CN109666068B

CN109666068B - Proline optical probe and preparation method and application thereof

Info

Publication number: CN109666068B
Application number: CN201910149337.1A
Authority: CN
Inventors: 杨弋; 赵玉政; 李写; 徐磊; 王巧慧
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2019-02-28
Filing date: 2019-02-28
Publication date: 2022-07-29
Anticipated expiration: 2039-02-28
Also published as: CN109666068A

Abstract

The invention relates to a proline optical probe and a preparation method and application thereof. In one aspect, the invention relates to an optical probe comprising a proline-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or the functional variant thereof is located within the sequence of the proline-sensitive polypeptide or the functional variant thereof. The invention also relates to a preparation method of the probe and application of the probe in detecting proline.

Description

Proline optical probe and preparation method and application thereof

Technical Field

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

Background

Proline is one of 20 natural amino acids and 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 proline content in the plants is increased dramatically, and the mechanism of this phenomenon and its physiological action in plants are still not clearly studied. In mammalian cells, the physiological role of proline as an unnecessary amino acid has not been widely recognized at first, but it is only a raw material for protein synthesis and plays an important role in maintaining the skeleton structure of proteins due to its structural specificity. However, in recent years, with the discovery of the regulation effect of proline metabolic pathway on intracellular ROS, people gradually focus on the research of proline and its metabolic pathway in mammalian cells again.

Proline metabolism in mammals occurs primarily in the cell cytosol and mitochondria, primarily in that proline enters mitochondria via a transport protein on the mitochondria, and oxidative dehydrogenation of proline by a proline oxidase (POX/PRODH) on the inner membrane of mitochondria results in Δ 1-pyrroline-5-carboxylic acid (P5C), while P5C shuttles back to the cytosol and is converted back to proline by PYCR (P5C reductase) in the cytosol. This forms a cycle of proline metabolism. Δ 1-pyrroline-5-carboxylic acid (P5C) is able to form a tautomeric equilibrium with GSA (glutamate- γ -semialdehyde) in the cytoplasm, and finally GSA can be metabolized by dehydrogenation of P5CDH to glutamate and ornithine to urea, respectively, in the tricarboxylic acid cycle and ornithine transaminase. In general, the metabolic pathway formed by POX and PYCR (P5CDH) regulation of interconversion between P5C and proline between cytosol and mitochondria is called the proline cycle (Liu W et al, Biofactors.2012,38(6): 398-.

In recent years, it has been found that the Proline Oxidase (POX) gene can negatively regulate the growth of tumor cells by promoting cell cycle arrest, inducing cell differentiation, promoting apoptosis, etc., and expression abnormality of the gene is found in various tumors. The oxidative dehydrogenation of proline to P5C under the action of POX also consumes ADP to generate ATP and a free electron, which is accompanied by the generation of ROS (reactive oxygen species) that is important for the apoptosis, proliferation and cell cycle regulation during the transmission in the electron respiratory chain in mitochondria (Liu Y et al, Cancer Res.2009,69(16): 6414; 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-.

It is because proline has the above-mentioned important functions, the detection of proline content is also particularly important. Common methods for detecting proline include acid ninhydrin color (Chen et al, applied Environmental Microbiology 2006,72: 4001-.

However, these methods are not suitable for the study of living cells, and have many disadvantages: time-consuming sample processing procedures such as cell disruption, separation, extraction and purification, etc. are required; in situ, real-time, dynamic, high-throughput and high spatial-temporal resolution detection in living cells and subcellular organelles is not possible. There remains a need in the art for methods for the real-time, localized, quantitative, and high-throughput detection of proline both in and out of cells.

Disclosure of Invention

The invention aims to provide a probe and a method for real-time positioning, high-flux and quantitative detection of proline in cells.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a proline optical probe, comprising a proline-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or the functional variant thereof is located within the sequence of the proline-sensitive polypeptide or the functional variant thereof. The proline-sensitive polypeptide or functional variant thereof is divided into a first part and a second part by the optically active polypeptide or functional variant thereof. In one embodiment, the optical probe of the present invention is sensitive to proline.

The invention provides a proline optical probe, which comprises a proline sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is positioned in the sequence of the proline sensitive polypeptide B, and the proline sensitive polypeptide B is divided into a first part B1 and a second part B2 to form a probe structure of a B1-A-B2 formula.

In one embodiment, the proline-sensitive polypeptide comprises a proline-binding protein or a functional variant thereof. In one embodiment, the proline binding protein is derived from an Agrobacterium, such as Agrobacterium fabrum. In one embodiment, the proline binding protein is derived from Atu2422-GABA receptor protein or an analog thereof. In one embodiment, the proline binding protein comprises Atu2422-GABA receptor protein or a functional variant thereof. In one embodiment, the proline binding protein has the sequence shown in SEQ ID NO. 1 or a functional variant thereof, or a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity thereto.

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 (mKate as shown in SEQ ID NO:4 or 8, mcherry as shown in SEQ ID NO: 5), green fluorescent protein (cpGFP as shown in SEQ ID NO: 6), blue fluorescent protein (cpBFP as shown in SEQ ID NO: 7), apple red fluorescent protein (cpapple as shown in SEQ ID NO: 9). Preferably, the optically active polypeptide is cpYFP. In one embodiment, the fluorescent protein has the sequence shown in any one 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 invention may be any amino acid sequence of any length. In one embodiment, the optically active polypeptide is flanked by linkers of no more than 5 amino acids, e.g., linkers of 0, 1, 2, 3, 4 amino acids. In one embodiment, the linker is located at the N-terminus and/or C-terminus of the optically active polypeptide. In one embodiment, the optical probe of the present invention does not comprise a linker. In one embodiment, the optical probe is as follows: first part of proline-sensitive polypeptide B1-optically active polypeptide A-second part of proline-sensitive polypeptide B2.

In one embodiment, the optical probes of the present invention further comprise a localization sequence for localizing the probe to a particular organelle of a cell, for example.

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

In one embodiment, the optically active polypeptide is located at a site of the proline-sensitive polypeptide 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 the site expressed in the form of "X/Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids described in the numbers. For example, a position at position 117/118 indicates that the optically active polypeptide is located between amino acids 117 and 118 of the proline-sensitive polypeptide. If two numbers in the position indicated in the form "X/Y" are not consecutive integers, this indicates that the optically active polypeptide replaces an amino acid between the amino acids indicated by the numbers. For example, position 249/259 indicates that the optically active polypeptide replaces amino acids 250-258 of the proline-sensitive polypeptide. Preferably, the optically active polypeptide is located at the following positions of the proline-sensitive polypeptide: 120/121, 121/122, 121/123, 324/330, 325/330, and 326/330.

In an exemplary embodiment, the optical probe of the invention of formula B1-A-B2 can be a probe formed when cpYFP is located at 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 of Atu2422, as shown in SEQ ID NO: 10-15. In one embodiment, the optical probe of the invention has or consists of the sequence shown in SEQ ID NO 10-15.

The invention also provides proline sensitive polypeptides having one or more mutations, including proline binding proteins having one or more mutations. The amino acid mutation comprises modification, substitution, deletion or truncation of the sequence of the amino acid. In one embodiment, the mutation may be selected from mutations at positions F77, a100, T102, D121, Y150, D226, G227, and Y275 of the proline-sensitive polypeptide. Illustratively, the mutation is selected from the group consisting of F77, a100, T102, D121, D226, Y226, D150, Y226, Y150, Y226, Y150, Y226, Y150, Y226, Y150, Y226, Y227, Y150, Y226, Y227, Y150, Y227, Y226, Y150, Y226, Y227, Y150, Y226, Y150, Y226, Y227, Y226, Y150, Y226, Y150, Y226, Y150, 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 above mutation is selected from F77L, a100G, D121E, D121S, D121T, D121V, D226E, D226N and Y275F. In one embodiment, a proline sensitive polypeptide as described herein comprises a mutation at a position selected from the group consisting of: f77, a100, T102, D121, Y150, D226 and Y275.

The proline sensitive polypeptide (e.g. proline binding protein) in the optical probe of the invention may comprise one or more amino acid mutations. In some embodiments, the proline sensitive polypeptide in the optical probe comprises a proline binding protein having one or more mutations described herein. The mutation includes modification, substitution, deletion or truncation of amino acid. In one embodiment, the mutation is selected from the group consisting of mutations at positions F77, a100, T102, D121, Y150, D226, G227 and Y275 of the proline sensitive polypeptide. Illustratively, the mutation is selected from the group consisting of F77, a100, T102, D121, D226, Y226, D150, Y226, Y150, Y226, Y150, Y226, Y150, Y226, Y150, Y226, Y227, Y150, Y226, Y227, Y150, Y227, Y226, Y150, Y226, Y227, Y150, Y226, Y150, Y226, Y227, Y226, Y150, Y226, Y150, Y226, Y150, 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: F77L, a100G, D121E, D121S, D121T, D121V, D226E, D226N and Y275F.

In one embodiment, the optical probe of the invention comprises a proline-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or the functional variant thereof is located within the sequence of the proline-sensitive polypeptide or the functional variant thereof, and the proline-sensitive polypeptide comprises a mutation selected from the group consisting of: f77, a100, T102, D121, Y150, D226 and Y275, the numbering corresponding to the full length of the proline sensitive polypeptide. In the above embodiments, the proline-sensitive polypeptide may comprise a mutation at a position selected from the group consisting of: f77, a100, T102, D121, Y150, D226 and Y275.

In an exemplary embodiment, the optical probe of the invention B1-A-B2 can be a probe formed when 121/122 site of Atu2422 is fused with cpYFP and has F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N or Y275F mutation, as shown in SEQ ID NO: 16-24. In an exemplary embodiment, the optical probe of the invention has or consists of the sequence shown in SEQ ID NO 16-24.

The optical probe provided by the invention comprises any one of amino acid sequences SEQ ID NO 10-24 or a variant thereof. In one embodiment, the invention provides an optical probe comprising a sequence that has 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of amino acid sequences SEQ ID NO 10-24. In a preferred embodiment, the present invention provides an optical probe comprising a sequence substantially similar or identical to any of the amino acid sequences SEQ ID NO 10-24. In a more preferred embodiment, the optical probe provided by the present invention comprises or consists of SEQ ID NO 23.

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 additional polypeptides fused thereto. Other polypeptides described herein do not affect the properties of the optical probe. In some embodiments, the additional polypeptide is located at the N-terminus and/or C-terminus of the optical probe. In some embodiments, the additional polypeptides include polypeptides that localize the optical probe to a different organelle or subcellular organelle, a tag for purification, or a tag for immunoblotting. The fusion polypeptide described herein may have a linker between the optical probe and the other polypeptide.

Subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, golgi apparatus, lysosomes, peroxisomes, and the like. In some embodiments, the tag for purification or the tag for immunoblotting comprises 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 the complement thereof. In one embodiment, the invention provides a nucleic acid sequence encoding an amino acid sequence set forth in any one of SEQ ID NOs 10-24. In one embodiment, the nucleic acid sequence of the invention comprises any one of the nucleotide sequences SEQ ID NO 25-26 or a variant thereof. In a preferred embodiment, the invention provides a nucleic acid sequence comprising a sequence having 99%, 95%, 90%, 80%, 70% or 50% identity to any one of the nucleotide sequences SEQ ID NO 25-26. 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 NO 25-26.

The present invention also relates to the complement of the above-described nucleic acid sequence or a variant thereof, which may comprise the nucleic acid sequence encoding the fragment, analog, derivative, soluble fragment and variant of the optical probe or fusion protein of the present invention or a complementary sequence thereof.

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

The invention also provides an expression vector comprising a nucleic acid sequence of the invention, or a complement thereof, encoding an optical probe or fusion polypeptide of the invention, operably linked to an expression control sequence. 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, the prokaryotic expression vector is obtained, for example, by operably linking plasmid pRSETb to a nucleic acid sequence described herein. In some embodiments, the expression control sequence includes an origin of replication, a promoter, an enhancer, an operator, a terminator, a ribosome binding site.

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

The invention also provides a detection kit comprising a proline optical probe or fusion polypeptide as described herein or a proline optical probe or fusion polypeptide prepared as described herein. The kit detects proline.

The present invention provides a method of making an optical probe as described herein, comprising: providing a cell comprising a vector expressing an optical probe or fusion polypeptide as described herein, culturing said cell under conditions in which said cell expresses, and isolating the optical probe or fusion polypeptide.

In one embodiment, a method of making a proline optical probe or fusion polypeptide as described herein comprises: 1) transferring an expression vector encoding the proline optical probe described herein into a host cell; 2) culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the proline optical probe.

The invention also provides a method for detecting proline in a sample, comprising: contacting an optical probe or fusion polypeptide as described herein or prepared as described herein with a sample and detecting a change in the optically active polypeptide. The detection may be performed in vivo, in vitro, subcellular, or in situ. Such as blood.

Also provided herein is a method of quantifying proline in a sample, comprising: contacting the optical probe or fusion polypeptide described herein or prepared as described herein with a sample, detecting a change in the optically active polypeptide, and quantifying proline in the sample from the change in the optically active polypeptide.

The invention also provides a method of screening a compound (e.g. a drug) comprising: contacting an optical probe or fusion polypeptide as described herein or prepared by a method as described herein with a candidate compound, detecting a change in the optically active polypeptide, and screening the compound for a change in the optically active polypeptide. The method allows for high throughput screening of compounds.

The invention also provides the use of a proline optical probe or fusion polypeptide as described herein or a proline optical probe or fusion polypeptide prepared as described herein for real-time proline localisation.

The invention has the beneficial effects that: the proline optical probe provided by the invention is easy to mature, has large fluorescence dynamic change and good specificity, can be expressed in cells by a gene operation method, can be used for real-time positioning, high-flux and quantitative detection of proline inside and outside the cells, and saves a time-consuming sample processing step. The experimental effect shows that the highest response of the proline optical probe provided by the application to proline is more than 4 times of that of a control, and the proline optical probe can be used for positioning, qualitatively and quantitatively detecting cells in subcellular structures such as cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosome, Golgi body and the like, and can be used for high-flux compound screening and quantitative detection of proline in blood.

Drawings

The invention is further illustrated by the following figures and examples.

FIG. 1 is a SDS-PAGE pattern of an exemplary proline optical probe described in example 1;

fig. 2 is a graph of the change in proline response to proline by an exemplary proline optical probe comprising cpYFP and proline binding protein described in example 2;

fig. 3 is a graph of the change in proline response to an exemplary proline optical probe comprising cpGFP and a proline binding protein as described in example 3;

FIG. 4 is a graph of the change in proline response to proline by an exemplary proline optical probe comprising a cPBF and proline binding protein as described in example 4;

FIG. 5 is a graph of the change in proline response to proline by an exemplary proline optical probe comprising cpmApple and proline binding protein, described in example 5;

fig. 6 is a titration curve of various concentrations of proline with an exemplary proline optical probe fused to cpYFP at positions 120/121, 121/122, 121/123, 324/330, 325/330, or 326/330 of the proline binding protein as described in example 6;

fig. 7A is a bar graph of the specific detection of 20 amino acids by an exemplary proline optical probe fused to cpYFP at position 121/122 of the proline binding protein, described in example 6;

FIG. 7B is a bar graph of the specificity for proline of the probe with fluorescent protein at position 121/122 of the proline binding protein compared to a fusion protein obtained by fusing fluorescent protein to the N-or C-terminus of the proline binding protein, as described in example 6;

FIG. 8 is a bar graph of the response of an exemplary proline optical probe described in example 7 to proline fused to cpYFP at position 121/122 of the proline binding protein and having a mutation at position F77, A100, T102, D121, Y150, D226, G227, or Y275;

FIG. 9 is a plot of the fluorescence spectra properties of an exemplary proline optical probe described in example 8;

FIG. 10 is a titration curve of the exemplary proline optical probe described in example 8 for different concentrations of proline;

FIG. 11 is a bar graph of the specific detection of 20 amino acids by the exemplary proline optical probe described in example 8;

FIG. 12 is a photograph of subcellular organelle localization in mammalian cells of the exemplary proline optical probe described in example 9;

FIG. 13 is a schematic illustration of the dynamic monitoring of proline transmembrane transport of an exemplary proline optical probe within different subcellular organelles in mammalian cells, as described in example 9;

FIG. 14 is a dot plot of high throughput compound screening at the viable cell level by the exemplary proline optical probe described in example 10;

FIG. 15 is a histogram of the quantification of proline in mouse and human blood by the exemplary proline optical probe described in example 11.

Detailed Description

As used herein, the term "about," when referring to a value or range, means that the value or range is within 20%, within 10%, and within 5% of the given value or range.

As used herein, the terms "comprising," including, "and their equivalents include the meaning of" containing "and" consisting of … …, e.g., a composition that "comprises" X may consist of X alone or may contain other materials, e.g., X + Y.

The term "proline-sensitive polypeptide" or "proline-responsive polypeptide" as used herein refers to a polypeptide that responds to proline, including any response in the chemical, biological, electrical or physiological parameters of the polypeptide associated with the interaction of the sensitive polypeptide. Responses include small changes, e.g., changes in the orientation of amino acids or peptide fragments of the polypeptide and, for example, changes in the primary, secondary, or tertiary structure of the polypeptide, including, for example, protonation, electrochemical potential, and/or conformational changes. "conformation" is the three-dimensional arrangement of the primary, secondary and tertiary structures of a molecule comprising pendant groups in the molecule; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include a transition from an alpha-helix to a beta-sheet or a transition from a beta-sheet to an alpha-helix. It is understood that the detectable change need not be a conformational change, so long as the fluorescence of the fluorescent protein moiety is changed. The proline sensitive polypeptide described herein may also include functional variants thereof. Functional variants of proline-sensitive polypeptides include, but are not limited to, variants that can interact with proline to produce the same or similar changes as the parent proline-sensitive polypeptide.

The proline-sensitive polypeptide of the invention includes, but is not limited to, proline-binding proteins or variants thereof having more than 90% homology. The amino acid binding proteins of the invention may be derived from Agrobacterium, such as Agrobacterium fabrum. An exemplary amino acid binding protein Atu2422 of the invention is the ABC transporter family, consisting of two domains linked by three flexible amino acid peptide chains. The proline-binding protein can sense the change of the concentration of proline, and the spatial conformation of the proline-binding protein can be changed in the process of dynamic change of the concentration of proline.

The term "optical probe" as used herein refers to a proline sensitive polypeptide fused to an optically active polypeptide. The inventors have found that a conformational change in a proline-sensitive polypeptide such as a proline-binding protein, upon binding of a physiological concentration of proline, specifically causes a conformational change in an optically active polypeptide (e.g. a fluorescent protein), which in turn results in a change in the optical properties of the optically active polypeptide. The presence and/or level of proline can be detected and analyzed by plotting a standard curve with the fluorescence of the fluorescent protein measured at different proline concentrations. An exemplary Atu2422 protein is shown in SEQ ID NO: 1. When describing the optical probe of the present invention (e.g., when describing the site or mutation site where the optically active polypeptide is located), the amino acid residue numbering referred to is with reference to SEQ ID NO: 1. However, the skilled person is aware of the corresponding residue numbering of other similar proline binding proteins.

In the optical probe of the present invention, an optically active polypeptide (e.g., a fluorescent protein) is operably fused to a proline sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide that has the ability to emit fluorescence. Fluorescence is an optical property of optically active polypeptides that can be used as a means to detect the responsiveness of the optical probes of the invention. As used herein, the term "fluorescence properties" refers to molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum efficiency, shape of excitation spectrum or emission spectrum, excitation wavelength maximum and emission wavelength maximum, amplitude of excitation at two different wavelengths, ratio of emission amplitudes at two different wavelengths, excited state lifetime, or fluorescence anisotropy. 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. The measurable difference can be determined by determining the amount of any quantitative fluorescent property, for example, the amount of fluorescence at a particular wavelength or the integral of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have a fluorescence characteristic that is readily distinguishable between 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 can undergo a change in the same or similar fluorescent property as a parent optically-active polypeptide.

"linker" or "connecting region" refers to an amino acid or nucleotide sequence that connects two moieties in a polypeptide, protein, or nucleic acid of the invention. Illustratively, the number of amino acids at the amino terminus of the connecting region of the proline-sensitive polypeptide and the optically active polypeptide of the invention is selected to be 0-3, and the number of amino acids at the carboxy terminus is selected to be 0-2; when the recombinant optical probe is linked as a basic unit to a functional protein, it may be fused to the amino acid or carboxyl terminus of the recombinant optical probe. The linker sequence may be a short peptide chain of one or more flexible amino acids.

As used herein, the terms "chromophore", "fluorophore" and "fluorescent protein" are synonymous and refer to a protein that fluoresces under excitation light. Fluorescent proteins are used as basic detection means in the field of bioscience, such as green fluorescent protein GFP commonly used in the field of biotechnology, and circularly rearranged blue fluorescent protein (cpBFP), circularly rearranged green fluorescent protein (cpGFP), circularly rearranged yellow fluorescent protein (cpYFP), and the like, which are derived from mutation of the protein; there are also the red fluorescent proteins RFP commonly used in the art, and circularly rearranged proteins derived from this protein, such as cpmApple, cpmOrange, cpmKate, etc. The sequence of an exemplary fluorescent protein is shown in any one of SEQ ID NOs 2-9.

The green fluorescent protein GFP was originally extracted from Victoria luminifera (Aequorea Victoria), and was composed of 238 amino acids and had a molecular weight of approximately 26 kDa. GFP is a unique barrel-shaped structure formed by 12 beta-pleated strands, and a chromogenic tripeptide (Ser65-Tyr66-Gly67) is wrapped in the GFP. When in the presence of oxygen, it spontaneously forms a chromophore structure of p-hydroxybenzylideneimidazolidinone to generate fluorescence. GFP does not require a cofactor for fluorescence, and fluorescence is very stable and a good imaging tool. GFP has two excitation peaks, the main peak at 395nm can generate 508nm emission, and the excitation light irradiation at the shoulder 475nm can generate 503nm emission. Exemplary cpGFP is shown in SEQ ID NO 6

The yellow fluorescent protein YFP is derived from green fluorescent protein GFP, the homology of the amino acid sequence of the yellow fluorescent protein YFP with GFP is more than 90 percent, and the key change of YFP compared with GFP is that the 203 th amino acid is mutated from threonine to tyrosine (T203Y). The wavelength red of the primary excitation peak of YFP was shifted to 514nm and the emission wavelength was changed to 527nm compared to the original AvGFP. On the basis, the fluorescence enhanced yellow fluorescent protein EYFP can be obtained by carrying out site-directed mutagenesis on the 65 th amino acid of YFP (S65T). The cpYFP is obtained by connecting the original N end and C end of GFP through a section of flexible short peptide chain, manufacturing a new N end and C end at the position of a near chromophore of the original GFP, taking the amino acid part at the positions 145-238 th position as the N end of a new protein, taking the amino acid at the positions 1-144 th position as the C end of the new protein, and connecting 5-9 sections of flexible short peptide chain. In the present invention, the proximal chromophore position is preferably at amino acids Y144 and N145; the short peptide chain with flexibility is preferably VDGGSGGTG or GGSGG. The sequence of an exemplary cpYFP is shown in SEQ ID NO 2.

The red fluorescent protein RFP is originally extracted from coral in the sea, the wild RFP is oligomeric protein which is not beneficial to the fusion expression of organisms, and then the red fluorescent protein with different color bands is further derived on the basis of the RFP, wherein the most common is mCheerry, mKate and the like. Exemplary cpmKate is shown in SEQ ID NOs 4 or 8. An exemplary mCherry is shown in SEQ ID NO 5.

In other embodiments, the fluorescent protein can also be one or more of a blue fluorescent protein cppBFP with an amino acid sequence shown in SEQ ID NO. 7, an orange fluorescent protein cpmOrange with an amino acid sequence shown in SEQ ID NO. 3, and an apple red fluorescent protein cpmApple with an amino acid sequence shown in SEQ ID NO. 9.

The proline optical probe of the invention comprises a proline-sensitive polypeptide B, such as a proline-binding protein or a variant thereof, and an optically active polypeptide A, such as a fluorescent protein. The optically active polypeptide A is inserted into a proline sensitive polypeptide B, and the B is divided into two parts, namely B1 and B2, so that a probe structure of a B1-A-B2 formula is formed; the interaction between the proline-sensitive polypeptide B and proline results in an increased optical signal of the optically active polypeptide A.

In the optical probe of the present invention, the optically active polypeptide may be located at or fused to any position of the proline-sensitive polypeptide. In one embodiment, the optically active polypeptide is positioned in the N-C direction anywhere on the proline sensitive polypeptide in the N-C direction. Specifically, the optically active polypeptide is located in a flexible region of the proline-sensitive polypeptide, wherein the flexible region refers to specific structures such as a ring-shaped domain existing in a higher-order structure of a protein, the specific structures have higher mobility and flexibility compared with other higher-order structures of the protein, and the flexible region can dynamically change the spatial structure conformation after the protein is combined with a ligand. The flexible region in the invention mainly refers to the region where the fusion site in the proline 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 site in the amino acid sequence of the proline 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 proline binding protein. Shown as SEQ ID NO 10-15.

The term "variant" or "mutant" as used herein in reference to a polypeptide or protein includes variants having the same function as the polypeptide or protein, but differing in sequence. These variants include, but are not limited to: a sequence obtained by deleting, inserting and/or substituting one or more (usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 5) amino acids in the sequence of the polypeptide or protein, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the carboxyl terminal and/or the amino terminal thereof. Without wishing to be bound by theory, amino acid residues are changed without changing the overall configuration and function of the polypeptide or protein, i.e., function conservative mutations. For example, in the art, substitutions with amino acids having similar or analogous properties will not generally alter the function of the polypeptide or protein. Amino acids with similar properties are often referred to in the art as families of amino acids with similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Also, for example, the addition of one or more amino acids at the amino-and/or carboxy-terminus will not generally alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of genetically coded amino acids. It is well known to those skilled in the art that in gene cloning procedures, it is often necessary to design appropriate cleavage sites, which may introduce one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. Also for example, to construct a fusion protein, to facilitate expression of a recombinant protein, to obtain a recombinant protein that is automatically secreted outside of a host cell, or to facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, for example, including, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, glutathione S-transferase (GST), maltose E binding protein, protein a, a tag such as 6His or Flag, or a proteolytic enzyme site for factor Xa or thrombin or enterokinase. Variants of a polypeptide or protein may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants may further comprise a polypeptide or protein having 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 99%, or 100% sequence identity to the polypeptide or protein.

The optical probe of the present invention may comprise a proline sensitive polypeptide having a mutation. The mutation is, for example, a mutation at a site selected from among F77, A100, T102, D121, Y150, D226, G227 and Y275. Illustratively, the mutation is selected from the group consisting of F77, a100, T102, D121, D226, Y227, Y226, Y150, Y227, Y150, Y227, Y150, 226, Y150, Y227, Y150, 226, Y150, Y227, 226, Y150, Y227, Y150, 226, Y227, Y150, 226, Y150, a 227, 226, Y150, Y227, Y150, a 227, 226, a 227, Y150, a 227, a 226, Y150, a 226, a 227, a 226, a 227, a100, a 226, 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 F77L, a100G, D121E, D121S, D121T, D121V, D226E, D226N, and Y275F.

In an exemplary embodiment, the optical probe of the invention, B1-A-B2, can be a probe formed when 121/122 site of Atu2422 is fused with cpYFP and has mutation selected from F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N and Y275F, as shown in SEQ ID NO: 16-24.

The optical probe provided by the invention comprises any one of amino acid sequences SEQ ID NO 10-24 or a variant thereof. In one embodiment, the invention provides an optical probe comprising a sequence that has 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of amino acid sequences SEQ ID NO 10-24. 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 NO 10-24. In a more preferred embodiment, the optical probe provided by the present invention comprises or consists of SEQ ID NO 23.

The term "identity" or "percent identity" in two or more polypeptide or nucleic acid molecule sequences refers to two or more sequences or subsequences that are the same or wherein a percentage of amino acid residues or nucleotides are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) over a window of comparison or designated region, when compared and aligned for maximum correspondence by manual alignment and visual inspection using methods known in the art, e.g., sequence comparison algorithms. 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, respectively.

The terms "functional variant", "derivative" and "analogue" as used herein refer to a protein that retains substantially the same biological function or activity as the original polypeptide or protein (e.g., proline-binding protein or fluorescent protein). A functional variant, derivative or analogue of a polypeptide or protein of the invention (e.g. a proline-binding protein or a fluorescent protein) may be (i) a protein in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein having a substituent group in one or more amino acid residues, or (iii) a protein in which the mature protein is fused to another compound (such as a compound that extends the half-life of the protein, e.g. polyethylene glycol), or (iv) a protein in which an additional amino acid sequence is fused to the sequence of the protein (such as a secretory sequence or a sequence or proprotein sequence used to purify the protein, or a fusion protein with an antigenic IgG fragment). Such functional variants, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

The analog may differ from the original polypeptide or protein by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques.

The analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proline-sensitive polypeptides of the invention are not limited to the representative proteins, variants, derivatives and analogs listed above. Modified (generally without altering 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 proteins that result from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

The invention also provides a preparation method of the proline optical probe, which comprises the following steps: 1) incorporating a nucleic acid sequence encoding a proline optical probe as described herein into an expression vector; 2) transferring the expression vector into a host cell; 2) culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the proline optical probe.

The term "nucleic acid" or "nucleotide" as used herein may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The term "variant" as used herein in reference to a nucleic acid may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substituted variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the protein encoded thereby. A nucleic acid of the invention can comprise a nucleotide sequence having 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 99%, or 100% sequence identity to the nucleic acid sequence. The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR).

The full-length sequence or a fragment thereof of the optical probe or fusion protein of the present invention can be obtained by PCR amplification, artificial synthesis, or recombinant methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, and the relevant sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the nucleotide sequence is more than 2500bp, 2-6 times of PCR amplification are preferably carried out, and then the amplified fragments are spliced together according to the correct sequence. The PCR amplification procedure and system of the present invention is not particularly limited, and conventional PCR amplification procedures and systems in the art may be used. The sequences of interest can also be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating and purifying the relevant polypeptide or protein from the proliferated host cells by conventional methods. In addition, the sequence can be synthesized by artificial synthesis, 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. The artificial synthesis method is a conventional artificial synthesis method of DNA in the field, and has no other special requirements. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, DNA sequences encoding the proteins of the invention (or functional variants, derivatives or analogues thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (e.g., 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.

After obtaining the nucleotide sequence for coding the optical probe, the invention brings the nucleotide sequence for coding the optical probe into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" are used interchangeably herein 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 adenoviruses, retroviruses, or other vectors, which are capable of replication and stable expression in a host, and one important feature of these recombinant vectors is that they typically contain expression control sequences. The term "expression control sequence" as used herein refers to an element which can be operably linked to a gene of interest to control transcription, translation and expression of the gene of interest, and may be an origin of replication, a promoter, a marker gene or a translation control element, including enhancers, operators, terminators, ribosome binding sites, and the like, and the choice of expression control sequence 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 the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. Those skilled in the art are familiar with methods which can be used to construct expression vectors containing the coding sequences of the fusion proteins of the present invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTR of retrovirus, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector may be a commercially available pRSETb vector without any other special requirement. Illustratively, the nucleotide sequence encoding the optical probe and the expression vector are subjected to double enzyme digestion by BamHI and EcoRI respectively, and then enzyme digestion products of the nucleotide sequence and the expression vector are connected to obtain the recombinant expression vector. The invention has no special restriction on the specific steps and parameters of enzyme digestion and connection, and the conventional steps and parameters in the field can be adopted.

After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including the fusion protein. Such transfer procedures may be carried out by conventional techniques known to those skilled in the art, such as transformation or transfection. The host cell of the invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, is a place for recombinant gene amplification, and an ideal receptor cell should meet two conditions of easy acquisition and proliferation. The "host cells" of the present invention may include prokaryotic and eukaryotic cells, including in particular bacterial cells, yeast cells, insect cells and mammalian cells. Specific examples thereof include bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, and the like, including but not limited to those host cells described above. The host cell is preferably a variety of cells that facilitate expression or fermentative production of the gene product, such cells being 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 one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

The methods of transfer to host cells described herein are conventional in the art and include calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemically mediated transfer, or electroporation. When the host is a prokaryote such as E.coli, the method is preferably CaCl ₂ Method or MgCl ₂ Methods, the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

After the expression vector is transferred into the host cell, the host cell transferred into the expression vector is subjected to amplification expression culture, and the proline optical probe is obtained by separation. The host cell is amplified and expressed by a conventional method. The medium used in the culture may be various conventional media depending on the kind of the host cell used. The culturing is performed under conditions suitable for growth of the host cell.

In the present invention, the optical probe is expressed in a cell, on a cell membrane, or secreted out of the cell. If desired, the recombinant protein can be isolated or purified by various separation methods using its physical, chemical and other properties. The method for separating the proline fluorescent protein is not particularly limited in the invention, and a conventional method for separating the fusion protein in the field can be adopted. Such methods are well known to those skilled in the art and include, but are not limited to: conventional renaturation treatment, salting-out method, centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof. In one embodiment, the isolation of the optical probes is performed using His-tagged affinity chromatography.

The invention also provides application of the proline optical probe in real-time positioning, quantitative detection and high-throughput compound screening of proline. In one aspect, the proline optical probe is preferably connected with signal peptides at different parts of a cell, transferred into the cell, and used for carrying out real-time positioning on proline by detecting the intensity of a fluorescent signal in the cell; and (4) carrying out quantitative detection on the corresponding proline through a proline standard dropping curve. The proline standard dripping curve is drawn according to fluorescence signals of a proline optical probe under the conditions of different concentrations of proline. The proline optical probe is directly transferred into cells, and a time-consuming sample processing process is not needed in the real-time positioning and quantitative detection process of proline, so that the proline optical probe is more accurate. When the proline optical probe is used for screening high-throughput compounds, different compounds are added into a cell culture solution, and the change of the proline content is measured, so that the compounds which have influence on the change of the proline content are screened. The application of the proline optical probe in real-time proline localization, quantitative detection and high-throughput compound screening is not diagnosis and treatment purposes and does not relate to diagnosis and treatment of diseases.

Concentrations, amounts, percentages, and other numerical values may be expressed herein in terms of ranges. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include the numerical values explicitly recited as the limits of the range, as well as to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

Examples

The proline optical probe provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

I. Test materials and reagents

In the examples, the conventional molecular biological cloning methods of genetic engineering and cell culture and imaging methods are mainly used, and these methods are well known to those skilled in the art, for example: briefly, Rous Kames et al, handbook of molecular biology laboratory references, J. SammBruk, D.W. Lassel, Huang Pentang et al: molecular cloning guidelines (third edition, 8 months 2002, published by scientific Press, Beijing); animal cell culture basic technical guidance (fifth edition), chapter calm, slow-release bolt, and so on, of Feremenoni et al; J.S. Bonifis Nong, M. Dasuo et al, eds of cell biology laboratory Manual, chapter Silent et al. Those of ordinary skill in the art will readily appreciate that modifications and variations may be made to the present invention as described in the following examples, and that such modifications and variations are within the scope of the claims of the present application.

The pRSETb-cpYFP, pRSETb-proline binding protein-based plasmid used in the examples was constructed by the protein laboratory of the university of east China's university and the pRSETb plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified correctly by Mass Spectrometry by Shanghai Czeri bioengineering technology, Inc. The expression plasmids constructed in the examples were subjected to sequencing, which was performed by Huada Gene Co and Jelie sequencing Co. Taq DNA polymerase used in each example was purchased from Dongpeng organisms, pfu DNA polymerase was purchased from Tiangen Biochemical technology (Beijing) Ltd, and primeSTAR DNA polymerase was purchased from TaKaRa, and the three polymerases were purchased with the corresponding polymerase buffer and dNTP. Restriction enzymes such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, and T4 phosphorylase (T4PNK) were purchased from Fermentas, and supplied with buffers. The transfection reagent Lip2000Kit was purchased from Invitrogen. Amino acids such as proline were purchased from Sigma. Unless otherwise stated, chemicals such as inorganic salts were purchased from Sigma-Aldrich. HEPES salts, ampicillin (Amp) and puromycin were purchased from Ameresco. A96-well detection blackboard and a 384-well fluorescence detection blackboard are purchased from Grenier company.

The DNA purification kit used in the examples was purchased from BBI (Canada) and the general plasmid minipump kit was purchased from Tiangen Biochemical technology (Beijing) Ltd. The clone strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and the desalting column packing were both 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 desk-top high-speed refrigerated centrifuge (Beckman, USA), PCR amplification instrument (Biometra, Germany), ultrasonication instrument (Ningbo Xinzhi Co.), nucleic acid electrophoresis instrument (Shenneng Bo Co.), fluorescence spectrophotometer (Varian, USA), CO ₂ Isothermal cell culture chamber (SANYO), inverted fluorescence microscope (japan nikon).

Methods of molecular biology and cell experiments

II.1 Polymerase Chain Reaction (PCR):

1. and (3) target fragment amplification PCR:

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

TABLE 3 Long fragment (>2500bp) amplification PCR reaction System

TABLE 4 Long fragment (>2500bp) amplification PCR amplification procedure

TABLE 5 Long fragment (>2500bp) amplification PCR amplification procedure

II.2 endonuclease cleavage reaction:

the system of double digestion of the plasmid vector is shown in Table 6, where n represents the amount of sterilized ultrapure water μ L to be added to bring the system to the total volume.

TABLE 6 plasmid vector Dual enzyme digestion System

II.3 phosphorylation of the 5' end of the DNA fragment

The ends of plasmids or genomes extracted from microorganisms contain phosphate groups, and PCR products do not contain phosphate groups, so that phosphate group addition reaction is needed to be carried out on 5' end bases of the PCR products, and only DNA molecules with phosphate groups at the ends can carry out ligation reaction. The phosphorylation reaction system is shown in Table 7, wherein T4PNK is abbreviated as T4 polynucleotide kinase, and is used for addition reaction to the 5' end phosphate group of DNA molecule.

TABLE 7 phosphorylation reaction System

II.4 ligation of the fragment of interest and the vector

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

1. Blunt-ended short fragment and blunt-ended ligation of linearized vector

The principle of the method is that after the blunt end product obtained by PCR is used for phosphorylation reaction on the 5' end of a DNA fragment under the action of T4PNK, the blunt end product is connected with a linearized vector under the action of PEG4000 and T4DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is shown in Table 8.

TABLE 8 blunt-ended fragment ligation reaction System

2. Ligation of DNA fragment containing cohesive Ends and vector fragment containing cohesive Ends

DNA fragments cut by restriction endonucleases will generally produce overhanging sticky ends and can therefore be ligated with sticky end vector fragments containing sequence complementarity 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 approximately between 2:1 and 6: 1. .

TABLE 9 cohesive end-joining reaction System

3. Ligation reaction of 5' end phosphorylated DNA fragment product self cyclization after introduction of site-directed mutagenesis by inverse PCR

And (3) carrying out self-cyclization ligation on the DNA fragment with 5 ' end phosphorylation to carry out ligation reaction on the 3 ' end and the 5 ' end of the linearized vector to obtain the recombinant plasmid. The self-cyclized ligation reaction system is shown in Table 10.

TABLE 10 self-cyclizing ligation reaction System

II.5 preparation and transformation of competent cells

Preparation of competent cells:

1. a single colony (e.g., Mach1) was picked and inoculated into 5mL LB medium and shaken overnight at 37 ℃.

2. 0.5-1mL of overnight-cultured broth was transferred to 50mL of LB medium and cultured at 37 ℃ and 220rpm for 3 to 5 hours until OD600 reached 0.5.

3. Cells were pre-cooled in an ice bath for 2 hours.

Centrifuge at 4000rpm for 10 minutes at 4.4 ℃.

5. Discard the supernatant, resuspend the cells with 5mL of pre-cooled buffer, add resuspension buffer until the final volume is 50mL after homogenization.

6. Ice-bath for 45 min.

Centrifugation at 4000rpm for 10 minutes at 7.4 ℃ resuspended the bacteria with 5mL of ice-chilled storage buffer.

8. Each EP tube was filled with 100. mu.L of the bacterial solution and frozen at-80 ℃ or with liquid nitrogen.

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

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

Transformation of competent cells:

1. 100 μ L of competent cells were thawed on an ice bath.

2. The appropriate volume of ligation product was added, gently whipped and mixed, and ice-cooled for 30 minutes. The ligation product is typically added in a volume less than 1/10 the volume of competent cells.

3. The bacterial liquid is put into a water bath with the temperature of 42 ℃ for 90 seconds through heat shock, and is quickly transferred into an ice bath for 5 minutes.

4. 500. mu.L of LB was added and incubated at 37 ℃ for 1 hour on a constant temperature shaker at 200 rpm.

5. The bacterial liquid is centrifuged for 3 minutes at 4000rpm, 200 mul of supernatant is left to evenly blow the thalli, the thalli are evenly coated on the surface of an agar plate containing proper antibiotics, and the plate is placed in a constant temperature incubator at 37 ℃ overnight.

II.6 expression, purification and fluorescence detection of proteins

1. An expression vector (e.g., a proline optical probe expression vector based on pRSETb) was transformed into JM109(DE3) cells, cultured overnight in an inverted state, picked from the plate and cloned into a 250ml Erlenmeyer flask, placed in a shaker at 37 ℃ and cultured at 220rpm until OD becomes 0.4-0.8, added with IPTG (1M) at 1/1000(v/v), and induced at 18 ℃ for 24-36 hours.

2. After induction expression is finished, centrifuging at 4000rpm for 30 minutes to collect bacteria, adding 50mM phosphate buffer solution to resuspend the bacteria sediment, and carrying out ultrasonic disruption until the bacteria are clear. 9600rpm, and centrifugation at 4 ℃ for 20 minutes.

3. The centrifuged supernatant is purified by a self-contained nickel column affinity chromatography column to obtain protein, and the protein after the nickel column affinity chromatography is subjected to a self-contained desalting column to obtain the protein dissolved in 20mM MOPS buffer (pH 7.4) or phosphate buffer PBS.

4. After SDS-PAGE identification of the purified proteins, the probes were diluted to a final concentration of 5-10. mu.M protein solution using assay buffer (100mM HEPES, 100mM NaCl, pH 7.3) or phosphate buffered saline PBS. Proline was formulated as a stock solution with a final concentration of 1M in assay buffer (20mM MOPS, pH 7.4) or phosphate buffered saline PBS.

5. 100 mul of 5 MuM protein solution is taken, incubated for 5 minutes at 37 ℃, added with proline respectively and mixed evenly until the final concentration is 100mM, and the light absorption of the protein under 340nm is measured by a multifunctional fluorescence microplate reader.

6. 100 μ l of 1 μ M protein solution was incubated at 37 ℃ for 5 minutes, and then titrated by adding proline to measure the fluorescence intensity emitted at 528nm after 485nm fluorescence excitation of the protein. The fluorescence excitation and emission measurement of the sample are completed by using a multifunctional fluorescence microplate reader.

7. Mu.l of a 1. mu.M protein solution was incubated at 37 ℃ for 5 minutes, proline was added, and the absorption spectrum and fluorescence spectrum of the protein were measured. The measurement of the absorption spectrum and the fluorescence spectrum of the sample is performed by a spectrophotometer and a fluorescence spectrophotometer.

II.7 transfection and fluorescence detection of mammalian cells

1. pCDNA3.1+ -based proline optical probe plasmid was transfected into HeLa by the transfection reagent Lipofectamine2000(Invitrogen) and placed at 37 ℃ with 5% CO ₂ Cultured in a cell culture box. And carrying out fluorescence detection after the exogenous gene is fully expressed for 24-36 h.

2. After the induction expression is finished, the adherent HeLa cells are washed three times by PBS and placed in HBSS solution for detection by a fluorescence microscope and a microplate reader respectively.

Example 1: proline binding protein plasmid

The proline binding protein (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, while the pRSETb vector was subjected to the corresponding double digestion. After ligation with T4DNA ligase, MachI was transformed with the product, and the transformed MachI was plated on LB plates (ampicillin 100ug/mL) and incubated overnight at 37 ℃. After plasmid extraction of the MachI transformant, PCR identification is carried out. And (4) carrying out subsequent plasmid construction after the positive plasmid is sequenced correctly.

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

In this example, the following site fusion cpYFP was selected based on pRSETb-Atu2422 to obtain the corresponding pRSETb-Atu2422-cpYFP plasmid: 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.

Generating a DNA fragment of cpYFP by utilizing PCR, inactivating the DNA fragment after using a phosphorization operation at the 5 'terminal, generating pRSETb-proline binding protein linearized vectors containing different fracture sites by reverse PCR amplification, connecting the linearized pRSETb-Atu2422 and the cpYFP fragment phosphorylated at the 5' terminal under the action of PEG4000 and T4DNA ligase to generate recombinant plasmids, placing the plates in a Kodak multifunctional living body imaging system, selecting a clone with yellow fluorescence under the excitation of a FITC channel, and completing sequencing by Shanghai branch company of great Gene science and technology Limited in Heixinghua Beijing.

After the sequencing was completed, the recombinant plasmid was transformed into JM109(DE3) to induce expression, and the protein was purified and electrophoresed to have a size of about 68Kda by SDS-PAGE. The size of the fusion protein is consistent with the size of Atu2422-cpYFP fusion protein containing His-tag purification label expressed by pRSETb-Atu 2422-cpYFP. The results are shown in FIG. 1.

The purified Atu2422-cpYFP fusion protein was subjected to proline response screening, and the detection signal of the fusion fluorescent protein containing 100mM proline was divided by the detection signal of the fusion fluorescent protein without proline. As shown in FIG. 2, the results of the assay showed that there were optical probes that responded to proline by more than 2-fold at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 (as shown in SEQ ID NO 10-15) or corresponding amino acid positions of the family proteins.

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

The proline green fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpGFP as in example 2. As shown in FIG. 3, the results of the examination revealed that there were optical probes which responded to proline by more than 2 times, and those fused at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 or the corresponding amino acid positions of the family proteins thereof.

Example 4: expression and detection of cppBFP optical probes at different fusion sites

The proline blue fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpBFP as in example 2. As shown in FIG. 4, the results of the examination revealed that there were optical probes which responded to proline by more than 2 times, and those fused at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 or the corresponding amino acid positions of the family proteins thereof.

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

The proline red fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpmpample as in example 2. As shown in FIG. 5, the results of the examination revealed that there were optical probes which responded to proline by more than 2 times, and those fused at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 or the corresponding amino acid positions of the family proteins thereof.

Example 6: performance of optical probe

For the optical probes obtained in example 2, which responded to proline by more than 2 times, i.e., 6 optical probes fused at sites 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330, concentration-gradient proline detection was performed, and changes in the ratio of fluorescence intensity at 528nm excitation at 420nm and fluorescence intensity at 528nm excitation at 485nm were detected. K for 6 proline optical probes with fusion sites 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 _d (binding constant) was 18.6mM, 0.2mM, 15.5mM, 0.95mM, 0.38mM and 0.65mM, respectively, and the ranges of variation were 3.0-fold, 4.5-fold, 2.0-fold, 2.9-fold, 2.6-fold and 2.0-fold, respectively, as shown in FIG. 6.

For the probe (Atu2422-121/122-cpYFP) with the fusion site of 121/122, specific detection of each amino acid was performed. Meanwhile, a fusion protein obtained by fusing a fluorescent protein cpYFP, cpGFP, cppBFP or cpmApple to the N-terminal or C-terminal of Atu2422 was used as a control, and the specificity of the probe having a fusion site of 121/122 for proline was compared.

The results showed that the probe with the fusion site of 121/122 had higher responses to all of alanine, proline, valine, serine, threonine, and cysteine, which were 4.3-fold, 4.5-fold, 4.0-fold, 3.2-fold, 2.4-fold, and 3.0-fold, respectively, as shown in FIG. 7A. Meanwhile, the fusion protein obtained by fusing the fluorescent protein to the N-terminal or C-terminal of Atu2422 did not respond to proline, while the probe with the fusion site 121/122 responded to proline by about 4.5 times, as shown in FIG. 7B.

Example 7: expression and detection of mutated cpYFP optical probes

An optical probe mutant is constructed on the basis of Atu 2422-121/122-cpYFP. The plasmid pRSETb-Atu2422-121/122-cpYFP is linearized by inverse PCR, a primer contains a base sequence of a site to be mutated, the obtained PCR product is added with phosphorus under the action of PNK, T4DNA ligase and PEG4000 for connection to obtain 8 sites of site-specific saturated mutation plasmids of F77, A100, T102, D121, Y150, D226, G227 and Y275, and sequencing is completed by Shanghai Branch of great Gene science and technology Co., Ltd, Heixian, Beijing.

The results are shown in FIG. 8. Fluorescence detection results show that F77L, A100G, D121E, D121S, D121T, D121V, D226E, D226N and Y275F mutants respond more than 2 times to proline.

EXAMPLE 8 Performance of optical Probe mutants

Illustratively, the fluorescence spectra were detected using a fluorescence spectrophotometer after subjecting purified proline optical probe Atu2422-121/122-cpYFP-D226N to 0mM and 500mM proline treatment for 10 minutes, respectively.

Measurement of excitation spectrum: the excitation spectrum was recorded with an excitation range of 350nm to 510nm and an emission wavelength of 530nm, read every 1 nm. The results showed that the probe had two excitation peaks at about 420 and 490nm, as shown in FIG. 9A.

Measurement of emission spectra: the fixed excitation wavelengths were 420nm and 490nm, respectively, and an emission spectrum of 505 and 600nm was recorded, read every 1 nm. The results show that the fluorescence intensity of the probe after adding 500mM proline is reduced to 1.2 times of that of the probe after adding 0mM proline under the excitation of 420 nm; the fluorescence intensity at 490nm excitation decreased 0.23 times that of the addition of 0mM proline. As shown in fig. 9B and 9C.

And carrying out proline detection on the purified Atu2422-121/122-cpYFP-D226N in a concentration gradient (0-100 mM). After 10 minutes of treatment of the purified probe, the change in the ratio of the fluorescence intensity at 528nm excitation at 420nm to the fluorescence intensity at 528nm excitation at 485nm was detected. The results are shown in FIG. 10, K for the proline optical probe _d (binding constant) 2.8mM, 5-fold amplitude of change.

Atu2422-121/122-cpYFP-D226N and 20 amino acids are subjected to reactivity detection, and the results show that the specificity is good, as shown in FIG. 11.

Example 9: subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

In this example, different localization signal peptides were used to fuse optical probe Atu2422-121/122-cpYFP-D226N to localize the optical probe to different organelles.

After HeLa cells were transfected with optical probe plasmids fused with different localization signal peptides for 36 hours, they were washed with PBS and placed in HBSS solution for fluorescence detection under FITC channel using an inverted fluorescence microscope. The results are shown in FIG. 12. The proline optical probe can be localized to subcellular organelles including the cytoplasm, mitochondria, nucleus, golgi apparatus, peroxisomes and lysosomes by fusing with different specific localization signal peptides. Fluorescence is shown in different subcellular structures and the distribution and intensity of fluorescence varies.

HeLa cells were transfected with the cytosolic-expressed optical probe plasmid for 36 hours, washed with PBS, and placed in HBSS solution to detect changes in the ratio of fluorescence intensity at 528nm excitation at 420nm to fluorescence intensity at 528nm excitation at 485nm over a 40min period. The results are shown in FIG. 13. 10mM proline was added and the assay was continued for 20 minutes. The 485/420 for the proline added sample increased gradually up to 2.3 fold, while 485/420 for the control without proline was 1.

Example 10: high throughput compound screening in living cells based on optical probes

In this example, we used HeLa cells that expressed cytosolic expression Atu2422-121/122-cpYFP-D226N for high-throughput compound screening.

Transfected HeLa cells were washed with PBS, treated in HBSS solution (without proline) for 1 hour, and then treated with 10. mu.M of the compound for 1 hour. Proline was added dropwise to each sample. The change of the ratio of the fluorescence intensity at 528nm excitation of 420nm to the fluorescence intensity at 528nm excitation of 485nm was recorded by a microplate reader. Samples not treated with any compound were used as controls for normalization. The results are shown in FIG. 14. Of the 2000 compounds used, the vast majority of compounds had minimal effect on proline entry into cells. 14 compounds can improve the proline uptake capacity of cells, and 10 compounds can obviously reduce the proline uptake of cells.

Example 11 quantitative determination of proline in blood with an optical Probe

In this example, proline in mouse and human blood supernatants was analyzed using purified Atu 2422-121/122-cpYFP-D226N.

Atu2422-121/122-cpYFP-D226N and diluted blood supernatant are mixed and treated for 10 minutes, and the ratio of fluorescence intensity at 528nm excitation of 420nm to fluorescence intensity at 528nm excitation of 485nm is detected by using a microplate reader. As shown in FIG. 15, the proline content in the blood of the mouse was about 140. mu.M, and the proline content in the blood of the human was about 160. mu.M.

The proline optical probe provided by the invention has the advantages that the protein molecular weight is relatively small and easy to mature, the fluorescence dynamic change is large, the specificity is good, the proline optical probe can be expressed in cells by a gene operation method, and the proline can be positioned and quantitatively detected inside and outside the cells in real time; and enables high throughput screening of compounds.

Other embodiments

This specification describes many embodiments. However, it will be understood that various modifications which do not depart from the spirit and scope of the invention as understood by those skilled in the art from this disclosure are intended to be included within the scope of the appended claims.

Sequence listing

<110> university of east China's college of science

<120> proline optical probe and preparation method and application thereof

<130> 191303 1CNCN

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<170> PatentIn version 3.5

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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 Asp Asp Gln Gln Gly Gly Ile Ala

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly

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

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His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val

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

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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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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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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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Pro Ser Lys Leu Gly His Lys Leu Asn

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

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Lys Lys Gly Leu Arg Leu Lys Asp Gly Gly His Tyr Ala Ala Glu Val

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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 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

1 5 10 15

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

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

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 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> 17

<211> 596

<212> PRT

<213> Artificial sequence

<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 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> 18

<211> 596

<212> PRT

<213> Artificial sequence

<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 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> 19

<211> 596

<212> PRT

<213> Artificial sequence

<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 Ser 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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 sequence

<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 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> 25

<211> 1791

<212> DNA

<213> Artificial sequence

<400> 25

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> 26

<211> 1791

<212> DNA

<213> Artificial sequence

<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 ggcaacggta 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

1. An optical probe comprising a proline-sensitive polypeptide or a functional variant thereof and an optically active polypeptide, wherein the optically active polypeptide is located at a site of the proline-sensitive polypeptide selected from the group consisting of: 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330, the proline-sensitive polypeptide being as set forth in SEQ ID NO:1, the functional variant of the proline-sensitive polypeptide comprising one or more mutations in the sequence as set forth in SEQ ID NO:1 selected from the group consisting of: F77L, a100G, D121E, D121S, D121T, D121V, D226E, D226N and Y275F.

2. A nucleic acid sequence encoding the optical probe of claim 1.

3. An expression vector comprising the nucleic acid sequence of claim 2 operably linked to an expression control sequence.

4. A host cell comprising the nucleic acid sequence of claim 2 or the expression vector of claim 3.

5. A method of making the optical probe of claim 1, comprising the steps of:

(1) transferring the expression vector of claim 2 into a host cell,

(2) Culturing said host cell under conditions suitable for expression of said expression vector, and

(3) isolating the optical probe from the host cell.

6. Use of an optical probe according to claim 1 or an optical probe prepared by the method according to claim 5 for the preparation of a kit for the detection of proline in a sample.

7. A detection kit comprising the optical probe of any one of claim 1 or the optical probe prepared by the method of claim 5.

8. A non-diagnostic method for detecting proline in a sample comprising: contacting the optical probe of claim 1 with a sample, detecting a change in the optically active polypeptide, and detecting proline in the sample based on the change in the optically active polypeptide.

9. A method of screening compounds comprising: contacting a candidate compound with a cell expressing the optical probe of claim 1, measuring a change in the optically active polypeptide, and screening for a compound having an effect on the change in proline content based on the change in the optically active polypeptide.