CN110003344A - Amino acid optical probe and its preparation method and application - Google Patents

Abstract

The present invention relates to a kind of amino acid optical probes and its preparation method and application.On the one hand, the present invention relates to a kind of optical probes, and comprising amino acid-sensitive polypeptide or its functional variety and optical activity polypeptide or its functional variety, wherein optical activity polypeptide or its functional variety are located in the sequence of amino acid-sensitive polypeptide or its functional variety.The present invention also relates to the preparation method of above-mentioned probe and its applications in detection amino acid.Amino acid optical probe molecular weight provided by the invention is relatively small and is easy to mature, and fluorescence dynamic change is big, and specificity is good, and can express in cell difference subcellular organelle, can high-throughput, quantitative detection amino acid outside in the cell.

Description

Amino acid optical probe and preparation method and application thereof

technical field

The invention relates to the technical field of optical probes, in particular to an amino acid optical probe and a preparation method and application thereof.

Background technique

Amino acids are compounds in which the hydrogen atoms on the carbon atoms of carboxylic acids are replaced by amino groups, and are organic compounds containing basic amino groups and acidic carboxyl groups. The amino acids obtained after proteolysis are all α-amino acids with amino groups attached to the α-carbon, and there are only two dozen of them. They are the basic units of protein. Amino acids play a role in the human body through metabolism, such as synthesizing tissue proteins; synthesizing acids, hormones, antibodies, creatine and other ammonia-containing substances; converting them into carbohydrates and fats; or oxidizing them into carbon dioxide, water and urea to generate energy.

As one of the 20 natural amino acids, alanine is the basic amino acid that constitutes protein. As a non-essential amino acid, alanine in cells mainly acts as an N source donor of glutamate and reversibly reacts with pyruvate through transamination. Pyruvate and glutamate are produced between alanine and α-ketoglutarate under the action of alanine aminotransferase (ALT). The metabolism of alanine is an integral part of the pyruvate synthesis pathway, so alanine is also considered to be one of the most important amino acids in the gluconeogenesis pathway. Intracellular ALT can perform the mutual transamination between pyruvate and alanine as required (Jojima T et al., Appl Microbiol Biotechnol. 2010, 87(1): 159-165; Garcia R F et al. Amino Acids. 2007, 33(1):151-155; Pukrittayakamee S et al Trop Med IntHealth. 2002, 7(11):911-918; Brosnan J T et al J Biol Chem. 2001, 276(34):31876-31882; Arguello J M et al Arch Biochem Biophys. 1999, 367(2):341-347). The reference value of ALT enzyme activity in the serum of an adult is: 8-50U/L. When various tissues and organs in the human body become diseased, ALT will be released into the blood with the death of diseased cells, thereby increasing the content of ALT in serum. When 1/1000 liver cells are inflamed, the serum ALT level will more than double. Therefore, the serum aminotransferase level is an important indicator of the degree of liver disease (Oka R et al., J Atheroscler Thromb. 2014, 21(6) : 582-592; Liu Z et al, Int J Med Sci. 2014, 11(9): 925-935; Kunutsor S K et al, PLoS One. 2014, 9(4): e96068). In addition, L-alanine is an important raw material for the synthesis of vitamin B6, and the amino acid injection with L-alanine as the main component has a certain therapeutic effect on liver and brain diseases. At the same time, L-alanine is also a kind of Good diuretic. A new study shows that alanine and glucose can enhance the killing of multidrug-resistant bacteria E. Thereby, the absorption of kanamycin is improved, and its lethality to bacteria is increased (Peng B et al., Cell Metab. 2015, 21(2):249-261).

It is precisely because alanine has the above-mentioned important role, so the detection of alanine content is particularly important. The commonly used detection method of alanine is capillary electrophoresis (Li X-t et al., Chem Res Chin Univ 2013, 29(3): 434-438; Meng J et al., The Analyst 2010, 135(7): 1592-1599), high-efficiency Liquid chromatography (Tateda N et al, Analytical sciences: the international journal of the Japan Society for Analytical Chemistry 2001, 17(6): 775-778), UV-Vis spectrophotometry (Du J et al, Chemical communications (Cambridge, England) 2013, 49(47):5399-5401; Engeser M et al. Chemical Communications 1999,(13):1191-1192) and fluorescence spectrometry (Engeser M et al. Chemical Communications 1999,(13):1191-1192).

Proline is widely distributed in various animals and plants. In plants, proline accumulation is a very common and important phenomenon. When plants are harmed by the external environment, the content of proline in plants will increase dramatically. The mechanism of this phenomenon and its physiological role in plants have not been clearly studied. In mammalian cells, the physiological role of proline as a non-essential amino acid was not widely recognized at the beginning. maintenance plays an important role. However, in recent years, with the discovery of the regulation of proline metabolism pathway on intracellular ROS, people have gradually refocused on the study of proline and its metabolic pathway in mammalian cells.

In mammals, proline metabolism is mainly carried out in the cytoplasm and mitochondria, mainly through the transport protein on the mitochondrial proline into the mitochondria, through a proline oxidase (POX/PRODH) on the mitochondrial inner membrane. ) oxidatively dehydrogenates proline to generate Δ1-pyrroline-5-carboxylic acid (P5C), which shuttles back to the cytoplasm and is converted back to proline by PYCR (P5C reductase) in the cytoplasm. This forms a cycle of proline metabolism. Δ1-pyrroline-5-carboxylic acid (P5C) can form a tautomeric equilibrium with GSA (glutamate-γ-semialdehyde) in the cytoplasm, and finally GSA can be dehydrogenated to glutamate through P5CDH on the one hand Entering the tricarboxylic acid cycle for metabolism, on the other hand, it can generate ornithine through ornithine aminotransferase and enter the urea cycle for metabolism. In general, it is the metabolic pathway formed by the regulation of P5C and proline interconversion between cytoplasm and mitochondria by POX and PYCR (P5CDH), which we call the proline cycle (Liu W et al., Biofactors. 2012, 38(6):398-406; Liu W et al. Autophagy. 2012, 8(9):1407-1409; Liu W et al. Cancer Res. 2012, 72(14):3677-3686).

In recent years, studies have found that proline oxidase (POX) gene can negatively regulate the growth of tumor cells by promoting cell cycle arrest, inducing cell differentiation and promoting cell apoptosis, and abnormal expression of its expression has been found in various tumors. . Proline is oxidatively dehydrogenated to P5C under the action of POX, and it also consumes ADP to generate ATP and a free electron, which is accompanied by the generation of ROS (reactive oxygen species) in the process of electron respiration chain transfer in mitochondria, ROS plays a crucial role in the regulation of cell apoptosis, proliferation and cell cycle (Liu Y et al., Cancer Res. 2009, 69(16): 6414-6422; Phang J M et al., Szabados L et al., Trends Plant Sci 2010, 15(2):89-97; Pandhare J et al, J Biol Chem. 2006, 281(4):2044-2052; Liu Y et al, Oncogene. 2006, 25(41):5640-5647).

Common detection methods for proline include acid ninhydrin chromogenic method (Chen et al., Applie Environmental Microbiology 2006, 72: 4001-4006), spectrophotometer (Hortala MA et al., J Am Chem So 2003, 125(1): 20 -21; Pu F et al., Anal Chem 2010, 82(19): 8211-8216), high performance liquid chromatography (Wadud S et al., Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2002, 767(2): 369-374), etc.

Valine, leucine, and isoleucine belong to branched-chain amino acids and are all essential amino acids for the human body. The three work together to promote normal body growth, repair tissue, regulate blood sugar, and provide needed energy. During intense physical activity, valine can provide the muscles with additional energy to produce glucose to prevent muscle weakness. It also helps remove excess nitrogen from the liver and transports nitrogen the body needs to various parts. Leucine increases the production of growth hormone and helps burn visceral fat, which, due to its internal body, is difficult to effect effectively through diet and exercise alone. Injections of branched-chain amino acids such as valine are commonly used to treat liver failure and damage to these organs caused by alcohol and drug abuse. In addition, it can also be used as a therapeutic agent to speed up wound healing. The commonly used detection methods for valine, leucine and isoleucine are acid-base titration and potentiometric titration.

Serine is a non-essential amino acid that plays a role in fat and fatty acid metabolism and muscle growth. Serine It helps in immune hemoglobin and antibody production and is also required for maintaining a healthy immune system. Serine plays a role in the manufacture of cell membranes, the synthesis of muscle tissue, and the sheath that surrounds nerve cells. Commonly used detection methods for serine include spectrophotometry, fluorescence quenching method, ninhydrin chromogenic method, high performance liquid chromatography, enzyme reaction detection method and capillary electrophoresis-electrochemiluminescence (CE-ECL) method (Li Zhongcai et al., Research progress of L-serine detection technology, Industrial Microbiology, 2016, (5): 61-65).

Threonine is also an essential amino acid, mainly used in medicine, chemical reagents, food fortifiers, feed additives and so on. The metabolic pathway of threonine in the body is different from other amino acids. It is the only one that does not undergo dehydrogenase and transamination, but through threonine dehydratase (TDH) and threonine dehydration (TDG) and aldehyde condensation. Amino acids that are converted into other substances catalyzed by enzymes. There are three main pathways: metabolized to glycine and acetaldehyde by aldolase; metabolized to aminopropionic acid, glycine, and acetyl COA by TDG; metabolized to propionic acid and α-aminobutyric acid by TDH. Threonine is an important nutritional fortifier. Like tryptophan, it has the effect of relieving human fatigue and promoting growth and development. In medicine, because the structure of threonine contains hydroxyl groups, it has a water-holding effect on human skin, combined with oligosaccharide chains, plays an important role in protecting cell membranes, and can promote phospholipid synthesis and fatty acid oxidation in the body. Commonly used detection methods for threonine include amino acid analyzer method, paper chromatography method, ninhydrin method and formaldehyde method.

Cysteine is the only amino acid with a reducing group sulfhydryl (-SH) among the more than 20 amino acids that make up proteins. It participates in the reduction process of cells and the synthesis of proteins and glutathione in vivo (Yang P. et al. , Sensitivechemiluminescencemethod for the determination of glutathione,L-cysteine and 6_mercaptopurine[J].Microchim Acta,2008(163):263-269). -SH can form insoluble thiolates with metal ions such as Ag+, Hg+, Cu2+, and condense with toxic aromatic compounds to form thioetheramines to detoxify, and -SH can interact with the disulfide bonds contained in cystine. Conversion, cysteine is easily oxidized to form a disulfide bond, usually the state of two cysteines that form a disulfide bond is called the oxidized state, and the cysteine that does not form a disulfide bond is called the reduced state , so L-cysteine has a strong reducibility. Since the isoelectric point of L-cysteine is close to neutral pH, the sulfhydryl group usually exists and has high activity, and therefore has many biological functions, such as enhancing liver function, reducing phlegm, and promoting hair. It is widely used in medicine, food, cosmetics and feed industries. Therefore, the study of L-cysteine, including quantitative analysis, has aroused widespread interest and is particularly important.

The determination methods of cysteine include the classical colorimetric method, titration method, and chromatography method (translated by Ma Jiasu et al. Analysis method of additives in food [M], edited by Food Chemistry Section, Environmental Health Bureau, Ministry of Health and Welfare, Beijing: Published by China Standard Press, 1988, 436), Optical Polarimetry (Zhang Weiguo, Zhai Li. Determination of L-cysteine Content by Optical Polarimetry [J]. Journal of North China Coal Medical College, 2001, 3(4):435), Fluorescence method (Cao Qiu'e, Li Fei. Study on flow injection fluorescence determination method of cysteine in drugs [J]. Journal of Yunnan University, 2003, 25(3): 266-268), single-scan polarography (Zhao Cheng, Cui Jimao, Chen Song. Determination of cystine and cysteine by single-scan polarography[J]. Occupational Health and Diseases, 2004, 19(3):206), Electrochemical Method (HsiaoY., Su ff ., Cheng J., et al. Electrochemical determination of cysteine based on conducting polymers gold nanopar—ticles hybridnanocomposites[J]. Electrochimica Acta, 2011, 56(20): 6887-6895), high performance liquid chromatography (Zheng Y., Liu H.,MaG.,et al.Determination of S-propar-gyl-cysteinein rat plasma by mixed-modereversed-phase and cation-exchange HPLC-MS/MS method and its application to pharmacokinetic studies[J].J Pharma BiomedAnal, 2011 , 54(5):1187-1191). The above methods either require complex sample extraction and refinement, or require high equipment and are expensive.

To sum up, these amino acid detection methods in the prior art are not suitable for living cell research, and have many defects: they need to go through time-consuming sample processing processes, such as cell disruption, separation, extraction and purification, etc.; they cannot be used in living cells and subcellular organelles. Perform in situ, real-time, dynamic, high-throughput, and high-temporal-resolution detection. There is still a need in the art for a method for real-time localization, quantification, and high-throughput detection of alanine in and out of cells.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide probes and methods for real-time localization, high-throughput, and quantitative detection of amino acids in and out of cells.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

In a first aspect, the present invention provides an amino acid optical probe, comprising an amino acid-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or a functional variant thereof is located in the amino acid-sensitive polypeptide or its function within the sequence of the variant. The amino acid-sensitive polypeptide or its functional variant is divided into a first part and a second part by the optically active polypeptide or its functional variant.

The present invention provides an amino acid optical probe, comprising an amino acid-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located in the sequence of the amino acid-sensitive polypeptide B, and the amino acid-sensitive polypeptide B is divided into a first part B1 and a second part B2 , forming a probe structure of the formula B1-A-B2.

In one embodiment, amino acid-sensitive polypeptides include amino acid binding proteins and functional variants thereof. In one embodiment, the amino acid binding protein is derived from Agrobacterium, eg, Agrobacterium fabrum. In one embodiment, the amino acid binding protein is derived from an Atu2422-GABA receptor protein or an analog thereof. In one embodiment, the amino acid binding protein comprises an Atu2422-GABA receptor protein or a functional variant thereof. In one embodiment, the amino acid binding protein comprises an alanine binding protein. In a specific embodiment, the amino acid binding protein has the sequence shown in SEQ ID NO: 1 or a functional variant thereof, or 35%, 40%, 50%, 60%, 70%, 80%, 85%, Sequences with 90%, 95%, 99% sequence identity.

In one embodiment, the optically active polypeptide is a fluorescent protein or a functional variant thereof. In one embodiment, the fluorescent protein is selected from the group consisting of yellow fluorescent protein (cpYFP as shown in SEQ ID NO: 2), orange fluorescent protein (cpmOrange as shown in SEQ ID NO: 3), red fluorescent protein (as shown in SEQ ID NO: 3) : mKate shown in 4 or 8, mcherry shown in SEQ ID NO:5), green fluorescent protein (cpGFP shown in SEQ ID NO:6), blue fluorescent protein (shown in SEQ ID NO:7) cpBFP), apple red fluorescent protein (cpmApple as shown in SEQ ID NO: 9). Preferably, the optically active polypeptide is cpYFP. In one embodiment, the fluorescent protein has the sequence set forth in any of SEQ ID NOs: 2-9.

In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the present invention can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide flanks comprise linkers of no more than 5 amino acids, eg, linkers of 0, 1, 2, 3, 4 amino acids. In one embodiment, the linker is located at the N-terminus and/or the C-terminus of the optically active polypeptide. In one embodiment, the optical probe of the present invention does not contain a linker. In one embodiment, the optical probe is as follows: the first part B1 of the amino acid sensitive polypeptide - the optically active polypeptide A - the second part B2 of the amino acid sensitive polypeptide.

In one embodiment, the optical probes of the present invention further comprise targeting sequences for targeting the probes to specific organelles such as cells.

The optically active polypeptides of the present invention may be located or fused to any position of the amino acid-sensitive polypeptides described herein. In one embodiment, the optically active polypeptide is located within a segment of an amino acid-sensitive polypeptide described herein selected from the group consisting of residues 117-123, residues 249-259, and residues 323-330, numbered corresponding to the amino acid-sensitive polypeptide's full length. In one embodiment, the optically active polypeptide replaces one or more amino acids within the segment selected from the group consisting of residues 117-123, residues 249-259, and residues 323-330 of the amino acid-sensitive polypeptides described herein, by numbering Corresponds to the full length of the amino acid-sensitive polypeptide.

In one embodiment, the optically active polypeptide is located in an amino acid-sensitive polypeptide described herein at a position selected from the group consisting of: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/ 121, 119/120, 119/121, 120/121, 120/122, 120/123, 121/122, 121/123, 122/123, 249/250, 249/251, 249/252, 249/253, 249/254, 249/255, 249/256, 249/257, 249/258, 249/259, 250/251, 250/252, 250/253, 250/254, 250/255, 250/256, 250/ 257, 250/258, 250/259, 251/252, 251/253, 251/254, 251/255, 251/256, 251/257, 251/258, 251/259, 252/253, 252/254, 252/255, 252/256, 252/257, 252/258, 252/259, 253/254, 253/255, 253/256, 253/257, 253/258, 253/259, 254/255, 254/ 256, 254/257, 254/258, 254/259, 255/256, 255/257, 255/258, 255/259, 256/257, 256/258, 256/259, 257/258, 257/259, 258/259, 323/330, 324/330, 325/330, 326/327, 326/328, 326/329, 326/330, 327/328, 327/329, 327/330, 328/329, 328/ 330 and 329/330. Herein, if two numbers in a position represented by "X/Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids indicated by the number. For example, at positions 117/118 means that the optically active polypeptide is located between amino acids 117 and 118 of the amino acid-sensitive polypeptides described herein. If the two numbers in a position represented by the form "X/Y" are not consecutive integers, it means that the optically active polypeptide replaces the amino acid between the amino acids indicated by the number. For example, positions at positions 249/259 indicate that the optically active polypeptide replaces amino acids 250-258 of the amino acid-sensitive polypeptides described herein. Preferably, the optically active polypeptide is located at the following positions of the amino acid-sensitive polypeptides described herein: 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330.

In an exemplary embodiment, the B1-A-B2 style optical probe of the present invention may be formed when cpYFP is located at 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 of Atu2422 The probes are shown in SEQ ID NOs: 10-15. In one embodiment, the optical probes of the present invention have or consist of the sequences shown in SEQ ID NOs: 10-15. In one embodiment, the optical probes described above are sensitive to amino acids selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine.

The invention also provides amino acid-sensitive polypeptides described herein with one or more mutations, including amino acid binding proteins with one or more mutations. The mutations include amino acid modifications, substitutions, deletions or truncations of sequences. Said mutation may be selected from mutations at F77, A100, T102, D121, Y150, D226, G227 and Y275 alleles of the amino acid-sensitive polypeptide. Exemplarily, the mutation is selected from F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A of the amino acid-sensitive polypeptide , A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100T, A100N, A100K, A100M, A100D, A100D, A100G, T102Y, T102C, T102L, T102H, T102H ，T102Q，T102W，T102R，T102I，T102A，T102N，T102K，T102M， T102V，T102F，T102D，T102E，T102G，D121S，D121Y，D121C，D121L，D121P， D121H，D121Q，D121W，D121I，D121T，D121N，D121K ，D121R，D121V， D121A，D121F，D121E，D121M，D121A，Y150S，Y150T，Y150C，Y150L， Y150P，Y150H，Y150Q，Y150W，Y150R，Y150I，Y150A，Y150N，Y150K， Y150M，Y150V，Y150F，Y150D，Y150E ，Y150G，D226S，D226T，D226C， D226L，D226P，D226H，D226Q，D226W，D226R，D226I，D226A，D226N， D226K，D226M，D226V，D226F，D226Y，D226E，D226G，G227S，G227T， G227C，G227L，G227P ，G227H，G227Q，G227W，G227R，G227I，G227A， G227N，G227K，G227M，G227V，G227F，G227Y，G227E，G227D，Y275S，Y275T，Y275C，Y275L，Y275P，Y275H，Y275Q，Y275W，Y275R，Y275I， Y275A , Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the aforementioned mutations are selected from the group consisting of F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F.

Amino acid-sensitive polypeptides (eg, amino acid binding proteins) in the optical probes of the present invention may contain one or more amino acid mutations. In some embodiments, the amino acid-sensitive polypeptide in the optical probe comprises an amino acid binding protein described herein with one or more mutations. In one embodiment, the mutation is, for example, selected from the mutations at F77, A100, T102, D121, Y150, D226, G227 and Y275 of the amino acid-sensitive polypeptide. In one embodiment, the mutation is selected from F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M of the amino acid-sensitive polypeptide , F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100T, A100N, A100M, A100V, A100D, A100E, A100G, T102Y, T102C, T102P, T102P, T102P, T102P, T102P, T102P, T102P ， T102H，T102Q，T102W，T102R，T102I，T102A，T102N，T102K，T102M，T102V， T102F，T102D，T102E，T102G，D121S，D121Y，D121C，D121L，D121P，D121H， D121Q，D121W，D121I，D121T，D121N ，D121K，D121R，D121V，D121A，D121F，D121E，D121M，D121A，Y150S，Y150T，Y150C，Y150L，Y150P，Y150H， Y150Q，Y150W，Y150R，Y150I，Y150A，Y150N，Y150K，Y150M，Y150V， Y150F，Y150D ，Y150E，Y150G，D226S，D226T，D226C，D226L，D226P，D226H， D226Q，D226W，D226R，D226I，D226A，D226N，D226K，D226M，D226V， D226F，D226Y，D226E，D226G，G227S，G227T，G227C，G227L ，G227P，G227H，G227Q，G227W，G227R，G227I，G227A，G227N，G227K，G227M，G227V， G227F，G227Y，G227E，G227D，Y275S，Y275T，Y275C，Y275L，Y275P，Y275H， Y275Q，Y275W，Y275R，Y275I , Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the amino acid-sensitive polypeptide in the optical probe of the present invention may comprise a mutation selected from the group consisting of F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F.

In an exemplary embodiment, the B1-A-B2 type optical probe of the present invention may be Atu2422 fused with cpYFP at site 121/122 and having F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S, or the probe formed when Y275F is mutated, as shown in SEQ ID NOs: 16-25. In an exemplary embodiment, the optical probe of the present invention has or consists of the sequences shown in SEQ ID NOs: 16-25. In one embodiment, the optical probe comprising one or more amino acid mutations is sensitive to alanine.

The optical probe provided by the present invention comprises any one of the amino acid sequences SEQ ID NO: 10-25 or a variant thereof. In one embodiment, the optical probe provided by the present invention comprises 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90% of the amino acid sequence SEQ ID NO: 10-25. Sequences with %, 95%, 99% sequence identity. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence substantially similar or identical to any of the amino acid sequences SEQ ID NOs: 10-25.

The present invention also provides detection kits comprising the amino acid optical probes or fusion polypeptides described herein, or amino acid optical probes or fusion polypeptides prepared as described herein. In one embodiment, the kit detects amino acids selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine.

The present invention provides methods of making the optical probes described herein, comprising: providing cells comprising a vector expressing the optical probes or fusion polypeptides described herein, culturing the cells under conditions for expression by the cells, and isolating the optical probes or fusion polypeptides. In one embodiment, a method of preparing an amino acid optical probe or fusion polypeptide described herein comprises: 1) transferring an expression vector encoding an amino acid optical probe described herein into a host cell; 2) expressing an expression vector suitable for the expression vector Cultivate the host cell under the condition of 3) isolate the amino acid optical probe.

The present invention also provides methods of detecting amino acids in a sample comprising: contacting an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared as described herein, with the sample, and detecting a change in the optically active polypeptide. The detection can be performed in vivo, in vitro, subcellular or in situ. The sample is eg blood. In one embodiment, the amino acids in the sample are selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine.

Also provided herein are methods of quantifying amino acids in a sample comprising: contacting an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared as described herein, with a sample, detecting a change in the optically active polypeptide, and detecting a change in the optically active polypeptide. Changes in active polypeptides quantify amino acids in a sample. In one embodiment, the amino acids in the sample are selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine.

The present invention also provides methods of screening compounds (eg, drugs) comprising: contacting an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared as described herein, with a candidate compound, and detecting changes in the optically active polypeptide , and screen compounds based on changes in optically active polypeptides. The method allows for high-throughput screening of compounds.

The present invention also provides the use of the amino acid optical probes or fusion polypeptides described herein, or the amino acid optical probes or fusion polypeptides prepared by the methods described herein, in real-time amino acid localization. In one embodiment, the real-time localization of amino acids includes real-time localization of amino acids selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine.

In a second aspect, the present invention provides an alanine optical probe, comprising an alanine-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or its functional variant is located in alanine within the sequence of an acid-sensitive polypeptide or functional variant thereof. The alanine-sensitive polypeptide or its functional variant is divided into a first part and a second part by the optically active polypeptide or its functional variant.

The present invention provides an alanine optical probe, comprising an alanine-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located in the sequence of the alanine-sensitive polypeptide B, and the alanine-sensitive polypeptide B is divided into For the first part B1 and the second part B2, a probe structure of the formula B1-A-B2 is formed.

In one embodiment, the alanine-sensitive polypeptide comprises an amino acid binding protein or a functional variant thereof. In one embodiment, the amino acid binding protein is derived from Agrobacterium, eg, Agrobacterium fabrum. In one embodiment, the amino acid binding protein is derived from an Atu2422-GABA receptor protein or an analog thereof. In one embodiment, the amino acid binding protein comprises an Atu2422-GABA receptor protein or a functional variant thereof. In one embodiment, the amino acid binding protein comprises an alanine binding protein. In a specific embodiment, the amino acid binding protein has the sequence shown in SEQ ID NO: 1 or a functional variant thereof, or 35%, 40%, 50%, 60%, 70%, 80%, 85%, Sequences with 90%, 95%, 99% sequence identity.

In one embodiment, the optically active polypeptide is a fluorescent protein or a functional variant thereof. In one embodiment, the fluorescent protein is selected from the group consisting of yellow fluorescent protein (cpYFP as shown in SEQ ID NO: 2), orange fluorescent protein (cpmOrange as shown in SEQ ID NO: 3), red fluorescent protein (as shown in SEQ ID NO: 3) : mKate shown in 4 or 8, mcherry shown in SEQ ID NO:5), green fluorescent protein (cpGFP shown in SEQ ID NO:6), blue fluorescent protein (shown in SEQ ID NO:7) cpBFP), apple red fluorescent protein (cpmApple as shown in SEQ ID NO: 9). Preferably, the optically active polypeptide is cpYFP. In one embodiment, the fluorescent protein has the sequence set forth in any of SEQ ID NOs: 2-9.

In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the present invention can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide flanks comprise linkers of no more than 5 amino acids, eg, linkers of 0, 1, 2, 3, 4 amino acids. In one embodiment, the linker is located at the N-terminus and/or the C-terminus of the optically active polypeptide. In one embodiment, the optical probe of the present invention does not contain a linker. In one embodiment, the optical probe is as follows: the first part B1 of the alanine-sensitive polypeptide - the optically active polypeptide A - the second part B2 of the alanine-sensitive polypeptide.

In one embodiment, the optical probes of the present invention further comprise targeting sequences for targeting the probes to specific organelles such as cells.

The optically active polypeptides of the present invention may be located or fused to any position of the alanine-sensitive polypeptide. In one embodiment, the optically active polypeptide is located within a segment of the alanine-sensitive polypeptide selected from the group consisting of residues 117-123, residues 249-259, and residues 323-330, numbered corresponding to the alanine-sensitive polypeptide the full length. In one embodiment, the optically active polypeptide replaces one or more amino acids within a segment of the alanine-sensitive polypeptide selected from the group consisting of residues 117-123, residues 249-259, and residues 323-330, numbered corresponding to for the full length of alanine-sensitive polypeptides.

In one embodiment, the optically active polypeptide is located on an alanine-sensitive polypeptide at a site selected from the group consisting of: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121 , 119/120, 119/121, 120/121, 120/122, 120/123, 121/122, 121/123, 122/123, 249/250, 249/251, 249/252, 249/253, 249 /254, 249/255, 249/256, 249/257, 249/258, 249/259, 250/251, 250/252, 250/253, 250/254, 250/255, 250/256, 250/257 , 250/258, 250/259, 251/252, 251/253, 251/254, 251/255, 251/256, 251/257, 251/258, 251/259, 252/253, 252/254, 252 /255, 252/256, 252/257, 252/258, 252/259, 253/254, 253/255, 253/256, 253/257, 253/258, 253/259, 254/255, 254/256 , 254/257, 254/258, 254/259, 255/256, 255/257, 255/258, 255/259, 256/257, 256/258, 256/259, 257/258, 257/259, 258 /259, 323/330, 324/330, 325/330, 326/327, 326/328, 326/329, 326/330, 327/328, 327/329, 327/330, 328/329, 328/330 and 329/330. Herein, if two numbers in a position represented by the form "X/Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids described by that number. For example, being located at positions 117/118 means that the optically active polypeptide is located between amino acids 117 and 118 of the alanine-sensitive polypeptide. If the two numbers in a position represented by the form "X/Y" are not consecutive integers, it means that the optically active polypeptide replaces the amino acid between the amino acids indicated by the number. For example, positions at positions 249/259 indicate that the optically active polypeptide replaces amino acids 250-258 of the alanine-sensitive polypeptide. Preferably, the optically active polypeptide is located at the following positions of the alanine-sensitive polypeptide: 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330.

In an exemplary embodiment, the B1-A-B2 style optical probe of the present invention may be formed when cpYFP is located at 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 of Atu2422 The probes are shown in SEQ ID NOs: 10-15. In one embodiment, the optical probes of the present invention have or consist of the sequences shown in SEQ ID NOs: 10-15.

The present invention also provides alanine-sensitive polypeptides with one or more mutations, including amino acid binding proteins with one or more mutations. The mutations include amino acid modifications, substitutions, deletions or truncations of sequences. Said mutation may be selected from mutations at F77, A100, T102, D121, Y150, D226, G227 and Y275 of the alanine-sensitive polypeptide. Exemplarily, the mutation is selected from F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M of the alanine-sensitive polypeptide , F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100T, A100N, A100M, A100V, A100D, A100E, A100G, T102Y, T102C, T102P, T102P, T102P, T102P, T102P, T102P, T102P ，T102H，T102Q，T102W，T102R，T102I，T102A，T102N，T102K，T102M， T102V，T102F，T102D，T102E，T102G，D121S，D121Y，D121C，D121L，D121P， D121H，D121Q，D121W，D121I，D121T，D121N ，D121K，D121R，D121V，D121A，D121F，D121E，D121M，D121A，Y150S，Y150T，Y150C，Y150L， Y150P，Y150H，Y150Q，Y150W，Y150R，Y150I，Y150A，Y150N，Y150K， Y150M，Y150V，Y150F，Y150D ，Y150E，Y150G，D226S，D226T，D226C， D226L，D226P，D226H，D226Q，D226W，D226R，D226I，D226A，D226N，D226K，D226M，D226V，D226F，D226Y，D226E，D226G，G227S，G227T， G227C，G227L ，G227P，G227H，G227Q，G227W，G227R，G227I，G227A， G227N，G227K，G227M，G227V，G227F，G227Y，G227E，G227D，Y275S， Y275T，Y275C，Y275L，Y275P，Y275H，Y275Q，Y275W，Y275R，Y275I , Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the mutation is selected from the group consisting of F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F.

Alanine-sensitive polypeptides (eg, amino acid binding proteins) in the optical probes of the present invention may contain one or more mutations. In some embodiments, the alanine-sensitive polypeptide in the optical probe comprises an amino acid binding protein described herein with one or more mutations. In one embodiment, the mutation is, for example, selected from the mutation of the F77, A100, T102, D121, Y150, D226, G227 and Y275 alleles of the alanine-sensitive polypeptide. In one embodiment, the mutation is selected from the group consisting of F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E of an alanine-sensitive polypeptide , F77M, F77A, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100I, A100T, A100N, A100M, A100V, A100D, A100G, T102Y, T102Y, T102L, T102L ，T102P，T102H， T102Q，T102W，T102R，T102I，T102A，T102N，T102K，T102M，T102V，T102F， T102D，T102E，T102G，D121S，D121Y，D121C，D121L，D121P，D121H，D121Q， D121W，D121I，D121T ，D121N，D121K，D121R，D121V，D121A，D121F，D121E，D121M，D121A，Y150S，Y150T，Y150C，Y150L，Y150P，Y150H， Y150Q，Y150W，Y150R，Y150I，Y150A，Y150N，Y150K，Y150M，Y150V， Y150F ，Y150D，Y150E，Y150G，D226S，D226T，D226C，D226L，D226P，D226H， D226Q，D226W，D226R，D226I，D226A，D226N，D226K，D226M，D226V， D226F，D226Y，D226E，D226G，G227S，G227T，G227C ，G227L，G227P，G227H， G227Q，G227W，G227R，G227I，G227A，G227N，G227K，G227M，G227V， G227F，G227Y，G227E，G227D，Y275S，Y275T，Y275C，Y275L，Y275P，Y275H， Y275Q，Y275W，Y275R , Y275I, Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the alanine-sensitive polypeptide in the optical probe of the present invention may comprise a mutation selected from the group consisting of F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F.

In an exemplary embodiment, the B1-A-B2 type optical probe of the present invention may be Atu2422 fused with cpYFP at site 121/122 and having F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S, or the probe formed when Y275F is mutated, as shown in SEQ ID NOs: 16-25. In one embodiment, the optical probe of the present invention has or consists of the sequences shown in SEQ ID NOs: 16-25.

The optical probe provided by the present invention comprises any one of the amino acid sequences SEQ ID NO: 10-25 or a variant thereof. In one embodiment, the optical probe provided by the present invention comprises 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90% of the amino acid sequence SEQ ID NO: 10-25. Sequences with %, 95%, 99% sequence identity. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence substantially similar or identical to any of the amino acid sequences SEQ ID NOs: 10-25. In a more preferred embodiment, the optical probe provided by the present invention comprises or consists of SEQ ID NO: 24.

The invention also provides fusion polypeptides comprising the optical probes described herein and other polypeptides. In some embodiments, the optical probes described herein further comprise other polypeptides fused thereto. The other polypeptides described herein do not affect the properties of the optical probe. In some embodiments, other polypeptides are located at the N-terminus and/or C-terminus of the optical probe. In some embodiments, other polypeptides include polypeptides that localize optical probes to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting. Linkers may be present between the optical probes and other polypeptides in the fusion polypeptides described herein.

The subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosome, and peroxisome, among others. In some embodiments, tags for purification or tags for immunoblotting include 6-Histidine (6*His), Glutathione S-transferase (GST), Flag.

The invention also provides nucleic acid sequences encoding the optical probes or fusion polypeptides described herein, or their complements. In one embodiment, the present invention provides a nucleic acid sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 10-25. In one embodiment, the nucleic acid sequence of the present invention comprises any one of the nucleotide sequences SEQ ID NOs: 26-27 or variants thereof. In a preferred embodiment, the present invention provides a nucleic acid sequence comprising 99%, 95%, 90%, 80%, 70% or 50% identity to any of the nucleotide sequences SEQ ID NOs: 26-27 the sequence of. In another preferred embodiment, the present invention provides a nucleic acid sequence comprising a nucleotide sequence substantially similar or identical to any of the nucleotide sequences SEQ ID NOs: 26-27.

The present invention also relates to complementary sequences of the above nucleic acid sequences or variants thereof, which may comprise nucleic acid sequences or complementary sequences thereof encoding fragments, analogs, derivatives, soluble fragments and variants of the optical probes or fusion proteins of the present invention.

The amino acid sequences and nucleic acid sequences of the present invention are preferably provided in isolated form, more preferably purified to homogeneity.

The present invention also provides an expression vector comprising the nucleic acid sequence of the present invention or a complementary sequence thereof operably linked to an expression control sequence, the nucleic acid sequence encoding the optical probe or fusion polypeptide of the present invention. In some embodiments, the expression vector is selected from the group consisting of prokaryotic expression vectors, eukaryotic expression vectors and viral vectors. In some embodiments, prokaryotic expression vectors are preferably derived from plasmid pRSETb operably linked to the nucleic acid sequences described herein. In some embodiments, expression control sequences include origins of replication, promoters, enhancers, operators, terminators, ribosome binding sites.

The present invention also provides cells comprising the expression vector of the present invention, the expression vector comprising the nucleic acid sequence of the present invention or its complement operably linked to an expression control sequence. The cells express the optical probes or fusion polypeptides described herein.

The present invention also provides detection kits comprising the alanine optical probes or fusion polypeptides described herein or the alanine optical probes or fusion polypeptides prepared as described herein.

The present invention provides methods of making the optical probes described herein, comprising: providing cells comprising a vector expressing the optical probes or fusion polypeptides described herein, culturing the cells under conditions for expression by the cells, and isolating the optical probes or fusion polypeptides. In one embodiment, the method of preparing the alanine optical probe or fusion polypeptide described herein comprises: 1) transferring an expression vector encoding the alanine optical probe described herein into a host cell; 2) in a suitable place for the The host cell is cultured under the condition that the expression vector is expressed, and 3) the alanine optical probe is isolated.

The present invention also provides a method of detecting alanine in a sample, comprising: contacting an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared as described herein, with the sample, and detecting a change in the optically active polypeptide . The detection can be performed in vivo, in vitro, subcellular or in situ. The sample is eg blood.

Also provided herein are methods of quantifying alanine in a sample comprising: contacting an optical probe or fusion polypeptide described herein or an optical probe or fusion polypeptide prepared as described herein with a sample, detecting a change in the optically active polypeptide, and Alanine in a sample is quantified based on changes in optically active polypeptides.

The present invention also provides methods of screening compounds (eg, drugs) comprising: contacting an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared as described herein, with a candidate compound, and detecting changes in the optically active polypeptide , and screen compounds based on changes in optically active polypeptides. The method allows for high-throughput screening of compounds.

The present invention also provides the use of the alanine optical probes or fusion polypeptides described herein, or the alanine optical probes or fusion polypeptides prepared by the methods described herein, in real-time alanine localization.

Beneficial effects of the present invention: the amino acid optical probe provided by the present invention is easy to mature, has large dynamic changes in fluorescence, good specificity, can be expressed in cells by genetic manipulation, and can be located in and out of cells in real-time, high-throughput, and quantitatively. Detects amino acids, eliminating time-consuming sample processing steps. The experimental results show that the highest response of the amino acid optical probe provided in this application to amino acids is more than 4 times that of the control, and it can be used in subcellular structures such as cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosome and Golgi apparatus. Cells can perform localization, qualitative and quantitative detection, and can perform high-throughput compound screening and quantitative detection of amino acids in blood.

Description of drawings

The present invention will be further described below with reference to the accompanying drawings and embodiments.

Fig. 1 is the SDS-PAGE graph of the exemplary amino acid optical probe described in Example 1;

Figure 2 is a graph of the response change of the exemplary amino acid optical probe comprising cpYFP and amino acid binding protein to alanine according to Example 2;

Figure 3 is a graph of the response change of the exemplary amino acid optical probe comprising cpGFP and amino acid binding protein to alanine according to Example 3;

Figure 4 is a graph of the response change of the exemplary amino acid optical probe comprising cpBFP and amino acid binding protein to alanine according to Example 4;

Figure 5 is a graph of the response change of the exemplary amino acid optical probe comprising cpmApple and amino acid binding protein to alanine according to Example 5;

6 is an exemplary amino acid optical probe fused with cpYFP at positions 120/121, 121/122, 121/123, 324/330, 325/330 or 326/330 of the amino acid binding protein described in Example 6 Titration curves for different concentrations of alanine;

7A is a bar graph of the specific detection of 20 amino acids by an exemplary amino acid optical probe fused to cpYFP at site 121/122 of the amino acid binding protein described in Example 6;

Figure 7B shows the fusion protein obtained by fusing the fluorescent protein to the N-terminus or C-terminus of the amino acid-binding protein as described in Example 6, the fluorescent protein is located at the amino acid-binding protein site 121/122 The probe for alanine histogram of acid specificity;

Figure 8 is an exemplary amino acid optical probe described in Example 7 fused to cpYFP at positions 121/122 of the amino acid binding protein and having mutations at F77, A100, T102, D121, Y150, D226, G227 or Y275. Histogram of response to alanine;

Figure 9 is a graph of the fluorescence spectrum properties of the exemplary amino acid optical probe described in Example 8;

Figure 10 is the titration curve of the exemplary amino acid optical probe described in Example 8 to different concentrations of alanine;

Figure 11 is a bar graph of the specific detection of 20 amino acids by the exemplary amino acid optical probe described in Example 8;

Figure 12 is a photograph of the subcellular organelle localization of the exemplary amino acid optical probe described in Example 9 in mammalian cells;

13 is a schematic diagram of the dynamic monitoring of alanine transmembrane transport of exemplary amino acid optical probes in different subcellular organelles in mammalian cells as described in Example 9;

Figure 14 is a dot plot of the exemplary amino acid optical probes described in Example 10 for high-throughput compound screening at the live cell level;

15 is a bar graph of the quantification of alanine in mouse and human blood by the exemplary amino acid optical probe described in Example 11. FIG.

Detailed ways

When a value or range is given, the term "about" as used herein means that the value or range is within 20%, within 10% and within 5% of the given value or range.

The terms "comprising", "including" and their equivalents as used herein include the meanings of "comprising" and "consisting of," eg, a composition "comprising" X may consist of X only or may contain other substances, eg, X+ Y.

As used herein, the term "amino acid-sensitive polypeptide" or "amino acid-responsive polypeptide" refers to a polypeptide that responds to an amino acid, including any response to a chemical, biological, electrical or physiological parameter of the polypeptide associated with the interaction of the sensitive polypeptide. The amino acid-sensitive polypeptide is sensitive to any amino acid that forms a protein. In one embodiment, the amino acid-sensitive polypeptide is sensitive to an amino acid selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine. Responses include small changes, eg, changes in the orientation of amino acids or peptide fragments of the polypeptide, as well as changes in, eg, primary, secondary, or tertiary structure of the polypeptide, including, eg, changes in protonation, electrochemical potential, and/or conformation. "Conformation" is the three-dimensional arrangement of the primary, secondary and tertiary structure of a molecule that contains pendant groups; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include switching from α-helix to β-sheet or from β-sheet to α-helix. It will be appreciated that as long as the fluorescence of the fluorescent protein moiety is altered, the detectable change need not be a conformational change. The amino acid-sensitive polypeptides described herein may also include functional variants thereof. Functional variants of amino acid-sensitive polypeptides include, but are not limited to, variants that can interact with amino acids to produce the same or similar changes as the parent amino acid-sensitive polypeptide. An "amino acid-sensitive polypeptide" can be any amino acid-sensitive polypeptide, such as an alanine-sensitive polypeptide, including amino acid binding proteins. Illustratively, the term "alanine-sensitive polypeptide" or "alanine-responsive polypeptide" as used herein refers to a polypeptide that responds to amino acids including alanine, "proline-sensitive polypeptide" or "proline-responsive polypeptide" "Polypeptide" refers to a polypeptide that responds to amino acids including proline, and so on.

The amino acid-sensitive polypeptides of the present invention include, but are not limited to, amino acid binding proteins (AABPs) or variants with more than 90% homology thereto. The amino acid binding protein of the present invention can be derived from Agrobacterium, such as Agrobacterium fabrum. The exemplary amino acid binding protein of the present invention, Atu2422, is a family of ABC transporters, consisting of two domains connected by three flexible amino acid peptide chains. Amino acid-binding proteins can sense changes in amino acid (eg, alanine) concentrations, and the spatial conformation of amino acid-binding proteins will also change during dynamic changes in amino acid (eg, alanine) concentrations.

The term "optical probe" as used herein refers to an amino acid-sensitive polypeptide fused to an optically active polypeptide. The inventors found that the conformational change produced by the binding of amino acid-sensitive polypeptides such as amino acid binding proteins to physiological concentrations of amino acids (such as alanine) can cause conformational changes of optically active polypeptides (such as fluorescent proteins), which in turn lead to optical activity of optically active polypeptides. change in nature. The presence and/or level of amino acids (eg, alanine) can be detected and analyzed by plotting a standard curve of the fluorescence of fluorescent proteins measured at different amino acid (eg, alanine) concentrations. An exemplary Atu2422 protein is shown in SEQ ID NO:1. When describing an optical probe of the invention (eg, when describing a site at which an optically active polypeptide is located or a site of mutation), reference to amino acid residue numbers is referenced to SEQ ID NO: 1. However, those skilled in the art are aware of corresponding residue numbers for other similar amino acid binding proteins. The amino acid optical probes described herein may be alanine optical probes.

In the optical probes of the present invention, an optically active polypeptide (eg, a fluorescent protein) is operably fused to an amino acid-sensitive polypeptide. Protein-based "optically active polypeptides" are polypeptides that have the ability to emit fluorescence. Fluorescence is an optical property of optically active polypeptides that can be used as a means of detecting the responsiveness of the optical probes of the present invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient at the appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation or emission spectrum, the excitation wavelength maximum and the emission wavelength maximum, the amplitude of excitation at two different wavelengths, The ratio of emission amplitudes, excited state lifetimes or fluorescence anisotropy for two different wavelengths. A measurable difference in any of these properties between the active and inactive states is sufficient for the utility of the fluorescent protein substrates of the invention in activity assays. A measurable difference can be determined by determining the amount of any quantitative fluorescent property, eg, the amount of fluorescence at a particular wavelength or the integration of the fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescent properties that are readily distinguishable in the unactivated and activated conformational states. The optically active polypeptides described herein may also include functional variants thereof. Functional variants of an optically active polypeptide include, but are not limited to, variants that undergo changes in the same or similar fluorescence properties as the parent optically active polypeptide.

A "linker" or "linking region" refers to an amino acid or nucleotide sequence that joins two moieties in a polypeptide, protein or nucleic acid of the invention. Exemplarily, in the present invention, the number of amino acids at the amino terminus of the linking region between the amino acid-sensitive polypeptide and the optically active polypeptide is 0-3, and the number of amino acids at the carboxyl terminus is 0-2; when the recombinant optical probe is used as the basic unit with When the functional protein is connected, it can be fused to the amino acid or carboxyl terminus of the recombinant optical probe. The linker sequence can be a short peptide chain consisting of one or more flexible amino acids.

As used herein, the terms "chromophore" and "fluorophore" are synonymous with "fluorescent protein" and refer to a protein that fluoresces upon irradiation with excitation light. Fluorescent proteins are used as basic detection methods in the field of biological sciences, such as the green fluorescent protein GFP commonly used in the field of biotechnology and the cyclically rearranged blue fluorescent protein (cpBFP), the cyclically rearranged green fluorescent protein derived from the mutation of this protein. (cpGFP), cyclic rearranged yellow fluorescent protein (cpYFP), etc.; there are also red fluorescent protein RFP commonly used in the art, and cyclic rearranged proteins derived from this protein, such as cpmApple, cpmOrange, cpmKate, etc. . The sequences of exemplary fluorescent proteins are set forth in any of SEQ ID NOs: 2-9.

The green fluorescent protein GFP was originally extracted from Aequorea Victoria and consists of 238 amino acids with a molecular weight of about 26kDa. GFP is composed of 12 β-folded strands forming a unique barrel-like structure, which is encapsulated by a chromophore tripeptide (Ser65-Tyr66-Gly67). In the presence of oxygen, it spontaneously forms the chromophore structure of p-hydroxybenzylidazolidinone and fluoresces. GFP does not require cofactors to generate fluorescence, and the fluorescence is very stable, making it a good imaging tool. GFP has two excitation peaks, the main peak at 395nm produces emission at 508nm, and the shoulder peak at 475nm produces emission at 503nm. An exemplary cpGFP is shown in SEQ ID NO:6

The yellow fluorescent protein YFP is derived from the green fluorescent protein GFP, and its amino acid sequence is more than 90% homologous to GFP. Compared with GFP, the key change of YFP is that the 203rd amino acid is mutated from threonine to tyrosine (T203Y). Compared with the original AvGFP, the wavelength of the main excitation peak of YFP is red-shifted to 514 nm and the emission wavelength is changed to 527 nm. On this basis, site-directed mutation (S65T) of the 65th amino acid of YFP can be obtained to obtain the fluorescence-enhanced yellow fluorescent protein EYFP. cpYFP connects the original N-terminus and C-terminus of GFP through a flexible short peptide chain, and creates a new N-terminus and C-terminus at the position near the chromophore of the original GFP, and uses the original 145-238 amino acids as a new protein. The N-terminus of the protein, the original amino acids 1-144 are used as the C-terminus of the new protein, and the two fragments are obtained by connecting 5-9 short peptide chains with flexibility. In the present invention, the positions near the chromophore are preferably Y144 and N145 amino acids; the flexible short peptide chain is preferably VDGGSGGTG or GGSGG. The sequence of an exemplary cpYFP is shown in SEQ ID NO:2.

The red fluorescent protein RFP was originally extracted from corals in the ocean. The wild RFP is an oligomeric protein that is not conducive to the fusion expression of organisms. Then, on the basis of RFP, red fluorescent proteins with different color bands were further derived. Among them The most commonly used are mCherry and mKate. Exemplary cpmKates are shown in SEQ ID NO:4 or 8. An exemplary mCherry is shown in SEQ ID NO:5.

In other embodiments, the fluorescent protein can also be a blue fluorescent protein cpBFP whose amino acid sequence is shown in SEQ ID NO: 7, an orange fluorescent protein cpmOrange whose amino acid sequence is shown in SEQ ID NO: 3, and an amino acid sequence such as SEQ ID NO: 9 One or more of the apple red fluorescent proteins cpmApple shown.

The amino acid optical probes of the present invention include amino acid-sensitive polypeptide B, such as amino acid binding protein or a variant thereof, and optically active polypeptide A, such as fluorescent protein. The optically active polypeptide A is inserted into the amino acid-sensitive polypeptide B, and B is divided into two parts B1 and B2, forming a probe structure of the formula B1-A-B2; the interaction between the amino acid-sensitive polypeptide B and the amino acid leads to the optical activity of the optically active polypeptide A The signal becomes stronger.

In the optical probe of the present invention, the optically active polypeptide can be located or fused to any position of the amino acid-sensitive polypeptide. In one embodiment, the optically active polypeptide is located anywhere in the N-C orientation of the amino acid-sensitive polypeptide in the N-C orientation. Specifically, the optically active polypeptide is located in the flexible region of the amino acid-sensitive polypeptide, and the flexible region refers to some specific structures such as loop domains that exist in the higher-order structure of the protein. Compared with other higher-order structures of the protein, these domains have Higher mobility and flexibility, and this region can dynamically change the spatial conformation of the protein and ligand after binding. The flexible region mentioned in the present invention mainly refers to the region where the fusion site in the amino acid binding protein is located, such as the regions of amino acid residues 117-123, 249-259 and 317-330. Illustratively, the optically active polypeptide is located at a position in the amino acid sequence of the amino acid binding protein selected from the group consisting of: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121 , 119/120, 119/121, 120/121, 120/122, 120/123, 121/122, 121/123, 122/123, 249/250, 249/251, 249/252, 249/253, 249 /254, 249/255, 249/256, 249/257, 249/258, 249/259, 250/251, 250/252, 250/253, 250/254, 250/255, 250/256, 250/257 , 250/258, 250/259, 251/252, 251/253, 251/254, 251/255, 251/256, 251/257, 251/258, 251/259, 252/253, 252/254, 252 /255, 252/256, 252/257, 252/258, 252/259, 253/254, 253/255, 253/256, 253/257, 253/258, 253/259, 254/255, 254/256 , 254/257, 254/258, 254/259, 255/256, 255/257, 255/258, 255/259, 256/257, 256/258, 256/259, 257/258, 257/259, 258 /259, 323/330, 324/330, 325/330, 326/327, 326/328, 326/329, 326/330, 327/328, 327/329, 327/330, 328/329, 328/330 and 329/330. In a preferred embodiment, the optically active polypeptide is located at 120/121, 121/122, 121/123, 324/330, 325/330 or 326/330 of the amino acid sequence of the amino acid binding protein. As shown in SEQ ID NOs: 10-15.

When referring to a polypeptide or protein, the terms "variant" or "mutant" as used herein include variants that have the same function of the polypeptide or protein but differ in sequence. These variants include, but are not limited to, deletions, insertions and/or substitutions of one or more (usually 1-30, preferably 1-20, more preferably 1- 10, optimally 1-5) amino acids, and one or more (usually within 20, preferably within 10, more preferably within 5) added to their carboxy-terminus and/or amino-terminus ) amino acid sequence obtained. Without wishing to be bound by theory, amino acid residues are altered without altering the overall configuration and function of the polypeptide or protein, ie, functionally conservative mutations. For example, in the art, substitution with amino acids with similar or similar properties usually does not change the function of the polypeptide or protein. In the art, amino acids with similar properties often refer to amino acid families with similar side chains, which have been clearly defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar Amino acids with side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g. threonine, valine, isoleucine ) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). As another example, the addition of one or more amino acids to the amino terminus and/or the carboxy terminus generally does not alter the function of the polypeptide or protein. Conservative amino acid substitutions for many common known non-genetically encoded amino acids are known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on a comparison of their physical properties with those of the genetically encoded amino acids. It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable restriction sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, which does not affect the purpose The activity of a polypeptide or protein. For another example, in order to construct a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted to the outside of the host cell, or facilitate the purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus or the recombinant protein of the recombinant protein. In other suitable regions within the protein, for example, including, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, such as 6His or Flag tags, or proteolytic enzyme sites for factor Xa or thrombin or enterokinase. Variants of polypeptides or proteins can include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants may also comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the polypeptide or protein %, at least about 99% or 100% of the polypeptide or protein.

The optical probes of the present invention may comprise amino acid-sensitive polypeptides with mutations. Said mutation is, for example, selected from mutations at F77, A100, T102, D121, Y150, D226, G227 and Y275 alleles. Exemplarily, the mutation is selected from F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A, A100S, A100Y , A100C, A100L, A100h, A100H, A100Q, A100W, A100R, A100T, A100N, A100K, A100M, A100F, A100D, A100G, A100G, T102Y, T102C, T102P, T102H, T102Q, T102Q ，T102R，T102I，T102A， T102N，T102K，T102M，T102V，T102F，T102D，T102E，T102G，D121S，D121Y， D121C，D121L，D121P，D121H，D121Q，D121W，D121I，D121T，D121N， D121K，D121R，D121V ，D121A，D121F，D121E，D121M，D121A，Y150S，Y150T，Y150C，Y150L，Y150P，Y150H，Y150Q，Y150W，Y150R，Y150I， Y150A，Y150N，Y150K，Y150M，Y150V，Y150F，Y150D，Y150E，Y150G， D226S ，D226T，D226C，D226L，D226P，D226H，D226Q，D226W，D226R， D226I，D226A，D226N，D226K，D226M，D226V，D226F，D226Y，D226E，D226G，G227S，G227T，G227C，G227L，G227P，G227H，G227Q ，G227W， G227R，G227I，G227A，G227N，G227K，G227M，G227V，G227F，G227Y， G227E，G227D，Y275S，Y275T，Y275C，Y275L，Y275P，Y275H，Y275Q，Y275W， Y275R，Y275I，Y275A，Y275N，Y275K , Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the mutation is selected from the group consisting of F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F.

In an exemplary embodiment, the B1-A-B2 type optical probe of the present invention may be Atu2422 fused with cpYFP at site 121/122 and having a compound selected from the group consisting of F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, Probes formed upon mutation of G227S and Y275F are shown in SEQ ID NOs: 16-25.

The optical probe provided by the present invention comprises any one of the amino acid sequences SEQ ID NO: 10-25 or a variant thereof. In one embodiment, the optical probe provided by the present invention comprises 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90% of the amino acid sequence SEQ ID NO: 10-25. Sequences with %, 95%, 99% sequence identity. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence substantially similar or identical to any of the amino acid sequences SEQ ID NOs: 10-25. In a more preferred embodiment, the optical probe provided by the present invention comprises or consists of SEQ ID NO: 24.

In two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percent identity" refers to a comparison window or specified region, by manual alignment and visual inspection using methods known in the art, such as sequence comparison algorithms Two or more sequences or subsequences are identical or in which a certain percentage of amino acid residues or nucleotides are identical in a given region (eg, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the same). For example, preferred algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403.

As used herein, the terms "functional variant," "derivative," and "analog" refer to proteins that retain substantially the same biological function or activity as the original polypeptide or protein (eg, amino acid binding protein or fluorescent protein). Functional variants, derivatives or analogs of polypeptides or proteins (eg, amino acid binding proteins or fluorescent proteins) of the present invention may (i) have one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) ) proteins that are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) have substitution groups in one or more amino acid residues, or (iii) mature A protein formed by fusion of a protein with another compound (such as a compound that prolongs the half-life of a protein, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the protein sequence (such as a secretion sequence or used for purification) The sequence of this protein or the pro-protein sequence, or a fusion protein with an antigenic IgG fragment). These functional variants, derivatives and analogs are well known to those skilled in the art in light of the teachings herein.

The difference between the analog and the original polypeptide or protein can be the difference in amino acid sequence, or the difference in modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to mutagens, as well as by site-directed mutagenesis or other known molecular biology techniques.

The analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the amino acid-sensitive polypeptides of the present invention are not limited to the above-listed representative proteins, variants, derivatives and analogs. Modified (usually without altering the primary structure) forms include chemically derivatized forms of the protein, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins modified to increase their resistance to proteolysis or to optimize solubility.

The present invention also provides a method for preparing the above amino acid optical probe, comprising the following steps: 1) incorporating the nucleic acid sequence encoding the amino acid optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; The host cell is cultured under conditions suitable for the expression of the expression vector, and 3) the amino acid optical probe is isolated.

The term "nucleic acid" or "nucleotide" as used herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be the coding or non-coding strand. The term "variant" as used herein in reference to a nucleic acid can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternate form of a nucleic acid, which may be a substitution, deletion or insertion of one or more nucleotides, but which does not substantially alter the function of the protein it encodes. Nucleic acids of the invention may comprise at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 98%, At least about 99% or 100% of the nucleotide sequence. The present invention also relates to nucleic acid fragments hybridizing to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (eg, PCR).

The full-length sequence or the fragment thereof of the optical probe or fusion protein of the present invention can usually be obtained by PCR amplification method, artificial synthesis method or recombinant method. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art is used as a template to amplify the relevant cDNAs. sequence. When the nucleotide sequence is greater than 2500 bp, preferably 2 to 6 times of PCR amplification are performed, and then the amplified fragments of each time are spliced together in the correct order. The present invention does not specifically limit the PCR amplification procedure and system, and conventional PCR amplification procedures and systems in the art may be used. Recombinant methods can also be used to obtain relevant sequences in large quantities. This is usually by cloning it into a vector, then transferring it into cells, and then isolating and purifying the relevant polypeptide or protein from the propagated host cells by conventional methods. In addition, synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. In the present invention, when the nucleotide sequence of the optical probe is less than 2500bp, it can be synthesized by artificial synthesis method. The artificial synthesis method is a conventional DNA artificial synthesis method in the art, and there are no other special requirements. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation. At present, DNA sequences encoding the proteins of the present invention (or functional variants, derivatives or analogs thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (eg, vectors) and cells known in the art. Mutations can be introduced into the protein sequences of the present invention by methods such as mutation PCR or chemical synthesis.

In the present invention, after obtaining the nucleotide sequence encoding the optical probe, the nucleotide sequence encoding the optical probe is incorporated into an expression vector to obtain a recombinant expression vector. As used herein, the terms "expression vector" and "recombinant vector" are used interchangeably and refer to prokaryotic or eukaryotic vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, Retroviral or other vectors capable of replication and stable expression in the host, an important feature of these recombinant vectors is that they usually contain expression control sequences. The term "expression control sequence" as used herein refers to an element that can be operably linked to a gene of interest that regulates the transcription, translation and expression of a gene of interest, and may be an origin of replication, promoter, marker gene or translational control element, including enhancers, operons , terminators, ribosome binding sites, etc., the choice of expression control sequences depends on the host cell used. Recombinant vectors suitable for use in the present invention include, but are not limited to, bacterial plasmids. In a recombinant expression vector, "operably linked" means that a nucleotide sequence of interest is linked to regulatory sequences in a manner that allows expression of the nucleotide sequence. Those skilled in the art are familiar with methods that can be used to construct expression vectors containing the fusion protein coding sequences of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the bacteriophage lambda PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the reverse The LTR of the transcription virus and some other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the commercial pRSETb vector can be used as the expression vector, without other special requirements. Exemplarily, the nucleotide sequence encoding the optical probe and the expression vector are respectively subjected to double digestion with BamHI and EcoRI, and then the two digestion products are ligated to obtain a recombinant expression vector. The specific steps and parameters of the enzyme cleavage and ligation are not particularly limited in the present invention, and conventional steps and parameters in the art can be used.

After obtaining the recombinant expression vector, the vector is transformed into host cells to produce proteins or peptides including fusion proteins. This transfer process can be performed using conventional techniques such as transformation or transfection, which are well known to those skilled in the art. The host cell in the present invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, and is a place for the amplification of recombinant genes. An ideal recipient cell should satisfy the two conditions of easy acquisition and proliferation. "Host cells" of the present invention may include prokaryotic cells and eukaryotic cells, specifically bacterial cells, yeast cells, insect cells and mammalian cells. Specifically, it can be Escherichia coli, Streptomyces, Salmonella typhimurium bacterial cells, fungal cells such as yeast, plant cells, Drosophila S2 or Sf9 insect cells, CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells animal cells, etc., including but not limited to those host cells mentioned above. The host cell is preferably a variety of cells that facilitate gene product expression or fermentative production, and such cells are well known and commonly used in the art. An exemplary host cell used in the examples of the present invention is Escherichia coli JM109-DE3 strain. It will be clear to those of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

The method for transferring to host cells described in the present invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical mediation induced transfer or electroporation. When the host is a prokaryotic organism such as Escherichia coli, the method is preferably CaCl ₂ method or MgCl ₂ method, and the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging and the like.

In the present invention, after the expression vector is transformed into the host cell, the host cell transformed into the expression vector is amplified, expressed and cultured, and the amino acid optical probe is obtained by separation. The host cell amplification, expression and culture can adopt conventional methods. The medium used in the culture may be various conventional mediums depending on the species of host cells used. Cultivation is carried out under conditions suitable for growth of the host cells.

In the present invention, the optical probe is expressed inside the cell, on the cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated or purified by various isolation methods utilizing their physical, chemical and other properties. The method for separating the amino acid fluorescent protein is not particularly limited in the present invention, and a conventional method for separating fusion proteins in the art can be used. These methods are well known to those skilled in the art, including but not limited to: conventional renaturation treatment, salting out method, centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange layer chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. In one embodiment, the separation of optical probes is performed using His-tagged affinity chromatography.

The invention also provides the application of the amino acid optical probe in real-time amino acid localization, quantitative detection and high-throughput compound screening. In one aspect, the amino acid optical probe is preferably connected with signal peptides in different parts of the cell and transferred into the cell, and the real-time positioning of the amino acid is carried out by detecting the intensity of the fluorescent signal in the cell; Quantitative detection of corresponding amino acids. The amino acid standard dropping curve of the present invention is drawn according to the fluorescent signals of amino acid optical probes under the condition of different concentrations of amino acids. The amino acid optical probe of the present invention is directly transferred into cells, and in the process of real-time positioning and quantitative detection of amino acids, time-consuming sample processing is not required, and it is more accurate. When the amino acid optical probe of the present invention performs high-throughput compound screening, different compounds are added to the cell culture medium to measure the change of amino acid content, so as to screen out compounds that have an impact on the change of amino acid content. The application of the amino acid optical probe described in the present invention in real-time amino acid localization, quantitative detection and high-throughput compound screening is not for the purpose of diagnosis and treatment, and does not involve the diagnosis and treatment of diseases. The aforementioned amino acids may be any amino acids.

Herein, concentrations, amounts, percentages, and other numerical values may be expressed in the form of ranges. It should also be understood that this range format is used for convenience and brevity only, and should be flexibly read to include the values expressly recited at the upper and lower limits of the range, as well as to include all individual values or subranges subsumed within the range.

Example

The amino acid optical probes provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

I. Experimental Materials and Reagents

In the examples, conventional genetic engineering molecular biology cloning methods, cell culture and imaging methods, etc. are mainly used, and these methods are well known to those of ordinary skill in the art, for example: "Molecular Biology Experiment Reference" by Jane Roskems et al. Handbook, J. Sambrook, D.W. Russell, translated by Huang Peitang et al.: "Molecular Cloning Experiment Guide" (Third Edition, August 2002, published by Science Press, Beijing); Animal Cell Culture: A Guide to Basic Techniques (Fifth Edition), translated by Zhang Jingbo, Xu Cunshuan et al; Those of ordinary skill in the art can successfully implement the present invention by making slight modifications and transformations according to the specific conditions according to the following embodiments, and these modifications and transformations all fall within the scope of the claims of the present application.

Based on pRSETb-cpYFP, the pRSETb-amino acid binding protein plasmid used in the examples was constructed by the Protein Laboratory of East China University of Science and Technology, and the pRSETb plasmid vector was purchased from Invitrogen Company. All primers used for PCR were synthesized, purified and correctly identified by mass spectrometry by Shanghai Jierui Bioengineering Technology Co., Ltd. The expression plasmids constructed in the examples were all subjected to sequence determination, and the sequence determination was completed by Huada Gene Company and Jie Li Sequencing Company. Taq DNA polymerase used in each embodiment was purchased from Dongsheng Bio, pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., primeSTAR DNA polymerase was purchased from TaKaRa Company, and three kinds of polymerases were purchased with corresponding gifts. Polymerase buffer and dNTPs. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, T4 phosphorylase (T4PNK) were purchased from Fermentas, and the corresponding buffer solution was attached to the purchase. The transfection reagent Lip2000Kit was purchased from Invitrogen Company. Amino acids such as alanine were purchased from Sigma Company. Unless otherwise stated, chemical reagents such as inorganic salts were purchased from Sigma-Aldrich Company. HEPES salts, ampicillin (Amp) and puromycin were purchased from Ameresco. The 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from Grenier Company.

The DNA purification kits used in the examples were purchased from BBI Company (Canada), and the common plasmid mini-pump kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and the desalting column packing material were obtained from GE healthcare.

The main instruments used in the examples include: Biotek Synergy 2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R desktop high-speed refrigerated centrifuge (Beckman, USA) , PCR Amplifier (German Biometra Company), Ultrasonic Breaker (Ningbo Xinzhi Company), Nucleic Acid Electrophoresis Instrument (Shenergy Gaming Company), Fluorescence Spectrophotometer (American Varian Company), CO ₂ Constant Temperature Cell Incubator (SANYO) , an inverted fluorescence microscope (Nikon, Japan).

II. Molecular biological methods and cell experimental methods

II.1 Polymerase Chain Reaction (PCR):

1. Target fragment amplification PCR:

This method is mainly used for gene fragment amplification and colony PCR to identify positive clones. The reaction system of the PCR amplification is shown in Table 1, and the amplification procedure is shown in Table 2.

Table 1. PCR amplification reaction system

Table 2. PCR Amplification Procedure

2. Long fragment (>2500bp) amplification PCR:

The long-fragment amplification used in the present invention is mainly an inverse PCR amplification vector, a technique used to obtain site-directed mutagenesis in the following examples. Reverse PCR primers were designed at the variant site, and the 5' end of one primer contained the variant nucleotide sequence. The amplified product contains the corresponding mutation site. The PCR reaction system for long fragment amplification is shown in Table 3, and the amplification procedure is shown in Table 4 or Table 5.

Table 3. PCR reaction system for long fragment (>2500bp) amplification

Table 4. PCR amplification procedure for long fragment (>2500bp) amplification

Table 5. PCR amplification procedure for long fragment (>2500bp) amplification

II.2 Endonuclease digestion reaction:

The system for double-enzyme digestion of the plasmid vector is shown in Table 6, where n represents the amount of sterilized ultrapure water μL added to make the system reach the total volume.

Table 6. Plasmid vector double enzyme digestion system

II.3 Phosphorylation reaction at the 5' end of DNA fragments

The ends of plasmids or genomes extracted from microorganisms contain phosphate groups, but PCR products do not. Therefore, it is necessary to perform phosphate group addition reaction on the 5'-end bases of PCR products. Only DNA molecules with phosphate groups at the ends can occur. ligation reaction. The phosphorylation reaction system is shown in Table 7, wherein T4PNK is the abbreviation of T4 polynucleotide kinase, which is used for the addition reaction of the phosphate group at the 5' end of the DNA molecule.

Table 7. Phosphorylation reaction system

II.4 Ligation reaction of target fragment and vector

The connection methods between different fragments and vectors are different, and three connection methods are used in the present invention

1. Blunt-end ligation of blunt-ended short fragments and linearized vectors

The principle of this method is that the blunt end product obtained by PCR is phosphorylated at the 5' end of the DNA fragment under the action of T4PNK, and then connected with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination connection system is shown in Table 8.

Table 8. Blunt-end fragment ligation reaction system

2. Ligation of DNA Fragments Containing Cohesive Ends and Vector Fragments Containing Cohesive Ends

DNA fragments cut by restriction endonucleases usually produce sticky ends that stick out, so they can be ligated with vector fragments containing complementary sticky ends to form recombinant plasmids. The ligation reaction system is shown in Table 9, wherein the mass ratio of the PCR product fragment to the vector double-enzyme digestion product is roughly between 2:1-6:1. .

Table 9. Sticky end ligation reaction system

3. Ligation reaction of self-circularization of 5'-end phosphorylated DNA fragment products after introducing site-directed mutagenesis by inverse PCR

The 5'-end phosphorylated DNA fragment is ligated to the 3'-end and 5'-end of the linearized vector through self-circularization ligation reaction to obtain a recombinant plasmid. The self-cyclization ligation reaction system is shown in Table 10.

Table 10. Self-cyclization ligation reaction system

II.5 Preparation and transformation of competent cells

Preparation of competent cells:

1. Pick a single colony (eg Mach1) and inoculate it in 5 mL of LB medium, shake at 37°C overnight.

2. Transfer 0.5-1 mL of the overnight cultured bacterial solution to 50 mL of LB medium, and cultivate at 37°C and 220 rpm for 3 to 5 hours until the OD600 reaches 0.5.

3. Pre-cool the cells in an ice bath for 2 hours.

4.4°C, 4000 rpm centrifugation for 10 minutes.

5. Discard the supernatant, resuspend the cells with 5 mL of pre-chilled buffer, and add resuspension buffer to a final volume of 50 mL after uniformity.

6. Ice bath for 45 minutes.

Centrifuge at 4000rpm at 7.4°C for 10 minutes, and resuspend the bacteria with 5mL of ice-cold storage buffer.

8. Put 100 μL of bacterial solution in each EP tube and freeze at -80°C or liquid nitrogen.

Resuspension buffer: CaCl ₂ (100 mM), MgCl ₂ (70 mM), NaAc (40 mM)

Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10×CaCl ₂ (1M), 1 mL 10× MgCl ₂ (700 mM), 1 mL 10× NaAc (400 mM), 4.6 mL ddH ₂ O

Transformation of competent cells:

1. Take 100 μL of competent cells and thaw on an ice bath.

2. Add an appropriate volume of ligation product, mix by gently blowing, and ice bath for 30 minutes. The volume of ligation product added is usually less than 1/10 of the volume of competent cells.

3. Put the bacterial solution in a 42°C water bath and heat shock for 90 seconds, then quickly transfer it to an ice bath for 5 minutes.

4. Add 500 μL of LB and incubate at 200 rpm on a constant temperature shaker at 37°C for 1 hour.

5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes, leave 200 μL of supernatant to blow the bacteria evenly, spread evenly on the surface of an agar plate containing appropriate antibiotics, and invert the plate overnight in a constant temperature incubator at 37°C.

II.6 Expression, purification and fluorescence detection of proteins

1. Transform the expression vector (such as pRSETb-based amino acid optical probe expression vector) into JM109 (DE3) cells, invert overnight culture, pick and clone from the plate into a 250ml Erlenmeyer flask, shake at 37°C bed, cultured at 220rpm to OD=0.4-0.8, added 1/1000 (v/v) IPTG (1M), and induced expression at 18°C for 24-36 hours.

2. After induction and expression, the bacteria were harvested by centrifugation at 4000 rpm for 30 minutes, 50 mM phosphate buffer was added to resuspend the bacterial pellet, and the bacteria were sonicated until the bacteria became clear. Centrifuge at 9600 rpm for 20 minutes at 4°C.

3. The centrifugation supernatant is purified by a self-packed nickel column affinity chromatography column to obtain the protein, and the protein after nickel column affinity chromatography is then passed through a self-packed desalting column to be dissolved in 20mM MOPS buffer (pH 7.4) or phosphate protein in buffer PBS.

4. After the purified protein was identified by SDS-PAGE, the probe was diluted with assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.3) or phosphate buffered saline PBS to a protein solution with a final concentration of 5-10 μM. Alanine was formulated as a final 1 M stock solution in assay buffer (20 mM MOPS, pH 7.4) or phosphate buffered saline PBS.

5. Take 100 μl of 5 μM protein solution, incubate at 37°C for 5 minutes, add alanine and mix well to reach a final concentration of 100 mM, and use a multifunctional fluorescence microplate reader to measure the light absorption of the protein at 340 nm.

6. Take 100 μl of 1 μM protein solution, incubate at 37° C. for 5 minutes, add alanine for titration, and measure the fluorescence intensity of the protein emitted at 528 nm after fluorescence excitation at 485 nm. The fluorescence excitation and emission measurement of the samples were completed by a multifunctional fluorescence microplate reader.

7. Take 100 μl of 1 μM protein solution, incubate at 37° C. for 5 minutes, add alanine, and measure the absorption spectrum and fluorescence spectrum of the protein. The determination of the absorption spectrum and fluorescence spectrum of the sample is completed by a spectrophotometer and a fluorescence spectrophotometer.

II.7 Transfection and Fluorescence Detection of Mammalian Cells

1. The pCDNA3.1+-based amino acid optical probe plasmid was transfected into HeLa by the transfection reagent Lipofectamine2000 (Invitrogen), and cultured in a cell incubator at 37° C., 5% CO ₂ . Fluorescence detection was performed 24-36 hours after the exogenous gene was fully expressed.

2. After induction and expression, the adherent HeLa cells were washed three times with PBS and placed in HBSS solution for fluorescence microscope and microplate reader detection respectively.

Example 1: Amino acid binding protein particles

The AABP (here Atu2422) gene in the Agrobacterium tumefaciens gene was amplified by PCR, the PCR product was recovered after gel electrophoresis, and digested with BamHI and EcoRI, and the pRSETb vector was subjected to corresponding double digestion. After ligation with T4 DNA ligase, MachI was transformed with the product, and the transformed MachI was spread on an LB plate (100 ug/mL of ampicillin) and cultured at 37°C overnight. After plasmid extraction of the growing MachI transformants, PCR identification was performed. After the positive plasmids were sequenced correctly, the subsequent plasmid construction was carried out.

Example 2: Expression and detection of cpYFP optical probes at different fusion sites

In this example, the following sites were selected to fuse cpYFP based on pRSETb-Atu2422 to obtain the corresponding pRSETb-Atu2422-cpYFP plasmids: 117/118, 117/119, 117/120, 117/121, 118/119, 118/ 120, 118/121, 119/120, 119/121, 120/121, 120/122, 120/123, 121/122, 121/123, 122/123, 249/250, 249/251, 249/252, 249/253, 249/254, 249/255, 249/256, 249/257, 249/258, 249/259, 250/251, 250/252, 250/253, 250/254, 250/255, 250/ 256, 250/257, 250/258, 250/259, 251/252, 251/253, 251/254, 251/255, 251/256, 251/257, 251/258, 251/259, 252/253, 252/254, 252/255, 252/256, 252/257, 252/258, 252/259, 253/254, 253/255, 253/256, 253/257, 253/258, 253/259, 254/ 255, 254/256, 254/257, 254/258, 254/259, 255/256, 255/257, 255/258, 255/259, 256/257, 256/258, 256/259, 257/258, 257/259, 258/259, 323/330, 324/330, 325/330, 326/327, 326/328, 326/329, 326/330, 327/328, 327/329, 327/330, 328/ 329, 328/330 or 329/330.

The DNA fragment of cpYFP was generated by PCR, and the DNA fragment was inactivated by adding phosphorus at the 5' end. At the same time, a pRSETb-amino acid binding protein linearized vector containing different breakpoints was generated by inverse PCR amplification. The linearized The pRSETb-Atu2422 and the phosphorylated cpYFP fragment at the 5' end were ligated under the action of PEG4000 and T4 DNA ligase to generate recombinant plasmids. These plates were used in the Kodak multifunctional in vivo imaging system, and the clones with yellow fluorescence under the excitation of the FITC channel were picked. , and the sequencing was completed by Beijing Liuhe Huada Gene Technology Co., Ltd. Shanghai Branch.

After being sequenced correctly, the recombinant plasmid was transformed into JM109(DE3) to induce expression, and the protein was purified, and the size was around 68Kda by SDS-PAGE electrophoresis. This size corresponds to the size of the Atu2422-cpYFP fusion protein containing the His-tag purified tag expressed by pRSETb-Atu2422-cpYFP. The results are shown in Figure 1.

The purified Atu2422-cpYFP fusion protein was screened for alanine response, and the detection signal of the fusion fluorescent protein containing 100 mM alanine was divided by the detection signal of the fusion fluorescent protein without alanine. The results are shown in Figure 2. The detection results show that the optical probes that respond to alanine more than 2 times are at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 ( As shown in SEQ ID NOs 10-15) or the corresponding amino acid positions of its family proteins are optical probes for fusion.

Example 3: Expression and detection of cpGFP optical probes at different fusion sites

The amino acid green fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpGFP according to the method in Example 2. As shown in Figure 3, the detection results showed that the optical probes with more than 2-fold response to alanine were found at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 or other Optical probes that implement fusions at the corresponding amino acid positions of the family proteins.

Example 4: Expression and detection of cpBFP optical probes at different fusion sites

The amino acid blue fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpBFP according to the method in Example 2. As shown in Figure 4, the detection results showed that the optical probes with more than 2-fold response to alanine were found at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 or their Optical probes that implement fusions at the corresponding amino acid positions of the family proteins.

Example 5: Expression and detection of cpmApple optical probes at different fusion sites

According to the method in Example 2, cpYFP was replaced with cpmApple to construct an amino acid red fluorescent protein fluorescent probe. As shown in Figure 5, the detection results showed that the optical probes with more than 2-fold response to alanine were found at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 or other Optical probes that implement fusions at the corresponding amino acid positions of the family proteins.

Example 6: Performance of Optical Probes

For the optical probes obtained in Example 2 that responded more than 2-fold to alanine, i.e. fused at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 Six kinds of optical probes were detected by alanine concentration gradient, and the change of the ratio of fluorescence intensity at 420nm excitation at 528nm emission and 485nm excitation at 528nm emission was detected. The K _d (binding constant) of the six amino acid optical probes with fusion sites of 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 were 8.5mM, 0.02mM, 5.6, respectively mM, 0.13 mM, 0.13 mM and 0.79 mM, the change amplitudes were 3.4 times, 4.3 times, 2.8 times, 2.4 times, 2.7 times and 2.0 times, respectively. The results are shown in FIG. 6 .

For the probe with fusion site 121/122 (Atu2422-121/122-cpYFP), its specific detection for each amino acid was performed. At the same time, the fusion protein obtained by fusing fluorescent proteins cpYFP, cpGFP, cpBFP or cpmApple to the N-terminus or C-terminus of Atu2422 was used as a control to compare the specificity of the probe with fusion site of 121/122 to alanine.

The results showed that the probe probe with fusion site 121/122 had higher response to alanine, proline, valine, serine, isoleucine, threonine and cysteine, respectively. were 4.3 times, 4.5 times, 4.0 times, 3.2 times, 2.4 times and 3.0 times, as shown in Figure 7A. At the same time, the fusion protein obtained by fusing the fluorescent protein to the N-terminus or C-terminus of Atu2422 did not respond to alanine, while the probe with fusion site 121/122 responded to alanine about 4.3 times, as shown in Figure 7B Show.

Example 7: Expression and detection of mutated cpYFP optical probes

Optical probe mutants were constructed on the basis of Atu2422-121/122-cpYFP. The plasmid pRSETb-Atu2422-121/122-cpYFP was linearized by reverse PCR, and the primers contained the base sequence of the desired mutation site, and the obtained PCR product was ligated with phosphorus under the action of PNK, T4DNA ligase and PEG4000 to obtain F77, A100, T102, D121, Y150, D226, G227, Y275 site-directed saturation mutant plasmids were sequenced by Beijing Liuhe Huada Gene Technology Co., Ltd. Shanghai Branch.

The results are shown in Figure 8. Fluorescence detection showed that F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F mutants responded more than 2-fold to alanine.

Example 8. Performance of optical probe mutants

Exemplarily, after the purified amino acid optical probe Atu2422-121/122-cpYFP-G227S was treated with 0 mM and 500 mM alanine for 10 minutes, respectively, the fluorescence spectrum was detected using a fluorescence spectrophotometer.

Determination of Excitation Spectra: Excitation spectra were recorded with an excitation range of 350 nm to 510 nm and an emission wavelength of 530 nm, reading every 1 nm. The results showed that the probe had two excitation peaks at about 420 and 490 nm, as shown in Fig. 9A.

Determination of emission spectra: The excitation wavelengths were fixed at 420 nm and 490 nm, respectively, and the emission spectra at 505-600 nm were recorded and read every 1 nm. The results showed that after the addition of 500 mM alanine, the fluorescence intensity of the probe was reduced by 0.78 times under the excitation of 420 nm, and the fluorescence intensity was reduced by 0.22 times under the excitation of 490 nm. As shown in Figures 9B and 9C.

The purified Atu2422-121/122-cpYFP-G227S was subjected to concentration gradient (0-100 mM) alanine detection. After the purified probe was treated for 10 minutes, the change in the ratio of the fluorescence intensity at 420 nm excitation at 528 nm emission and the fluorescence intensity at 485 nm excitation at 528 nm emission was detected. As a result, as shown in Fig. 10, the K _d (binding constant) of the amino acid optical probe was 2.8 mM, and the variation was 4-fold.

The reactivity of Atu2422-121/122-cpYFP-G227S with 20 amino acids was detected, and the results showed that it had good specificity, as shown in FIG. 11 .

Example 9: Subcellular Organelle Localization of Optical Probes and Performance of Optical Probes in Subcellular Organelles

In this example, different localization signal peptides were used to fuse the optical probe Atu2422-121/122-cpYFP-G227S to localize the optical probe into different organelles.

HeLa cells were transfected with optical probe plasmids fused with different localization signal peptides for 36 hours, rinsed with PBS, placed in HBSS solution, and subjected to fluorescence detection under the FITC channel under an inverted fluorescence microscope. The results are shown in Figure 12. Amino acid optical probes can be localized to subcellular organelles including cytoplasm, mitochondria, nucleus, lysosome, endoplasmic reticulum, and Golgi apparatus by fusing with different specific localization signal peptides. Fluorescence was shown in different subcellular structures, and the distribution and intensity of fluorescence varied.

HeLa cells were transfected with the cytoplasmic-expressed optical probe plasmid for 36 hours, rinsed with PBS, and placed in HBSS solution to detect the change in the ratio of the fluorescence intensity at 420nm excitation at 528nm emission to that at 485nm excitation at 528nm emission within 40min. The results are shown in Figure 13. The 485/420 gradually decreased as alanine was depleted. Then alanine was added, and the detection was continued for 43 min. The 485/420 of the alanine-supplemented samples gradually increased, while the 485/420 of the control group continued to decrease until unchanged.

Example 10: Optical Probe-Based High-Throughput Compound Screening in Living Cells

In this example, we used HeLa cells expressing Atu2422-121/122-cpYFP-G227S in the cytoplasm for high-throughput compound screening.

Transfected HeLa cells were rinsed with PBS, treated in HBSS solution (without alanine) for 1 hour, and then treated with 10 [mu]M of compound for 1 hour. Alanine was added dropwise to each sample. Use a microplate reader to record the ratio of the fluorescence intensity at 420nm excitation at 528nm emission to that at 485nm excitation at 528nm emission. Normalization was performed with samples not treated with any compound as controls. The results are shown in Figure 14. Of the 2,000 compounds used, the vast majority had minimal effect on the entry of alanine into cells. Eleven compounds increased cellular uptake of alanine, while 9 compounds significantly decreased cellular uptake of alanine.

Example 11: Quantitative detection of alanine in blood with optical probes

In this example, purified Atu2422-121/122-cpYFP-G227S was used for analysis of alanine in mouse and human blood supernatants.

After mixing Atu2422-121/122-cpYFP-G227S with diluted blood supernatant for 10 minutes, the ratio of fluorescence intensity at 420nm excitation at 528nm emission and 485nm excitation at 528nm emission was detected using a microplate reader. As a result, as shown in FIG. 15 , the alanine content in mouse blood was about 600 μM, and the alanine content in human blood was about 360 μM.

It can be seen from the above examples that the amino acid optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature, has a large dynamic change in fluorescence, and has good specificity. Localization and quantitative detection of amino acids; and high-throughput compound screening.

Other implementations

This specification describes a number of implementations. It should be understood, however, that various modifications that those skilled in the art will come to know from reading this specification without departing from the spirit and scope of the invention should also be included within the scope of the appended claims.

sequence listing

<110> East China University of Science and Technology

<120> Amino acid optical probe and its preparation method and application

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<170> PatentIn version 3.5

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Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

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Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr Val Met Tyr Glu Trp Lys

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Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser

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Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

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Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp

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Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly

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Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys

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Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu

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Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro

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Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr

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Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu

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Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr

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Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg

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Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly

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Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr

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Ile Val Asp Ile Lys Leu Asp Ile Val Ser His Asn Glu Asp Tyr Thr

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Ile Val Glu Gln Cys Glu Arg Ala Glu Gly Arg His Pro Thr Gly Gly

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Arg Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Leu Val Ser Lys

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Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys

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Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly

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Glu Gly Glu Gly Arg Pro Tyr Glu Ala Phe Gln Thr Ala Lys Leu Lys

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Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro

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Gln Phe Thr Tyr Gly Ser Lys Ala Tyr Ile Lys His Pro Ala Asp Ile

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Pro Asp Tyr Phe Lys Leu Ser Phe Pro Glu Gly Phe Arg Trp Glu Arg

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Val Met Asn Phe Glu Asp Gly Gly Ile Ile His Val Asn Gln Asp Ser

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Ser Leu Gln Asp Gly Val Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr

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Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

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Met Val Ser Lys Gly Glu Glu Leu Ile Lys Glu Asn Met His Met Lys

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Val Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr

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Ser Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile

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Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg

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Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr

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Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val

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Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp

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Arg Ser Asp Met Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys

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Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His

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Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe

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Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val

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Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys

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Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly

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Asp Asn His Tyr Leu Ser Val Gln Ser Ile Leu Ser Lys Asp Pro Asn

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Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly

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Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser

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Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Gln

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Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

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His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro

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Asp Asn His Tyr Leu Ser Val Gln Ser Ile Leu Ser Lys Asp Pro Asn

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Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly

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Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser

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Glu Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro

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Ile Gln Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val

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Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys

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Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val

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Thr Thr Leu Ser His Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His

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Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Gly Gly Tyr Ile

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Met Ser Glu Leu Ile Thr Glu Asn Met His Met Lys Leu Tyr Met Glu

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Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly

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Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly

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Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr

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Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe

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Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr

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Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp

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Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser

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Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr

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Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Ala Asp Met

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Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr

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Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val

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Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu

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Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu

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Val Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Ser Glu Ile

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Ile Val Asp Ile Lys Leu Asp Ile Val Ser His Asn Glu Asp Tyr Thr

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Ile Val Glu Gln Cys Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly

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Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Leu Val Ser Lys

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Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys

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Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly

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Glu Gly Glu Gly Arg Pro Tyr Glu Ala Phe Gln Thr Ala Lys Leu Lys

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Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro

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Gln Phe Met Tyr Gly Ser Lys Ala Tyr Ile Lys His Pro Ala Asp Ile

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Pro Asp Tyr Phe Lys Leu Ser Phe Pro Glu Gly Phe Arg Trp Glu Arg

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Val Met Asn Phe Glu Asp Gly Gly Ile Ile His Val Asn Gln Asp Ser

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Ser Leu Gln Asp Gly Val Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr

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Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

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Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

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Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

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Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

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Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

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Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

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Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

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Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

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Asn Thr Phe Arg Thr Cys Gly Arg Tyr Asn Ser Asp Asn Val Tyr Ile

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Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg

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His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln

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Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr

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Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp

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His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly

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Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser

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Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu

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Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu

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Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr

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Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr

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Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp

290 295 300

Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile

305 310 315 320

Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe

325 330 335

Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe

340 345 350

Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 11

<211> 596

<212> PRT

<213> Artificial sequences

<400> 11

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 12

<211> 595

<212> PRT

<213> Artificial sequences

<400> 12

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gln

355 360 365

Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala

370 375 380

Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala

385 390 395 400

Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met

405 410 415

Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser

420 425 430

Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His

435 440 445

Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu Lys

450 455 460

Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser

465 470 475 480

Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp

485 490 495

Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala

500 505 510

Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met

515 520 525

Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu

530 535 540

Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu Gly

545 550 555 560

Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr Val

565 570 575

Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln

580 585 590

Gln Gly Ser

595

<210> 13

<211> 591

<212> PRT

<213> Artificial sequences

<400> 13

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

115 120 125

Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

130 135 140

His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

145 150 155 160

Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

165 170 175

Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

180 185 190

Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

195 200 205

Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu Lys Ala Lys Leu Val Ser

210 215 220

Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

225 230 235 240

Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

245 250 255

Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

260 265 270

Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

275 280 285

Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

290 295 300

Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu Gly Glu Ile Ser Phe Asp

305 310 315 320

Glu Lys Gly Asp Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys

325 330 335

Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu

340 345 350

Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile

355 360 365

Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln

370 375 380

Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu

385 390 395 400

Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu

405 410 415

Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu

420 425 430

Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val

435 440 445

Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr

450 455 460

Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro

465 470 475 480

Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys

485 490 495

Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser

500 505 510

Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp

515 520 525

Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr

530 535 540

Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly

545 550 555 560

Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Val Met Tyr Glu Trp

565 570 575

Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly Ser

580 585 590

<210> 14

<211> 592

<212> PRT

<213> Artificial sequences

<400> 14

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

115 120 125

Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

130 135 140

His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

145 150 155 160

Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

165 170 175

Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

180 185 190

Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

195 200 205

Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu Lys Ala Lys Leu Val Ser

210 215 220

Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

225 230 235 240

Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

245 250 255

Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

260 265 270

Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

275 280 285

Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

290 295 300

Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu Gly Glu Ile Ser Phe Asp

305 310 315 320

Glu Lys Gly Asp Pro Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp

325 330 335

Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val

340 345 350

Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro

355 360 365

Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe

370 375 380

Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val

385 390 395 400

Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu

405 410 415

Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu

420 425 430

Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp

435 440 445

Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala

450 455 460

Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu

465 470 475 480

Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys

485 490 495

Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys

500 505 510

Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys

515 520 525

Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp

530 535 540

Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp

545 550 555 560

Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Val Met Tyr Glu

565 570 575

Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly Ser

580 585 590

<210> 15

<211> 593

<212> PRT

<213> Artificial sequences

<400> 15

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

115 120 125

Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

130 135 140

His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

145 150 155 160

Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

165 170 175

Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

180 185 190

Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

195 200 205

Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu Lys Ala Lys Leu Val Ser

210 215 220

Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

225 230 235 240

Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

245 250 255

Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

260 265 270

Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

275 280 285

Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

290 295 300

Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu Gly Glu Ile Ser Phe Asp

305 310 315 320

Glu Lys Gly Asp Pro Lys Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala

325 330 335

Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn

340 345 350

Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr

355 360 365

Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser

370 375 380

Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met

385 390 395 400

Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp

405 410 415

Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly

420 425 430

Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly

435 440 445

Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp

450 455 460

Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys

465 470 475 480

Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu

485 490 495

Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe

500 505 510

Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe

515 520 525

Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly

530 535 540

Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu

545 550 555 560

Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Val Met Tyr

565 570 575

Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly

580 585 590

Ser

<210> 16

<211> 596

<212> PRT

<213> Artificial sequences

<400> 16

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Ala Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 17

<211> 596

<212> PRT

<213> Artificial sequences

<400> 17

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Leu Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 18

<211> 596

<212> PRT

<213> Artificial sequences

<400> 18

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Gly Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 19

<211> 596

<212> PRT

<213> Artificial sequences

<400> 19

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Glu Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 20

<211> 596

<212> PRT

<213> Artificial sequences

<400> 20

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Thr Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 21

<211> 596

<212> PRT

<213> Artificial sequences

<400> 21

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Val Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 22

<211> 596

<212> PRT

<213> Artificial sequences

<400> 22

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Glu Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 23

<211> 596

<212> PRT

<213> Artificial sequences

<400> 23

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asn Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 24

<211> 596

<212> PRT

<213> Artificial sequences

<400> 24

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Ser Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 25

<211> 596

<212> PRT

<213> Artificial sequences

<400> 25

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Phe Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 26

<211> 1791

<212> DNA

<213> Artificial sequences

<400> 26

atggatgtcg tgatcgctgt cggcgcaccg ctgaccggcc cgaacgctgc tttcggcgct 60

cagatccaga agggtgccga acaggctgcg aaagacatca atgctgccgg cggtatcaat 120

ggcgagcaga ttaagatcgt gctgggcgac gacgtatccg accccaagca gggtatttcg 180

gttgccaaca aattcgttgc tgacggcgtg aaattcgttg tcggtcactt caactcgggt 240

gtttccattc cggcatcgga agtttatgcc gaaaacggca ttctcgaaat cacgcccgct 300

gcgaccaacc cggtctttac cgagcgtggc ctgtggaaca ccttccgcac ctgcggccgt 360

gactacaaca gcgacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc 420

aacttcaaga tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga ccactaccag 480

cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcttc 540

cagtccgtcc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 600

gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaacgtgga tggcggtagc 660

ggtggcaccg gcagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 720

ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 780

acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg 840

cccaccctcg tgaccaccct cggctacggc ctgaagtgct tcgcccgcta ccccgaccac 900

atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 960

atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1020

accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1080

gggcacaagc tggagtacaa cgaccagcag ggcggcattg ccggcaagta cctggccgat 1140

catttcaagg acgccaaggt cgccatcatt cacgacaaga cgccttatgg tcagggtctt 1200

gccgatgaaa ccaaaaaggc tgccaatgct gccggcgtga ctgaggtcat gtatgaaggc 1260

gtcaacgtcg gcgacaagga cttctccgcg ctgatctcga agatgaagga agccggcgtt 1320

tccatcatct actggggcgg cctgcacacc gaagccggcc tgatcatccg ccaggcggct 1380

gaccagggtc tgaaggccaa gctcgtttcg ggcgacggta ttgtctcgaa cgaacttgct 1440

tccatcgccg gcgacgccgt cgagggcacg ctgaacacct tcggccctga tccgacgctg 1500

cgcccggaaa acaaggaact ggtagagaag ttcaaggccg ccggcttcaa cccggaagcc 1560

tacacgctct actcctatgc cgcgatgcag gcgattgcag gcgcagcgaa ggctgcgggt 1620

tccgtggagc cggaaaaggt tgccgaagcc ctgaagaagg gctccttccc gaccgcactc 1680

ggcgaaatct ccttcgatga gaagggcgac ccgaagcttc ccggctacgt catgtacgaa 1740

tggaagaagg gtccggacgg caagttcacc tacatccagc agggcagcta a 1791

<210> 27

<211> 1791

<212> DNA

<213> Artificial sequences

<400> 27

atggatgtcg tgatcgctgt cggcgcaccg ctgaccggcc cgaacgctgc tttcggcgct 60

cagatccaga agggtgccga acaggctgcg aaagacatca atgctgccgg cggtatcaat 120

ggcgagcaga ttaagatcgt gctgggcgac gacgtatccg accccaagca gggtatttcg 180

gttgccaaca aattcgttgc tgacggcgtg aaattcgttg tcggtcactt caactcgggt 240

gtttccattc cggcatcgga agtttatgcc gaaaacggca ttctcgaaat cacgcccgct 300

gcgaccaacc cggtctttac cgagcgtggc ctgtggaaca ccttccgcac ctgcggccgt 360

gactacaaca gcgacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc 420

aacttcaaga tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga ccactaccag 480

cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcttc 540

cagtccgtcc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 600

gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaacgtgga tggcggtagc 660

ggtggcaccg gcagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 720

ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 780

acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg 840

cccaccctcg tgaccaccct cggctacggc ctgaagtgct tcgcccgcta ccccgaccac 900

atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 960

atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1020

accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1080

gggcacaagc tggagtacaa cgaccagcag ggcggcattg ccggcaagta cctggccgat 1140

catttcaagg acgccaaggt cgccatcatt cacgacaaga cgccttatgg tcagggtctt 1200

gccgatgaaa ccaaaaaggc tgccaatgct gccggcgtga ctgaggtcat gtatgaaggc 1260

gtcaacgtcg gcgacaagga cttctccgcg ctgatctcga agatgaagga agccggcgtt 1320

tccatcatct actggggcgg cctgcacacc gaagccggcc tgatcatccg ccaggcggct 1380

gaccagggtc tgaaggccaa gctcgtttcg ggcgactcta ttgtctcgaa cgaacttgct 1440

tccatcgccg gcgacgccgt cgagggcacg ctgaacacct tcggccctga tccgacgctg 1500

cgcccggaaa acaaggaact ggtagagaag ttcaaggccg ccggcttcaa cccggaagcc 1560

tacacgctct actcctatgc cgcgatgcag gcgattgcag gcgcagcgaa ggctgcgggt 1620

tccgtggagc cggaaaaggt tgccgaagcc ctgaagaagg gctccttccc gaccgcactc 1680

ggcgaaatct ccttcgatga gaagggcgac ccgaagcttc ccggctacgt catgtacgaa 1740

tggaagaagg gtccggacgg caagttcacc tacatccagc agggcagcta a 1791

Claims (10)

1. a kind of optical probe, comprising amino acid-sensitive polypeptide or its functional variety and optical activity polypeptide or its functional variety, Wherein optical activity polypeptide or its functional variety are located in the sequence of amino acid-sensitive polypeptide or its functional variety.

2. optical probe as described in claim 1, wherein amino acid-sensitive polypeptide has sequence shown in SEQ ID NO:1, Or have the sequence of 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity with it, and/ Or in which optical activity polypeptide is located in the section selected from the following of amino acid-sensitive polypeptide: residue 117-123, residue 249- 259 and residue 323-330, number correspond to the overall length of amino acid-sensitive polypeptide.

3. optical probe as described in any one of the preceding claims, wherein the optical probe is to selected from alanine, dried meat ammonia The amino acid-sensitive of acid, valine, serine, isoleucine, threonine and cysteine.

4. optical probe as described in any one of the preceding claims, the optical probe includes prominent at following sites Become: F77, A100, T102, D121, Y150, D226, G227 and Y275, number correspond to the overall length of amino acid-sensitive polypeptide.

5. a kind of nucleic acid sequence encodes optical probe of any of claims 1-4.

6. a kind of expression vector, it includes the nucleic acid sequences as claimed in claim 5 being operatively connected with expression control sequence.

7. a kind of host cell, it includes described in claim 5 nucleic acid sequence or expression vector as claimed in claim 6.

8. a kind of method for preparing optical probe of any of claims 1-4, comprising the following steps:

(1) expression vector described in claim 5 is transferred in host cell,

(2) host cell is cultivated under conditions of being suitble to the expression vector to express, and

(3) optical probe is separated by the host cell.

9. optical probe of any of claims 1-4 or the optical probe of claim 8 the method preparation are being examined The application in amino acid in sample.

10. a kind of detection kit, it includes described in optical probe of any of claims 1-4 or claim 8 The optical probe of method preparation.