the separated fusion molecules.

4. The method according to any one of the preceding claims, characterized in that that a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide, known on one or both sides of known Flanked nucleotide sequences is used. 5. The method according to any one of the preceding claims, characterized in that a library of fusion molecules consisting of a peptide that is linked to a poly-A nucleic acid sequence via a puromycin the region encoding the peptide and one or more known ones Connect nucleic acid sequences is used. 6. The method according to any one of the preceding claims, characterized in that fusion molecules containing peptides from at least 4 variables Amino acids can be used. 7. The method according to any one of the preceding claims, characterized in that that fusion molecules containing peptides from 7 to 11 variables Amino acids can be used. 8. The method according to any one of the preceding claims, characterized in that that fusion molecules are used that contain peptides next to the variable sequence area at least one constant sequence area have. 9. The method according to any one of the preceding claims, characterized in that that fusion molecules with labeled nucleic acid component and / or Peptide ingredient can be used.   10. The method according to claim 9, characterized in that fusion molecules with a magnetic label, a radioactive label, one Fluorescent labeling of the nucleic acid component and / or Peptide component or a label with a specific known Nucleic acid sequence and / or a known specific Amino acid sequence can be used. 11. The method according to any one of the preceding claims, characterized in that that the library's fusion molecules are fixed on a solid surface become. 12. The method according to any one of the preceding claims, characterized in that the library's fusion molecules are fixed on solid particles. 13. The method according to any one of the preceding claims, characterized in that that the known nucleic acid sequences with restriction enzymes be cut and only the cut or cut nucleic acid part continues to be used. 14. The method according to any one of claims 4 to 13, characterized in that after isolation of the cut from the proteolytically active sample Fusion molecule components for the amplification of the nucleic acid components a PCR is carried out. 15. The method according to claim 14, characterized in that the amplified Nucleic acid to create a new library from fusion molecules is used and with this library again a process cycle according to of claims 1 to 3 is carried out. 16. The method according to claim 14, for in vitro protein evolution, thereby characterized that the PCR, the in vitro transcription and / or the in vitro Translation runs with an increased error rate.   17. The method according to any one of the preceding claims, characterized in that as a proteolytically active sample, preliminary sample and comparative sample Whole cell extract, a cytosolic extract, a membrane extract, a extracellular extract, a subcellular extract, a combination of these Extracts or a solution from known proteases is used. 18. The method according to any one of the preceding claims, characterized in that disease-specific as a sample, pre-sample or comparative sample and / or tissue-specific and / or oganspecific and / or organism-specific extracts can be used. 19. The method according to any one of the preceding claims, characterized in that the proteolytically active sample and / or preliminary sample under physiological Conditions used in the process. 20. The method according to any one of the preceding claims, characterized in that additional auxiliaries are added to the sample and / or pre-sample. 21. The method according to claim 20, characterized in that as auxiliary substances specifically acting protease inhibitors and / or nuclease inhibitors be added. 22. The method according to any one of the preceding claims, characterized in that that potential protease inhibitors or activators are added to the sample become. 23. The method according to any one of the preceding claims, characterized in that that the process steps are repeated or different Repeated incubation period and / or different sample concentration be performed. 24. Use of the method according to claims 1 to 23, determined nucleic acid sequences for the production of specific proteolytically fissile substances.   25. Specifically proteolytically cleavable substances containing a peptide obtainable by a process according to any one of claims 1 to 23. 26. Specifically proteolytically cleavable substance according to claim 25 containing a chemical agent. 27. Specifically proteolytically cleavable substance according to claim 26, characterized characterized that the chemical agent by proteolytic Release can be activated. 28. Specifically proteolytically cleavable substance according to one of claims 25 to 27, characterized in that the substance further auxiliary and Additives are added. 29. Use of the specifically proteolytically cleavable substances according to Claims 25 to 28 for the manufacture of medicaments for asthma, Osteoporosis, cancer, stroke, neuronal disease, Arthritis, pancreatitis, high blood pressure, thrombosis, colds or Schistosmiasis. 30. Diagnostic markers containing proteolytically cleavable peptides are available by a method according to any one of claims 1 to 23. 31. A method for the detection of specifically acting proteases according to claim 1 to 23 containing diagnostic markers according to claim 30. 32. Use of substances containing specifically proteolytically cleavable Peptides determined by a method according to claims 1 to 23 Identification of the proteases cleaving these peptides. 33. Use of specifically proteolytically cleavable peptides determined according to a method of claims 1 to 23 for the identification of Protease inhibitors.   34. Use of specific proteolytically cleavable peptide sequences determined according to a method of claims 1 to 23 for the production of Inhibitors.