Gene-encoded potassium ion indicators
阅读说明：本技术 基因编码的钾离子指示剂 (Gene-encoded potassium ion indicators ) 是由 E.埃罗格鲁 H.比斯科夫 W.格莱尔 R.马利 M.沃尔德克-韦尔迈尔 于 2018-05-09 设计创作，主要内容包括：本发明涉及包含至少一个信号传导结构域和钾传感器的多肽,所述多肽能够结合K<Sup>+</Sup>,并且当K<Sup>+</Sup>与钾传感器结合后,第一信号传导结构域能够生成可检测的信号。本发明还涉及编码所述多肽的多核苷酸以及多肽在用于检测K<Sup>+</Sup>的多种应用中的用途。(The present invention relates to a polypeptide comprising at least one signalling domain and a potassium sensor, said polypeptide being capable of binding to K + And when K is + Upon binding to the potassium sensor, the first signaling domain is capable of generating a detectable signal. The invention also relates to polynucleotides encoding said polypeptides and the use of the polypeptides for detecting K + To a variety of applications.)
1. A polypeptide, comprising:
a) a first signaling domain, and
b) a potassium sensor, the potassium sensor comprising:
b1) a first domain of a potassium sensor comprising an amino acid sequence showing at least 70% identity to a sequence according to SEQ ID No. 1; and
b2) a second domain of a potassium sensor comprising an amino acid sequence showing at least 70% identity to the sequence of SEQ ID NO. 2;
wherein the potassium sensor is capable of binding positively charged potassium ions, and the first signaling domain is capable of generating a detectable signal upon binding of positively charged potassium ions to the potassium sensor.
2. The polypeptide of claim 1, comprising:
a) a first signaling domain, and
b) a potassium sensor, the potassium sensor comprising:
b1) a first domain of a potassium sensor comprising an amino acid sequence showing at least 70% identity to the sequence of SEQ ID No. 1; and
b2) a second domain of a potassium sensor comprising an amino acid sequence showing at least 70% identity to the sequence of SEQ ID NO. 2; and
c) (ii) a second signaling domain which is,
wherein the potassium sensor is capable of binding positively charged potassium ions, and the first signaling domain and the second signaling domain are capable of collectively generating a detectable signal upon binding of positively charged potassium ions to the potassium sensor.
3. The polypeptide of any one of claims 1 or 2, wherein the potassium sensor comprises the amino acid sequence of SEQ ID No. 3.
4. The polypeptide of any one of claims 1-3, wherein the potassium sensor comprises the amino acid sequence of the first domain of the potassium sensor of SEQ ID NO 1 with at least one of the following amino acid substitutions: D41N, D43N, D51N, D59N, E64Q, D83N, D84N, Q26R, N35Q, N75Q or G52D.
5. The polypeptide of claim 4, wherein the potassium sensor comprises the amino acid sequence of the first domain of the potassium sensor of SEQ ID NO 1 with the following substitutions: Q26R, N35Q, N75Q, G52D.
6. The polypeptide of any one of claims 1-5, wherein the potassium sensor comprises the amino acid sequence of the second domain of the potassium sensor of SEQ ID NO 2 with at least one of the following amino acid substitutions: D104N, E125Q, D135N, N116Q, N118Q, N121Q, or N127Q.
7. The polypeptide of claim 6, wherein the potassium sensor comprises the amino acid sequence of the second domain of the potassium sensor of SEQ ID NO. 2 with the following substitutions: D104N, E125Q, D135N.
8. The polypeptide of any one of claims 1-7, wherein the polypeptide further comprises at least one linker amino acid sequence-GGGG-or at least one linker sequence of formula (I):
-(GGS)x(GGGGS)y(GG)z- (I)
wherein
x is an integer of 0 or 1,
y is an integer of 1 to 6,
z is an integer of 0 or 1.
9. The polypeptide of claim 8, wherein y is 2, 3 or 5.
10. The polypeptide of any one of claims 8 or 9, wherein the linker amino acid sequence is preceded by the amino acid sequence of the first domain of the potassium sensor and followed by the sequence of the second domain of the potassium sensor.
11. The polypeptide of any one of claims 2-10, wherein the first signaling domain and the second signaling domain are collectively selected from the group consisting of: a Fluorescence Resonance Energy Transfer (FRET) -donor-acceptor pair, a cleavage enzyme pair, or a cleavage fluorescent protein pair, wherein said first signaling domain and said second signaling domain are paired respective parts, preferably wherein said first signaling domain and said second signaling domain are a FRET-donor-acceptor pair.
12. The polypeptide of any one of claims 2-11, wherein the FRET-donor-acceptor pair is a Cyan Fluorescent Protein (CFP) domain and a Yellow Fluorescent Protein (YFP) domain, such as a circularly permuted venus (cpv).
13. The polypeptide of any one of claims 2-11, wherein the FRET-donor-acceptor pair is Clover and mRuby 2.
14. The polypeptide of claims 1-10, wherein the first signaling domain is a fluorescent protein domain, preferably a CFP domain.
15. A polynucleotide encoding the polypeptide of any one of claims 1-14.
16. The polynucleotide of claim 15, wherein the polynucleotide comprises a sequence that shows at least 80% identity to a sequence according to SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 12.
17. A vector suitable for eukaryotic or prokaryotic gene expression encoding the polypeptide of any one of claims 1-14.
18. A cell comprising the polynucleotide of claim 15 or 16, the vector of claim 17 and/or the polypeptide of any one of claims 1-14.
19. A method of detecting positively charged potassium ions in a sample, the method comprising the steps of:
a) providing the polypeptide of any one of claims 1-14;
b) contacting the polypeptide of any one of claims 1-14 with the sample;
c) measuring a signal generated by the first signaling domain; and/or
d) Measuring a signal generated by the first and second signalling domains together;
wherein a change in signal intensity upon contact with the sample indicates the presence of potassium ions in the sample.
20. The method of claim 19, wherein the signal measured in step c) and/or d) is a fluorescent signal, a colorimetric signal or a FRET signal.
21. The method of any one of claims 19 or 20, wherein the measured signal is a FRET signal.
22. The method of claim 21, wherein the FRET signal is measured after excitation with light of a wavelength in the range of about 470nm to about 490nm and/or light emission in the range of about 510nm to 520nm and 590nm to about 610 nm.
23. The method of any one of claims 19-22, wherein in step a), providing the polypeptide comprises: (i) transfecting at least one eukaryotic cell or transformed prokaryotic cell outside the human or animal body with a polynucleotide according to any one of claims 15 or 16 or a vector according to claim 17, or (ii) providing a cell according to claim 18.
24. Use of a polypeptide according to any of claims 1 to 14 for detecting positively charged potassium ions in a sample.
25. A kit for detecting positively charged potassium ions, the kit comprising at least one of:
a) the polypeptide of any one of claims 1-14;
b) the polynucleotide of any one of claims 15 or 16 or the vector of claim 17; and/or
c) The cell of claim 18.
Background
Potassium ion (K)+) Is necessary for the proper functioning of all cell types. Electrochemical K across plasma and organelle membranes+Gradient driven K+Flow to control various cellular functions. It is well known that extracellular and intracellular K+Fluctuations in concentration control muscle contraction, neurotransmitter and hormone release, neuronal excitability, cell volume, cell proliferation and cell death. It is therefore not surprising that K+Imbalances in homeostasis have profound effects at both the cellular and organism level and are associated with a number of pathological conditions, including neurological, cardiovascular, renal, immune, muscle and metabolic disorders, and cancer. K across biological membranes+Flow and transport through a plurality of selective K+Channels, exchangers and pumps, which have become promising therapeutic drug targets for the treatment of many diseases. However, due to the lack of investigation of K at high spatial and temporal resolution+Suitable probes for kinetics, we are now directed to intracellular and extracellular K+The perception of the fluctuations is very limited.
Surprisingly, emerging evidence suggests that K is present in cells+The concentration controls the key signal events and is independent of its effect on membrane potential.
In a recent study, increased intracellular K has been shown+The levels enhanced the activity of the phosphatase PP2A in T-cells (Eil et al, Nature 537, 539-543 (22September 2016)). The Akt-mTOR complex is therefore lowly phosphorylated and inhibits T-cell effector function. This study revealed K+How tightly the ions control the basic cell function, independent of their contribution to the membrane potential. Furthermore, K has not been studied thoroughly to date+Distribution in organelles and how dynamically and strongly the internal organelles K are affected under certain physiological and pathological conditions+And (4) concentration. Our knowledge in this respect is very poor,this is mainly due to the lack of a system that allows the quantification of K in real time at the level of individual cells and organelles+Suitable methods and means for flowing. At present, K is generally used+Sensitive electrode for measuring extracellular K+Fluctuating and typically require relatively large sample volumes of at least 1 ml. These electrodes are for K+Are highly selective, but they are difficult to use to detect K+Fluctuation and intracellular K+The spatio-temporal dynamics of the signal. Several small chemiluminescent K's have been developed+Sensor to extracellular K+Fluctuations or intracellular K+And (4) change imaging. However, these fluorescent ion indicators have many limitations because they typically have K+Has a poor selectivity, shows a low dynamic range, is K in the non-physiological range+Sensitive, difficult to load into cells and organelles, and in some cases difficult to obtain.
Due to the fluorescence K+So much of the serious limitations of probes that meaningful quantification of K using fluorescence microscopy and/or fluorometry is currently not possible+And (6) imaging.
Ashraf et al disclose that potassium binding proteins (Kbp) can be used in vivo as cytoplasmic potassium sensors in e.coli at high K+Required for normal growth at concentration (Structure 2016, May 3; 24(5) 741-9).
WO 01/04623 a1 discloses fluorescently-labeled cyclic depsipeptides (depsipeptides) and their use for the optical determination of the potassium ion concentration in a sample.
US 2013/244891 a1 discloses biosensors comprising activatable acceptor fluorophores linked via a linker to an environmentally sensitive donor interacting with the analyte.
WO 2012/112440 a2 discloses fluorescent copolymers useful as potassium ion sensors.
US 2003/119195 a1 discloses fluorescent anthracene (anthrazene) based ionotropic fluorophores as potassium sensors.
In view of this prior art, there remains a need to provide other Ks+A sensor. Developing such sensors is challenging because, for example, the construction based on nearby probes is challenging. So far as the method for preparing the high-purity sodium silicate,it has not been reliably predicted whether spatial effects in gene-encoded sensors due to ligand binding can be induced by, for example, fluorescence quenching or
It is therefore an object of the present invention to provide a method suitable for detecting K+The novel reagent of (1).
Furthermore, it is another object of the present invention to provide a method for detecting K in a sample+The novel process of (1).
Disclosure of Invention
The object of the invention is solved by a polypeptide comprising:
a) a first signaling domain, and
b) a potassium sensor, comprising:
b1) a first domain of a potassium sensor comprising an amino acid sequence showing at least 70% identity to a sequence according to SEQ ID No. 1; and
b2) a second domain of a potassium sensor comprising an amino acid sequence showing at least 70% identity to the sequence of SEQ ID NO. 2;
wherein the potassium sensor is capable of binding positively charged potassium ions and the first signaling domain is capable of generating a detectable signal upon binding of the positively charged potassium ions to the potassium sensor.
In one aspect, the invention relates to a code suitable for detecting K+A polynucleotide of (4).
In one aspect, the invention relates to a vector encoding a polypeptide of the invention suitable for eukaryotic or prokaryotic gene expression.
In another aspect, the invention relates to a cell comprising a polynucleotide, vector or polypeptide of the invention.
In a further aspect, the present invention relates to a method of detecting positively charged potassium ions in a sample, said method comprising the steps of:
a) providing a polypeptide of the invention;
b) contacting a polypeptide of the invention with a sample;
c) measuring a signal generated by the first signaling domain; and/or
d) Measuring a signal generated by the first signaling domain and the second signaling domain together;
wherein a change in signal intensity upon contact with the sample indicates the presence of potassium ions in the sample.
In a further aspect, the invention relates to the use of a polypeptide according to the invention for detecting positively charged potassium ions in a sample.
In another aspect, the invention relates to a kit for detecting positively charged potassium ions.
Brief Description of Drawings
FIG. 1 shows that when K+Schematic representation of the conformational change in a polypeptide upon binding to a BON domain, which increases the detectable FRET signal.
Fig. 2 shows a schematic representation of FRET-based polypeptides according to the invention, in particular (a) two polypeptides designated GEPII1.0 and R-GEPII1.0, (b) the expected 3D structure of R-GEPII1.0, (c) HeLa cells expressing GEPII1.0 (left two panels) or R-GEPII1.0 (right two panels). Scale bar represents 10 μm, (d) when different K's are added and removed+After concentration, ECFP, FRET signal (left panel) and FRET ratio signal (middle panel) of GEPII1.0 as a function of time. The right panel shows the concentration response curve of GEPII1.0 in permeabilized (3. mu.M digitonin + 2. mu.M valinomycin) HeLa cells; EC (EC)502.04(1.716 to 2.413) mM; n-10. (e) Representative time-dependent FRET ratios of R-GEPII1.0 in HeLa cells treated with valinomycin (10. mu.M) (-left panel), -Clover- (middle panel) and FRET signals (right panel).
FIG. 3 illustrates a+Predicted 3-D structure of the bound BON domains (a, b) with highlighted acidic amino groupsAnd (4) acid. Under (c), FIG. 3 shows the distance between the two closest acidic amino acids. Under (d), FIG. 3 shows a band with K+Expected pore size of the ion, then shows K with or without hydration under (e)+The radius of the ion.
Fig. 4 shows confocal images of GEPII1.0 variants expressed in HeLa cells without and with different target sequences, the scale bar in fig. a representing 10 μm: (a) GEPII1.0 without any target sequence is shown, (b) GEPII1.0 with nuclear export sequence NES, (C) GEPII1.0 with nuclear leader sequence NLS, (d) GEPII1.0 with mitochondrial target sequence (tandem dimeric repeat of COX 8), (e) GEPII with ER target sequence (from calreticulin on N-terminus) + KDEL retention sequence on C-terminus, (f) GPI-anchored GEPII1.0, (g) GEPII1.0 targeted to the periphery of the nucleus by fusing it to the C-terminus of ivermectin (emerin), and (h) CAAX-GEPII1.0 in the cytoplasmic membrane region is monitored. The images show that the polypeptide is located in a target organelle or subdomain (sub-domain) of the cell.
FIG. 5 shows that purified GEPII1.0 responds to 3mM K, respectively+、Na+、Ca2+、Rb+Or the maximum Δ FRET-ratio signal of Cs. K+The highest ratio is obtained. Na (Na)+And Ca2+The lowest ratio is shown. Experiments were performed using a CLARIOstar fluorescent microplate reader (BMG LabtECh, Germany). In the absence or presence of 3mM KCl, NaCl, CaCl2Analysis of purified GEPII at 200nM in HEPES buffer solution containing 0.05% triton X100 (pH: 7.3.) 80. mu.l of the solution containing GEPII were transferred to multiwell plates (96 wells for fluorescence analysis, Greiner Bio-One, Kremsm ü nster, Austria) and irradiated at 430nM + -10 nM emission was collected at 475nM + -10 nM and 525nM + -10 nM, respectively, FRET ratio values were calculated (F.sub.525/F475) And correlating it to the respective FRET ratio values of GEPII in the absence of monovalent and divalent ions.
FIG. 6 illustrates possible uses of the polypeptides of the invention. Available no K+Diluting the potassium concentration in a small biological sample (e.g., a human or mouse sample), followed by addition of the polypeptide of the present invention, and detecting potassium by FRETIon concentration, for example, in a multiwell plate (A). Detection of K in Small blood samples (. about.30. mu.l) Using purified GEPII+Serum concentration, the small blood sample was taken from the facial vein or orbital (B) of laboratory mice without sacrifice of the animals. K was determined from the corresponding GEPII FRET ratio values using the linear calibration fit shown in FIG. C+Serum levels. As shown, 6 defined Ks in HEPES buffer solution were used+Concentration a calibration curve was obtained. Mouse sera were diluted 1:12.5 with HEPES buffer and mixed with GEPII solution in 96 wells to give a final dilution of 1: 25. The FRET ratio signals of these samples were measured using a CLARIOstar fluorescence microplate reader (BMG LabtECh, Germany). (D) K determined in 4 different mice using purified GEPII1.0+Serum values (in mM), blood was collected from the facial veins (red circles) or the orbit (blue squares) of the four different mice. The blood sampling mode does not influence K+The value is obtained. (E) The results shown in panel D are plotted according to the blood sampling regime. (F) K determined in sera of 5 different mice using GEPII1.0 shortly after blood sampling, 2 hours and 4 hours thereafter+The value is obtained. The data show that GEPII1.0 can remain functional in mouse serum for hours. (G) The results shown in panel F are plotted against the time of FRET measurement after blood collection.
Panel A of FIG. 7 schematically illustrates dynamic recording of extracellular K in (multi-well) cell culture plates using purified GEPII+The change in concentration over time is an effective measure of cell viability. Cells supplied with substrate (such as glucose) survived and remained at-5 mM K+Extracellular sum of-130 mM K+Intracellular physiology of+And (4) gradient. Under these conditions, only a very few cells die and release K+. In contrast, cells treated with toxic antimetabolites, such as 2-deoxyglucose (2-DG), die and release K+And thus it is increased in the supernatant. (B) K of extracellular matrix measured in 96-well plates using purified GEPII1.0 (c ═ 500nM)+Cloned pancreatic β cells (INS-1) were stored in the presence of 10mM glucose (blue curve, control) or 10mM 2-DG (red curve) as indicated,control cells were treated with 50 μ M digitonin at the 14 hour time point, which released cell K to the maximum extent+. The GEPII FRET ratio signal was measured as described above using a Clariostar fluorescent microplate reader (BMGLAbtECh, Germany).
Detailed Description
Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Although several documents are cited throughout this specification and are incorporated herein by reference in their entirety, nothing herein is to be construed as an admission that the invention is not entitled to limit such disclosure by virtue of prior invention.
K+The binding protein (Kbp) (also known as YgaU) is a soluble 6-kDa cytoplasmic protein from E.coli. It is highly specific K+Bind proteins and are high levels of external K+Required for normal growth in the presence. Potassium ions bind exclusively to the BON domain (SEQ ID NO:1), which undergoes a conformational change upon binding. Kbp further comprises a LysM domain (SEQ ID NO:2) that can interact with the BON domain.
The BON domain, LysM domain and full-length Kbp amino acid sequences are shown in Table 1 below.
Sequences of BON and LysM domains of Table 1, KbP
It has surprisingly been found that K+Can be detected by a fusion protein comprising:
a) a first signaling domain, and
b) the potassium sensor comprises
b1) A first domain of a potassium sensor comprising an amino acid sequence showing at least 70% identity to a sequence according to SEQ ID No. 1; and
b2) a second domain of a potassium sensor comprising an amino acid sequence showing at least 70% identity to the sequence of SEQ ID NO. 2;
wherein the potassium sensor is capable of binding positively charged potassium ions and the first signaling domain is capable of generating a detectable signal upon binding of the positively charged potassium ions to the potassium sensor.
K+Binding to a potassium sensor can induce a conformational change in the potassium sensor, which can then be detected via a signaling domain. This principle is illustrated in figure 1 for one polypeptide of the invention. The polypeptide according to the invention is also referred to herein as a gene-encoded potassium ion indicator (GEPII).
In a preferred embodiment, the first signaling domain is a fluorescent protein domain. Even more preferably, the first signaling domain is a cyan fluorescent protein, preferably a cyan fluorescent protein comprising the amino acid sequence of SEQ ID No. 6 or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95% or 100% identity to the sequence according to SEQ ID No. 6.
In a preferred embodiment, the first signaling domain is a fluorescent protein domain. Without being bound by theory, the detectable signal generated by the first signal may be a quenching of the fluorescent signal of the fluorescent protein domain. In a preferred embodiment, the amino acid sequence of the polypeptide of the invention comprises from N-terminus to C-terminus:
i) a first domain of a potassium sensor;
ii) a first signaling domain; and
iii) a second domain of a potassium sensor.
The first signaling domain may optionally be after and/or before the linker sequence.
In another preferred embodiment, the amino acid sequence of the polypeptide of the invention comprises, from N-terminus to C-terminus: i) a first domain of a potassium sensor;
i) a second domain of a potassium sensor;
ii) a first signaling domain; and
iii) a first domain of a potassium sensor.
The first signaling domain may optionally be after and/or before the linker sequence.
The first domain of the potassium sensor is based on the Kbp BON domain.
In a preferred embodiment, the first domain of the potassium sensor comprises an amino acid sequence showing at least 75% identity to the sequence according to SEQ ID No. 1. In a preferred embodiment, the first domain of the potassium sensor comprises an amino acid sequence showing at least 80% identity to the sequence according to SEQ ID No. 1. In a preferred embodiment, the first domain of the potassium sensor comprises an amino acid sequence showing at least 85% identity to the sequence according to SEQ ID No. 1. In a preferred embodiment, the first domain of the potassium sensor comprises an amino acid sequence showing at least 90% identity to the sequence according to SEQ ID No. 1. In a preferred embodiment, the first domain of the potassium sensor comprises an amino acid sequence showing at least 95% identity with the sequence according to SEQ id No. 1. In another preferred embodiment, the first domain of the potassium sensor comprises an amino acid sequence showing at least 100% identity to the sequence according to SEQ ID No. 1.
In another preferred embodiment, the first domain of the potassium sensor comprises the amino acid sequence of SEQ ID NO. 1 with at least one amino acid substitution. Preferably, the amino acid sequence of SEQ ID NO. 1 comprises from about 1 to about 11 substitutions. In a preferred embodiment, the at least one amino acid substitution is selected from the group consisting of: D41N, D43N, D51N, D59N, E64Q, D83N, D84N, Q26R, N35Q, N75Q or G52D. In a particularly preferred embodiment, the potassium sensor comprises the amino acid sequence of the first domain of the potassium sensor of SEQ ID No. 1 with the following substitutions: Q26R, N35Q, N75Q, G52D (SEQ ID NO: 5).
The second domain of the potassium sensor is based on the Kbp LysM domain.
In a preferred embodiment, the second domain of the potassium sensor comprises an amino acid sequence showing at least 75% identity to the sequence according to SEQ ID No. 2. In a preferred embodiment, the second domain of the potassium sensor comprises an amino acid sequence showing at least 80% identity to the sequence according to SEQ ID No. 2. In a preferred embodiment, the second domain of the potassium sensor comprises an amino acid sequence showing at least 85% identity to the sequence according to SEQ ID No. 2. In a preferred embodiment, the second domain of the potassium sensor comprises an amino acid sequence showing at least 90% identity to the sequence according to SEQ ID No. 2. In a preferred embodiment, the second domain of the potassium sensor comprises an amino acid sequence showing at least 95% identity with the sequence according to SEQ id No. 2. In another preferred embodiment, the second domain of the potassium sensor comprises an amino acid sequence showing at least 100% identity to the sequence according to SEQ ID No. 2.
In another preferred embodiment, the second domain of the potassium sensor comprises the amino acid sequence of SEQ ID NO. 2 with at least one amino acid substitution. In a preferred embodiment, the second domain of the potassium sensor comprises the amino acid sequence of SEQ ID NO. 2 comprising about 1 to about 11 substitutions. In a preferred embodiment, the at least one amino acid substitution is selected from the group consisting of: D104N, E125Q, D135N, N116Q, N118Q, N121Q, and N127Q. In a particularly preferred embodiment, the potassium sensor comprises the amino acid sequence of the second domain of the potassium sensor of SEQ ID No. 2 with the following substitutions: D104N, E125Q, D135N (SEQ ID NO: 4). The coding of the amino acids in the sequence is based on the Kbp wild type sequence (SEQ ID NO: 3).
Table 2 summarizes the corresponding amino acid sequences of Kbp and the variants of the BON and Lys domains.
Table 2: kbp and BON and Lys domain variants
The present invention also relates to a polypeptide comprising:
a) a first signaling domain, and
b) a potassium sensor comprising
b1) A first domain of a potassium sensor comprising an amino acid sequence having at least 70% identity to a sequence according to SEQ ID No. 1; and
b2) a second domain of a potassium sensor comprising an amino acid sequence having at least 70% identity to a sequence according to SEQ ID NO. 2; and
c) (ii) a second signaling domain which is,
wherein the potassium sensor is capable of binding positively charged potassium ions and the first signaling domain and the second signaling domain are capable of collectively generating a detectable signal upon binding of the positively charged potassium ions to the potassium sensor. Preferably, may be at K+Binding to the potassium sensor generates a detectable signal. K+Can, for example, induce a conformational change in the potassium sensor and subsequently bring the first and second signaling domains closer to or further away from each other, thereby at least aiding in the generation of a detectable signal. In a preferred embodiment, the first signaling domain and the second signaling domain are collectively selected from the group consisting of: a FRET-donor-acceptor pair, a cleavage enzyme pair, or a cleavage fluorescent protein pair, wherein the first signaling domain and the second signaling domain are respective portions of a pair (e.g., two halves of a cleavage enzyme or two halves of a cleavage fluorescent protein). Preferably, K+Binding to a potassium sensor can induce a conformational change in the polypeptide, and the first signaling domain and the second signaling domain can then generate a detectable signal, e.g., a FRET-donor-acceptor pair can generate a detectable FRET signal, the two halves of the cleavage enzyme can be functional and catalyze a reaction that can generate a detectable signal, or the two halves of the cleavage fluorescent protein will be capable of emitting a light having a specific wavelength if excited with light having a wavelength within a suitable rangeAs a detectable signal.
In a preferred embodiment, the first signaling domain and the second signaling domain are a FRET-donor-acceptor pair. Preferably, the donor may be a Cyan Fluorescent Protein (CFP) domain and the acceptor may be a Yellow Fluorescent Protein (YFP) domain. More preferably, the first signaling domain may be a donor and the second signaling domain may be an acceptor. In an even more preferred embodiment, the first signaling domain is a donor CFP domain and the second signaling domain is an acceptor YFP domain. Also preferably, the CFP domain may comprise the amino acid sequence of SEQ ID No. 6 or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95% or 100% identity to the sequence according to SEQ ID No. 6, wherein the excitation wavelength and the fluorescence emission wavelength are identical or substantially identical to the CFP domain according to SEQ ID No. 6, i.e. having an excitation peak at a wavelength of about 436nm and an emission peak at a wavelength of about 477 nm. Preferably, the YFP domain may be a circularly arranged venus (cpv) protein, even more preferably comprising the amino acid sequence of SEQ ID No. 7 or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95% or 100% identity to the sequence according to SEQ ID No. 7, wherein the excitation wavelength and the fluorescence emission wavelength are identical or substantially identical to the YFP domain according to SEQ ID No. 7, i.e. an excitation peak at a wavelength of about 514nm and an emission peak at about 527 nm.
In another preferred embodiment, the donor may be a Clover domain and the acceptor may be a mRuby2 domain. More preferably, the first signaling domain may be a donor and the second signaling domain may be an acceptor. It is also preferred that the Clover domain may comprise the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95% or 100% identity to the sequence according to SEQ ID NO:8, wherein the excitation wavelength and the fluorescence emission wavelength are identical or substantially identical to the Clover domain according to SEQ ID NO:8, i.e. having an excitation peak at a wavelength of about 505nm and an emission peak at a wavelength of about 515 nm. Preferably, the mRuby2 domain comprises the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95% or 100% identity to the sequence according to SEQ ID NO:9, wherein the excitation wavelength and the fluorescence emission wavelength are identical or substantially identical to the mRuby2 domain according to SEQ ID NO:9, i.e. an excitation peak at a wavelength of about 559nm and an emission peak at about 600 nm.
Table 3 shows the amino acid sequences of SEQ ID NOS 6 to 9.
Table 3: amino acid sequence of a signaling domain
In another preferred embodiment, the polypeptide according to the invention comprises the amino acid sequence of GEPII1.0 (SEQ ID NO:13) or R-GEPII1.0 (SEQ ID NO: 21).
In another preferred embodiment, the first signaling domain and the second signaling domain comprise a post-translational modification, such as a conjugated fluorescein molecule or other small molecule fluorophore or detectable moiety, which can facilitate detection at K+Upon binding to the potassium sensor, the first signaling domain and the second signal together generate a detectable signal.
In another preferred embodiment, the potassium sensor comprises an amino acid sequence of Kbp. In a preferred embodiment, the potassium sensor comprises an amino acid sequence showing at least 75% identity to the sequence according to SEQ ID No. 3. In a preferred embodiment, the potassium sensor comprises an amino acid sequence showing at least 80% identity to the sequence according to SEQ ID No. 3. In a preferred embodiment, the potassium sensor comprises an amino acid sequence showing at least 85% identity to the sequence according to SEQ ID No. 3. In a preferred embodiment, the potassium sensor comprises an amino acid sequence showing at least 90% identity to the sequence according to SEQ ID No. 3. In a preferred embodiment, the potassium sensor comprises an amino acid sequence showing at least 95% identity with the sequence according to SEQ id No. 3. In a preferred embodiment, the potassium sensor comprises an amino acid sequence showing at least 100% identity to the sequence according to SEQ ID No. 3.
In another preferred embodiment, the amino acid sequence of the polypeptide of the invention comprises, from N-terminus to C-terminus:
i) a first signaling domain;
ii) a potassium sensor, wherein preferably the first domain of the potassium sensor precedes the second domain of the potassium sensor; and
iii) a second signaling domain.
In another preferred embodiment, the polypeptide according to the invention may further comprise at least one linker sequence. The linker preferably has a flexible, i.e. non-rigid, structure which changes the sensitivity of the detectable signal. The linker amino acid sequence may be located in the amino acid sequence of the polypeptide between any two of: a first signaling group, a first domain of a potassium sensor, a second domain of a potassium sensor, and a second signaling domain. In a preferred embodiment, the linker amino acid sequence follows the amino acid sequence of the first domain of the potassium sensor and precedes the second domain of the potassium sensor. However, it is also obvious that the linking domain may be located between the potassium sensor and the first signalling domain or between the potassium sensor and the second signalling domain.
In one embodiment, the linker comprises the amino acid sequence-GGGG-.
In a preferred embodiment, the polypeptide further comprises at least one linker amino acid sequence of formula (I):
-(GGS)x(GGGGS)y(GG)z- (I)
wherein
x is an integer of 0 or 1,
y is an integer of 1 to 6,
z is an integer of 0 or 1.
In a preferred embodiment, y is not 4.
In a preferred embodiment, y is 2, 3 or 5.
In a preferred embodiment, x is 0, y is 1 and z is 1. It is further preferred in this embodiment that the polypeptide comprises the amino acid sequence of SEQ ID NO. 14.
In another preferred embodiment, x is 0, y is 2 or 3, and z is 0. It is further preferred in this embodiment that the polypeptide comprises the amino acid sequence of
In yet another preferred embodiment, x is 0, y is 4 and z is 1. It is further preferred in this embodiment that the polypeptide comprises the amino acid sequence of SEQ ID NO 17.
In another preferred embodiment, x is 1, y is 5 and z is 0. It is further preferred in this embodiment that the polypeptide comprises the amino acid sequence of
Extracellular K in known physiological systems+Levels are typically in the range of about 1mM to about 10 mM. In contrast, intracellular cytoplasm and organelles K+The concentration is typically in the range of about 100mM to 300 mM. As will be apparent from the examples, the polypeptides of the invention may provide for K+May depend on the presence of a linker sequence or amino acid substitution in SEQ ID NO 1 and/or
Thus, in a preferred embodiment, the polypeptides of the invention provide an EC of about 10 to about 300mM50The value is obtained. Such polypeptides may be particularly suitable for use in the detection of intracellular K+。
In another preferred embodiment, the polypeptides of the invention provide an EC of about up to 20mM50Value, preferably about 5mM, to measure extracellular K+。
In another preferred embodiment, the polypeptide further comprises a targeting sequence. Targeting sequences are amino acid sequences that direct the polypeptide to a cell or to a target organelle or subdomain secreted extracellularly.
Targeted organelles and subdomains can include, for example, the nucleus, mitochondria, Endoplasmic Reticulum (ER), cell surface, nuclear membrane, and subcortical regions. The targeting sequence may be located at the N-terminus or C-terminus of the polypeptide. The addition of a nuclear export sequence (e.g., amino acid sequence LPPLERLTL) to the C-terminus of a polypeptide can result in localization of the polypeptide only in the cytosol. The addition of the C-terminal nuclear leader sequence (KRSWSMAFC) results in targeting of the nucleus. Mitochondria can be targeted by a mitochondrial targeting sequence, such as the tandem dimeric repeat of
In a preferred embodiment, the polypeptide of the invention is an isolated polypeptide.
In another aspect, the invention relates to a polynucleotide encoding a polypeptide according to the invention.
It will be apparent to those skilled in the art that a given polypeptide according to the invention may be encoded by a different nucleotide sequence due to the degeneracy of the genetic code.
In a preferred embodiment, the polynucleotide according to the invention has a length of less than 9000 nucleotides, less than 8000 nucleotides, less than 7000 nucleotides, less than 6000 nucleotides, less than 5000 nucleotides, less than 4000 nucleotides, less than 3000 nucleotides, less than 2000 nucleotides, less than 1000 nucleotides or less than 500 nucleotides.
In a further preferred embodiment, the isolated polynucleotide according to the invention has a length of at least 24 to 9000 nucleotides, preferably at least 24 to 8000 nucleotides, more preferably at least 24 to 7000 nucleotides, more preferably at least 24 to 6000 nucleotides, more preferably at least 24 to 5000 nucleotides, even more preferably at least 24 to 4000 nucleotides. In another preferred embodiment, the polynucleotide according to the invention has a length of at least 60 to 9000 nucleotides, preferably at least 60 to 8000 nucleotides, more preferably at least 60 to 7000 nucleotides, more preferably at least 60 to 6000 nucleotides, more preferably at least 60 to 5000 nucleotides, even more preferably at least 60 to 4000 nucleotides. In a further preferred embodiment, the polynucleotide according to the invention has a length of at least 90 to 9000 nucleotides, preferably at least 90 to 8000 nucleotides, more preferably at least 90 to 7000 nucleotides, more preferably at least 90 to 6000 nucleotides, more preferably at least 90 to 5000 nucleotides, even more preferably at least 90 to 4000 nucleotides. In yet another preferred embodiment, the polynucleotide according to the invention has a length of at least 120 to 9000 nucleotides, preferably at least 120 to 8000 nucleotides, more preferably at least 120 to 7000 nucleotides, more preferably at least 120 to 6000 nucleotides, more preferably at least 120 to 5000 nucleotides, even more preferably at least 120 to 4000 nucleotides. In yet another preferred embodiment, the isolated polynucleotide according to the invention has a length of at least 300 to 9000 nucleotides, preferably at least 300 to 8000 nucleotides, more preferably at least 300 to 7000 nucleotides, more preferably at least 300 to 6000 nucleotides, more preferably at least 300 to 5000 nucleotides, even more preferably at least 300 to 4000 nucleotides.
In another preferred embodiment, the polynucleotide according to the invention has a length of at least 300 nucleotides, at least 400 nucleotides, at least 1000 nucleotides, at least 2000 nucleotides or at least 2500 nucleotides.
In a preferred embodiment, the polynucleotide according to the invention comprises or consists of a sequence showing at least 80% identity with the sequence according to SEQ ID No. 10. In a further preferred embodiment, the polynucleotide according to the invention comprises a nucleotide sequence identical to the nucleotide sequence according to SEQ ID NO:10, or a sequence consisting of a sequence that shows at least 85%, preferably at least 90%, more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or even more preferably at least 99% identity to a sequence according to SEQ ID NO:10, preferably at least 90%, more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or even more preferably at least 99% identical. In a particularly preferred embodiment, the polynucleotide according to the invention comprises or consists of a sequence showing at least 85%, preferably at least 90%, more preferably at least 95% or even more preferably at least 98% identity with the sequence according to SEQ ID No. 10.
In a preferred embodiment, the polynucleotide according to the invention comprises or consists of a sequence showing at least 80% identity with the sequence according to SEQ ID NO. 11. In a further preferred embodiment, the polynucleotide according to the invention comprises a nucleotide sequence identical to the nucleotide sequence according to SEQ ID NO:11, or a sequence consisting of a sequence that shows at least 85%, preferably at least 90%, more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or even more preferably at least 99% identity to a sequence according to SEQ ID NO:11, exhibits a sequence composition of at least 85%, preferably at least 90%, more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or even more preferably at least 99% identity.
In a preferred embodiment, the polynucleotide according to the invention comprises or consists of a sequence showing at least 80% identity to the sequence according to SEQ ID NO. 12. In a further preferred embodiment, the polynucleotide according to the invention comprises a nucleotide sequence identical to the nucleotide sequence according to SEQ ID NO:12, or a sequence showing at least 85%, preferably at least 90%, more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or even more preferably at least 99% identity to a sequence according to SEQ ID NO:12, shows a sequence composition showing at least 85%, preferably at least 90%, more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% or even more preferably at least 99% identity.
The sequences of SEQ ID NO 10-12 are shown below in Table 4.
Table 4: nucleotide sequences encoding a BON domain, a LysM domain and a Kbp
The polynucleotides according to the invention may be single-stranded or double-stranded RNA or DNA molecules.
In some embodiments, an isolated polynucleotide according to the present invention may be inserted into a vector (such as an expression vector). The expression vector may, for example, be a prokaryotic or eukaryotic expression vector, such as, for example, an isolated plasmid, a minichromosome, a cosmid, a phage, a retroviral vector, or any other vector known to those of skill in the art. The person skilled in the art will be familiar with how to select suitable vectors according to specific needs. In a preferred embodiment, the expression vector is an isolated plasmid.
The invention therefore also relates to an expression vector comprising a polynucleotide according to the invention.
In one aspect, the invention relates to a cell comprising a polypeptide, a polynucleotide expression vector encoding a polypeptide and/or a plasmid according to the invention. The cell is not a human embryonic stem cell. Examples of cells include, but are not limited to, cell lysates of in vitro cell culture cells or eukaryotic cells, such as mammalian, human or plant cells or prokaryotic cells, each of which can optionally be genetically modified by methods generally known to those skilled in the art, such as by transfection or transformation of the cells.
In one aspect, the present invention relates to a method of detecting positively charged potassium ions in a sample, said method comprising the steps of:
a) providing a polypeptide of the invention;
b) contacting a polypeptide of the invention;
c) measuring a signal generated by the first signaling domain;
wherein a change in signal intensity upon contact with the sample indicates the presence of potassium ions in the sample.
In one aspect, the present invention relates to a method of detecting positively charged potassium ions in a sample, the method comprising the steps of:
a) providing a polypeptide according to the invention;
b) contacting a polypeptide according to the invention;
c) measuring a signal generated by the first signaling domain; and/or
d) Measuring a signal generated by the first signaling domain and the second signaling domain together;
wherein a change in signal intensity upon contact with the sample indicates the presence of potassium ions in the sample.
The step a) of providing the polypeptide takes place outside the human body.
In one embodiment of the method, a change in signal intensity after contact with the sample as compared to the polypeptide signal in the absence of the sample indicates the presence of K in the sample+。
In another preferred embodiment, the method of the invention is a (quantitative) in vivo imaging method.
In a preferred embodiment, the signal measured is a fluorescent signal, a colorimetric signal or a FRET signal. Preferably, may be at K+Binding to the potassium sensor of the polypeptide of the invention generates a signal. In preferred embodiments, the signal may be generated by a FRET-donor-acceptor pair, a cleavage enzyme pair, or a cleavage fluorescent protein pair. Detection can be carried out by methods generally known to those skilled in the art.
In one embodiment, the measured signal is a fluorescent signal. In a preferred embodiment, the fluorescent signal is passed through K+Binding to the potassium sensor domain quenches.
In a more preferred embodiment, the measured measurable signal is a FRET signal. Preferably, a FRET signal is generated by the first signaling domain and the second signaling domain. More preferably, the FRET signal is generated by a FRET-donor-acceptor pair, preferably YFP (such as CPV) and CFP. One skilled in the art knows how to measure the FRET signal. Preferably, FRET between YFP and CFP is measured after excitation with light having a wavelength in the range of about 420nm to about 450 nm. More preferably, the measurement is performed after excitation with light of about 440 nm. It is also preferred to measure the light emission at a wavelength in the range of about 525nm to about 545nm, more preferably about 535 nm.
In another preferred embodiment, the FRET pair is Clover and mRuby2, rather than YFP and CFP. In this case, the FRET signal is measured after excitation with 470nm to about 490nm and/or light emission in the range of about 510nm to 520nm (emission 1) and 590nm to about 610nm (emission 2).
In a preferred embodiment, K is detected+Step a) of the method of (1), providing a polypeptide of the invention, may comprise the use of a polynucleotide, plasmid and/or table encoding a polypeptide of the inventionThe expression vector transfects at least one cell outside the human or animal body. The polypeptide according to the invention can then be provided by protein synthesis of the cell. The polypeptide of the invention may then be isolated from the cell, secreted by the cell, or left in the cell. In another preferred embodiment, the polypeptide according to the invention may be provided in step a) of the method according to the invention by providing a cell according to the invention.
In a preferred embodiment, the method according to the invention can detect the presence of potassium ions in any kind of sample. More preferably the sample is selected from the group consisting of: a biological sample or a liquid sample or a combination thereof. Even more preferred samples are cell cultures, cell aggregates, cell lysates, human or animal tissue samples, K-containing samples+Blood or liquid. In one embodiment, the sample may also include biological samples, such as cell cultures, monolayer cultures or 3-D cell cultures comprising cells, cell suspensions, cell aggregates, cell lysates, human or animal tissue samples, and K-containing samples+The liquid sample of (1). Preferably, the method according to the invention can then be used to detect K by+And optionally in combination with determining other relevant parameters of the biological sample (such as apoptosis, cell signaling, cellular gene expression, etc.) to characterize K+Effect on biological samples.
Thus, in one aspect, the polypeptides of the invention may be used to detect K in a sample as described above+. In a preferred embodiment, the use according to the invention comprises the use of a polypeptide according to the invention for (quantitative) in vivo imaging. The term "in vivo imaging" refers to imaging in living cells outside the human body, such as microscopy of isolated in vitro cultured living cells. The intracellular polypeptides according to the invention may indicate K in the cytosol, the subplasmic region, the nucleus, the endoplasmic reticulum, the nuclear membrane and the mitochondria, e.g. in response to a well-defined stimulus or stress+A change in level.
In another preferred embodiment, the isolated polypeptide of the invention can be based on a biological sample in a suitable container (e.g., a multiwell plate) and the potassium concentration can be measured directly, e.g., using a plate fluorescence reader.One advantage of using a polypeptide according to the invention is that a small amount of sample (only about 5-10. mu.l) will be required compared to the amount of biological sample required to measure potassium concentration using an electrode (typically about 100-. Therefore, by using such K+Electrode assay of Small laboratory animals [ which have a Total blood volume (6-8% of body weight) of 1.5-2.5 ml%]Serum K of (1)+Concentration usually requires the animal to be sacrificed for maximum blood concentration.
In another preferred embodiment, the polypeptide according to the invention is useful in an in vitro cell death assay. In virtually all cell types, especially in excitable cells, enormous energy is required to pass Na+/K+ATPase to maintain Na across the plasma membrane+And K+And (4) gradient. Under stress conditions, cells cannot gain enough energy, so they will have reduced viability and eventually undergo cell death. In this embodiment of the invention, the isolated polypeptide according to the invention is added to the medium in which the cells are cultured and can be used to monitor K in the medium+The change in concentration with time. K in the culture medium when cell viability is reduced or incidence of cell death is increased+The concentration will increase. This can then be detected using the polypeptides according to the invention. The use of a polypeptide according to the invention in this assay provides the following advantages: cell death/viability assays with other prior art (e.g., MTT assays or resazurin-based assays, such as
In another embodiment, the polypeptide according to the invention is used in a cell growth assay. It is known that K is generated when cells grow+The concentration is reduced. When the isolated polypeptide according to the present invention is added to the medium in which the cells are cultured, the expansion of the cell growth can be monitored again. The assay can be used, for example, to distinguish between growing bacterial cell cultures and dead cells containing predominantly no growth and replicationBacterial cell cultures of cells. This cannot be determined by existing standard methods to monitor bacterial growth, for example by measuring the optical density (OD600) of a bacterial cell culture.
In another embodiment, the polypeptide according to the invention can be used as extracellular K in living animals using in vivo microscopy+Fluctuations (e.g., extracellular K of brain or muscle+Wave) real-time visualization. In the present application, the polypeptides according to the invention can be applied, for example, topically to animals. In this context, the polypeptides according to the invention may for example be used as research tools for the study of cancer or neurological diseases (such as epilepsy, migraine or on the craniocerebral bed) for animal model studies.
In one aspect, the invention also relates to a method for detecting K in a sample+The kit of (a), comprising at least one of:
a) a polypeptide of the invention;
b) a polynucleotide of the invention or a vector of the invention; and/or
c) The cells of the invention.
Examples of such kits include kits for determining cell death or cell viability as described above. Such a kit may, for example, comprise, in addition to the polypeptide of the invention, at least one of:
a) suitable K-free for diluting samples+A buffer solution;
b) as a positive control with a known K+At least one standard solution of concentration;
c) if the kit contains more than one solution, the standard solution may contain K at different concentrations+So as to obtain a calibration curve using the standard solution; or
d) Suitable buffers for diluting the polypeptides of the invention.
Kits comprising a polynucleotide or vector according to the invention may further comprise a plasmid encoding one or both of the signaling domains or only the potassium sensor to generate a control sample. The kit may further comprise a suitable buffer for diluting the polynucleotide or vector.
The kit comprising the cells according to the invention may further comprise a suitable medium for the cells and/or a cryopreservation medium.
In yet another embodiment, the polypeptide according to the invention may also be used for portable K+A rapid detection kit. In such tests, the polypeptide according to the invention may be present in solution, preferably in a potassium-free solution, or it may be immobilized on a solid phase or bead.
Definition of
The following definitions are introduced. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
It is to be understood that the terms "comprises" and variations thereof (such as "comprises" and "comprising") are not limiting. In the context of the present invention, the term "consisting of … …" is to be taken as the preferred form of the term "comprising.
If in the following a group is defined comprising at least a certain number of embodiments, this means that it also comprises a group preferably consisting of only these embodiments.
The terms "about" and "approximately" or "substantially the same" refer in the context of the present invention to an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term generally encompasses deviations of ± 10%, preferably ± 5%, from the indicated value.
As used herein, the term "domain" refers to a component of a polypeptide or fusion protein. The term domain thus encompasses a portion of a polypeptide that is capable of folding, functioning and/or existing independently of the remainder of the polypeptide chain or structure. For example, when the cyan fluorescent protein is part of a fusion protein, it is considered a domain. Furthermore, as used herein, the term domain also encompasses each portion of a cleavage enzyme or cleavage fluorescent protein, wherein each portion is considered a domain even though the two domains of the cleavage enzyme or cleavage fluorescent protein can only fold and function together.
As used herein, the terms "polypeptide" and "protein/protein" are used interchangeably herein to describe a protein molecule that may comprise a partial or full-length protein. The term includes "fusion proteins" comprising proteins or polypeptides having amino acid sequences derived from two or more proteins. Fusion proteins may also include amino acid junction regions between amino acid portions of the proteins with individual atoms.
The term "detectable signal" as used herein refers to an increase or decrease in signal in the technical field commonly used in biochemical, chemical, medical or diagnostic techniques. Examples of detectable signals include, but are not limited to, electrical signals (e.g., capacitance), mechanical signals, optical signals, acoustic signals, or thermal signals. Preferably, the optical signal may be a fluorescent signal, a FRET signal, a colorimetric signal or an electrochemiluminescent signal. It is also preferred that the signal may be detectable, i.e. the signal and corresponding signal changes may be monitored using suitable technical equipment. Preferably, the detectable signal may be a signal that is generated or altered in a proximity-based manner, e.g., induced by a conformational change in the polypeptide.
As used herein, the term "FRET" refers to the intermolecular or intramolecular fluorescence resonance energy transfer. In the FRET method, one fluorophore can serve as an energy donor and the other an energy acceptor. These are sometimes referred to as reporters and quenchers, respectively. The donor may be excited with light of a particular wavelength at which it will typically exhibit a fluorescence emission wavelength. The acceptor may also be excited at a wavelength such that it can accept the emission energy of the donor molecule through a variety of distance-dependent energy transfer mechanisms. Typically, the acceptor receives the emission energy of the donor when the acceptor is in close proximity to the donor. The donor and acceptor may be different molecules or may be different parts of the same molecule, such as two different domains of a polypeptide. FRET measurement techniques are well known in the art.
As used herein, the term "FRET-donor-acceptor pair" refers to a fluorophore representing an energy donor and an energy acceptor capable of FRET, as described above. In this context, the term "fluorophore" refers to a moiety of a molecule that causes the molecule to fluoresce. Which is the portion of the molecule that absorbs a particular wavelength and re-emits light at a different (but also particular) wavelength. The amount and wavelength of light emitted depends on the isomer and the chemical environment of the isomer. Fluorophores include, but are not limited to, Fluorescein Isothiocyanate (FITC) (reactive derivative of fluorescein), rhodamine (TRITC), coumarin, anthocyanin dyes (Cy) (e.g., anthocyanin 3,
As used herein, the term "cleaving enzyme" refers to a biologically active enzyme that cleaves into at least two moieties with at least reduced or no biological activity.in this context, the term "cleaving enzyme pair" refers to at least two enzyme moieties that are at least partially inactivated.when brought into proximity, the enzyme moieties interact to form a biologically active enzyme that can be detected using conventional enzyme detection techniques.cleaving enzyme techniques are also further described in WO 2005/094441A2 examples of cleaving enzymes include, but are not limited to, renilla luciferase, which can be recovered and monitored via bioluminescence, complementary cleaving β -galactosidase, wherein activity can be monitored via colorimetric chemiluminescence or fluorescence detection, cleaving β -lactamase, whose complementarity can be determined by color change of the nitrocefin the enzyme after hydrolysis or by fluorescence via CCF-2/AM, GTPase (charge change), peroxidase (chroma), ribozymes (endo and exo), restriction endonucleases (sequence-specific endonucleases), proteases (protein cleavage), lipases (linked nucleic acid oligomers), and thiol-disulfide oxidoreductases (via change in disulfide conformation).
As used herein, the term "split-fluorescent protein (SFP) pair" refers to at least two portions of a fluorescent protein. SFPs consist of multiple peptides or polypeptide fragments that are individually non-fluorescent but complementary to form a functionally responsive molecule. For example, split green fluorescent protein (split GFP) is SFP. Some engineered split GFP molecules are self-assembling. (see, e.g., U.S. patent application publication No. 2005/0221343 and PCT publication No. WO/2005/074436; Cabantous et al, nat. BiotEChnol.,23: 102-854,2005; Cabantoundsand Waldo, nat. methods,3:845-854,2006). US2012282643 also describes split yellow and split cyan fluorescent protein variants.
As used herein, the determination of "% identity" between two sequences is preferably performed using the mathematical algorithm of Karlinanda Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877. Such algorithms are incorporated, for example, into the BLASTn and BLASTp programs of Altschul et al, (1990) J.MoI.biol.215: 403-.
The determination of percent identity is preferably performed using the standard parameters of the BLASTn and BLASTp programs.
BLAST polynucleotide searches are preferably performed using the BLASTn program.
For general parameters, the "Max Target sequences" box may be set to 100, the "Short queries" box may be a check, the "ExpECt Threshold" box may be set to 10, and the "Word Size" box may be set to 28. For the scoring parameter, "Match/mismatch Scocres" may be set to 1-2, and the "Gap Costs" block may be set to linear. For the filter and Mask parameters, the "Low Complexity regions" box may be unchecked, the "spaces-spacing templates" box may be unchecked, the "Mask for lookup table only" box may be unchecked, and the "Mask lower templates" box may be unchecked.
BLAST protein searching is preferably performed using the BLASTp program.
For general parameters, the "Max Target Sequences" box may be set to 100, the "Short Sequences" box may be a check, the "ExpECt threshold" box may be set to 10, and the "Word Size" box may be set to "3". For the scoring parameter, the "Matrix" box may be set to "BLOSUM 62", the "Gap Costs" box may be set to "Existence: 11Extension: 1", and the "Compositional additions" box may be set to "Compositional information Matrix addition". For the filter and Mask parameters, the "LowComplexityregions" box may be unchecked, the "Mask for lookup table only" box may be unchecked, and the "Mask lowercase letters" box may be unchecked.
Percent identity is determined over the full length of the corresponding reference sequence, i.e. over the full length of the sequence according to the sequences SEQ ID NO or SEQ ID NOs as described in the corresponding context. For example, an amino acid sequence showing at least 80% identity to a sequence according to SEQ ID NO. 1 shows at least 80% identity to SEQ ID NO. 1 over the entire length of SEQ ID NO. 1. In another example, a sequence that shows at least 80% identity to a sequence according to SEQ ID NO. 3 shows at least 80% identity to SEQ ID NO. 3 over the full length of SEQ ID NO. 3.
In the context of the present invention, the term "isolated" means that a polypeptide or polynucleotide has been removed from its natural environment and/or is present in a form that is not naturally found. An "isolated" polypeptide or "isolated" polynucleotide may also be one that is produced in vitro.
As used herein, the term "amino acid substitution" refers to a substitution in an amino acid sequence according to conservative or non-conservative substitutions, preferably conservative substitutions. In some embodiments, the substitution further comprises exchanging a natural amino acid for an unnatural amino acid. Conservative substitutions include the substitution of an amino acid for another amino acid having similar chemical properties as the amino acid being substituted. Preferably, the conservative substitutions are substitutions selected from the group consisting of:
(i) replacing the basic amino acid with a different basic amino acid;
(ii) replacing the acidic amino acid with a different acidic amino acid;
(iii) replacing an aromatic amino acid with another, different aromatic amino acid;
(iv) replacing a non-polar aliphatic amino acid with another, different non-polar aliphatic amino acid; and
(v) the polar uncharged amino acid is substituted with another different polar uncharged amino acid.
The basic amino acid is preferably selected from arginine, histidine and lysine. The acidic amino acid is preferably aspartate or glutamate.
The aromatic amino acid is preferably selected from phenylalanine, tyrosine and tryptophan. The non-polar aliphatic amino acid is preferably selected from the group consisting of glycine, alanine, valine, leucine, methionine and isoleucine. The polar uncharged amino acids are preferably selected from the group consisting of serine, threonine, cysteine, proline, asparagine and glutamine. Unlike conservative amino acid substitutions, non-conservative amino acid substitutions are those in which the amino acid is exchanged for any amino acid that does not fall within conservative substitutions (i) to (v) outlined above.
As used herein, the term "biological sample" refers to a sample of tissue (e.g., tissue biopsy), organ, cell lysate, or bodily fluid (blood, urine, saliva, bile, serum, cerebrospinal fluid, etc.) outside of a human or animal body. Furthermore, the term "biological sample" also includes in vitro cell culture cells or cell lysates of eukaryotic cells (such as mammalian cells, human cells or plant cells) or prokaryotic cells, which may optionally be genetically modified according to methods generally known to the person skilled in the art, including transfection and transformation methods.
As used herein, the term "binding" refers to an attractive interaction between two molecules that results in a stable association of the molecules in close proximity to each other. The result of the association is sometimes the formation of a molecular complex in which the attractive forces holding the moieties together are generally non-covalent and therefore generally weaker in energy than covalent bonds.
Examples
1. Cell culture, cell transfection, chemicals, and buffers
HeLa cells were grown in Dulbeccosfs modified Eagle Medium (DMEM, Sigma Aldrich) containing 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin. At 60-80% fusion, cells in 30-mm imaging dishes were transfected with 1ml of serum-and antibiotic-free medium mixed with 1.5. mu.g of the appropriate plasmid DNA and 3. mu.g of TransFastTM transfection reagent (Promega). The cells were kept in a humidified incubator (37 ℃, 5% CO2, 95% air) for 16-20 hours and then changed back to the corresponding medium. All experiments were performed 24 hours after transfection. Valinomycin was purchased from Sigma Aldrich and used at a final concentration of 7 μ M. The experimental buffer used had the following composition (in g/L): 8.0 or 7.6NaCl, 1.44Na2HPO4，0.12NaH2PO4The pH was adjusted to 7.40 using NaOH with or without 0.4 KCl.
2. Live cell imaging
Fluorescence imaging was performed using TiLL iMIC (Till Photonics, Graefelfing, Germany, a digital wide area fluorescence imaging system). The red-shifted FRET-based R-GEPII was excited at 480nm and the emission was captured at 510-540nm (Clover, FRET donor) and 560-610nm (FRET from Clover to mRuby 2), respectively. The CFP/YFP based GEPII1.0 was excited at 430nm and the emission was recorded at 480nm and 535nm, respectively. Data acquisition and control of the digital fluorescence microscope was performed using field acquisition software version 2.0.0.12 (Till Photonics).
3. Computer modeling
Using the online tool Phyre2(Protein Homology/Homology RECognition Engine V2.0;http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi？id＝index) Models predicting the BON domain and full length Kbp. The predicted chimera 3D structure was further analyzed using the software PyMol viewer. PoreWalker 1.0 (using online tool)http://www.ebi.ac.uk/thorntonsrv/software/PoreWalker/) And performing channel prediction.
4. Detection of K by FRET+
Generation of GEPII1.0 andplasmid DNA encoding the R-GEPII1.0 polypeptide (FIGS. 2a and b). GEPII1.0 comprises an optimized CFP/YFP (sECFP as FRET donor, cpV as FRET acceptor) FRET pair (fig. 2a, top panel). The red-shifted R-GEPII1.0 comprises Clover and mRuby2 (fig. 2a lower panels and b), which are bright green and red FP variants, respectively, optimized to generate a red FRET-based probe with improved kinetics. Both probes were detected in HeLa cells expressing CFP/YFP-based GEPII1.0 (FIG. 2c, left panel s) or red-shifted R-GEPII1.0 (FIG. 2c, right panel) after transfection with the corresponding plasmids. To control cytosol K+Concentration ([ K ]+]cyto) With digitonin and K+Cells were permeabilized with mixtures of ionophores valinomycin (FIG. 2d) or with valinomycin alone (FIG. 2 e). In fact, the FRET ratio signal of GEPIIs responds to K in a concentration-dependent manner+Increased by increasing (FIG. 2d) while removing K+FRET fluorescence immediately thereafter decreased (FIGS. 2d and e). These experiments confirm that the design and generation of FP and kbp-based chimeric constructs provides K+Altered functional FRET-based probes read in real time.
In situ, half maximal most Effective Concentrations (EC) of CFP/YFP-based GEPII1.0 and corresponding R-GEPII1.0 were found50) 2.04(1.72-2.41) mM (fig. 2d, right panel) and 4.11(3.25-5.19) mM (n ═ 8), respectively. For in situ determination of EC50Values (cultured HeLa cells alone), cells expressing GEPIIs in the absence of K+The solution was permeabilized with 5. mu.M digitonin for 10 min. Digitonin was applied to cells on a microscope using a semi-automated perfusion system. K of probe study using continuous FRET ratio imaging over time+Sensitivity. Addition of different K in the range of 0.01mM to 100mM via a perfusion system+Concentration until the FRET ratio signal rises and remains stable. Maximum Δ FRET ratio value relative to logarithm K+Concentrations were plotted and fitted using the Sigmoidal concentration response equation. Data analysis was performed using
5. Rational design of polypeptide variants
K for controlling GEPIIs+Sensitivity, we rationally based on sequence analysis and 3D homology modeling using Phyre-2 and PyMol softwareThe wild type kbp was redesigned. Our predictions and preliminary data indicate that mutations encoding charged and polar amino acids in the BON and LysM domains significantly reduce the K of the corresponding FRET-based GEPIIs+Sensitivity.
Wild type K at kbp+In the bound BON domain, 8 acidic amino acid positions can be identified: d41, D43, D51, D59, E64, E67, D83 and D84 (fig. 3a and b, red region). Interestingly, 3-D modeling of the BON domain using Phyre2 and PyMol predicted a pore or channel-like structure (fig. 3 a). The minimum distance between the two acidic amino acids in the well was around 680-800pm (FIG. 3 c). Considering the minimum pore size around 660pm (FIG. 3d), acidic amino acids near and in the pores may significantly interfere with and bind to the hydrated K+Ions (fig. 3 e).
Furthermore, sequence analysis combined with 3D modeling of the BON domain (fig. 3) predicted that Q (glutamine) at position 27, N (asparagine) at position 35, N (asparagine) at position 75, and G (glycine) at position 53 were also considered for K in addition to all acidic amino acids+Perception is important.
The three negatively charged amino acids at positions 105 (D), 126 (E) and 408 (D) in the LysM domain are considered to be linked to K+Bound BON Domain interaction, on K+The conformational changes of the bound protein also have a significant effect.
6. FRET detection using polypeptide variants
Site-directed mutagenesis was used to generate plasmid DNA encoding the GEPII1.0 mutant. Here, primers containing the designed single nucleotide polymorphisms were subjected to PCR using herculase II polymerase (Agilent Techologies, Santa Clara, USA). The corresponding PCR products were subsequently subcloned into pcDNA3.1(-) mammalian expression vectors using the corresponding restriction enzymes. Hela cells were subsequently transfected with the corresponding plasmid DNA and the EC determined as described above in example 450. The results for the corresponding polypeptide variants are summarized in table 5.
Table 5: sensitivity of GEPII1.0 variants with amino acid substitutions
7. FRET detection of polypeptide variants using different linker molecules between a first domain of a potassium sensor and a second domain of a potassium sensor
Forward and reverse primer pairs designed to encode the amino acids forming the corresponding linkers were used to generate plasmids encoding polypeptides according to the invention comprising a linker sequence comprising glycine and serine residues. The forward primer was designed to extend the wild-type LysM domain with a 5' overhang by overhang extension PCR to form a linker. The reverse primer pair was designed to bind to the wild-type BON domain and form the same linker at the 3' end. These two PCR products were then fused by additional PCR and subcloned into pcdna3.1(-) vectors flanked by nucleotide sequences encoding MSECfp and cpV, respectively. The final construct encodes a novel CFP/YFP FRET-based GEPII variant with a flexible linker between the Kbp BON and LysM domains.
Cells were subsequently transfected with plasmid DNA and EC determined as described above in example 450. The results for the corresponding polypeptide variants are summarized in table 6.
Table 6: sensitivity of GEPII1.0 variants in which BON and LysM are linked by different linker sequences
Polypeptides comprising linking molecules exhibit increased EC compared to GEPII1.050。
8. FRET detection of polypeptide variants comprising a linker between the BON and LysM domains
Plasmids encoding polypeptides according to the invention comprising amino acid substitutions and linker sequences were generated using designed forward and reverse primer pairs encoding the amino acids forming the corresponding linkers. The forward primer was designed to extend the Δ LysM domain with a 5' overhang (see Δ LysM GEPII1.0) by overhang extension PCR to form a linker. The reverse primer pair was designed to bind to the wild-type BON domain and form the same linker at the 3' end. These two PCR products were then fused by additional PCR and subcloned into pcdna3.1(-) vectors flanked by nucleotide sequences encoding msECFP and cpV, respectively. The final construct encodes a novel CFP/YFP FRET-based GEPII variant with a flexible linker between the BON and Δ LysM domains.
Cells were subsequently transfected with plasmid DNA and EC determined as described above in example 450. The results for the corresponding polypeptide variants are summarized in table 7.
Table 7: sensitivity of GEPII1.0 variants comprising amino acid substitutions and linker sequences
9. Targeting organelles and subdomains of cells expressing a polypeptide of the invention
One great advantage of gene-coded probes is that they can be precisely targeted to cellular organelles and subdomains. Thus, targeting GEPIIs will enable quantitative measurement of K with high spatial and temporal resolution+Horizontal and kinetic. Due to lack of targeting K+Probe, we are currently about subcellular K+The idea of flow is very vague.
The corresponding DNA plasmid encoding GEPII1.0 with an N-terminal or C-terminal target sequence was cloned using general molecular biology methods known to the skilled worker. Experiments targeting GEPII1.0 polypeptides (see fig. 2) to the nucleus (fig. 4c), mitochondria (fig. 4d), endoplasmic reticulum (ER, fig. 4e), cell surface (fig. 4f), nuclear membrane (fig. 4g) and subcolasmic reticulum area (fig. 4h) were analyzed by fluorescence microscopy. GEPII1.0 without any targeting sequence was localized in the cytosol and nucleus (fig. 4 a). Adding a core export sequence (NES; LPPLERLTL) to the GEPII1.0C-terminal leads to K+The probe was localized only in the cytosol (FIG. 4 b). The target probe will allow subcellular K+Time-lapse (time-lapse) fluorescence imaging of flow.
10. Characterization of isolated Polypeptides
Cloning different GEPII variants into petM11 bacterial expression vectors after transformation of the bacterial expression plasmids encoding GEPIIs into chemically active DH5 α bacteria, the cells were cultured on LB agar plates to receive individual colonies, pre-cultures containing individual colonies were cultured overnight and then inoculated into 1L of fresh LB medium at 37 ℃whenan optical density of 0.7 was observed, expression was induced by addition of IPTG (isopropyl β -D-1-thiogalactopyranoside) to a final concentration of 0.5mM and the cells were further cultured at room temperature+And Ca2+Influence. In contrast, corresponding to the addition of 3mN corresponding ions, K+Relative to Rb+And Cs+The FRET ratio signal of the purified GEPII was increased (see FIG. 5).
11. Determination of K in biological samples+Concentration of
In one set of preliminary experiments, we used GEPII1.0 purified as described above, and measured K in mouse serum in the experimental setup shown in fig. 6A+. We determined serum K+The concentration was 6.63 ± 0.34mM (SD; n ═ 5; fig. 6), which is in good agreement with published data. Importantly, reproducibility and reproducibility independent of the blood sampling regime (facial vein or orbital) and GEPII stability in mouse serum were very high (fig. 6), indicating GEPII-based K in the bioprobe(s)+The assay shows a robust and accurate method.
12. Cell viability/death assay
In addition, purified GEPII1.0 was used to dynamically record extracellular K in (multi-well) cell culture plates+Concentration as a measure of cell viability and cell death. The FRET ratio signal of extracellular-localized recombinant GEPII1.0 was measured every hour while the cells were maintained in a medium containing glucose or 2-deoxyglucose (2-DG). As shown in FIG. 7, extracellular K in the supernatant of control cells in the presence of 10nM glucose+The concentration was kept constant over time until the cells were permeabilized with 50 μ M digitonin. In contrast, if cells were treated with 2-DG, the FRET ratio signal of purified GEPII1.0 increased dramatically over time, indicating a rapid induction of intracellular K+Metabolic crisis of loss. These findings further emphasize the measurement of K in culture+Release represents a real-time reading of cell viability.
Sequence listing
<110> university of glauz medical
<120> Gene-encoded Potassium ion indicator
<130>M11159
<160>21
<170>PatentIn version 3.5
<210>1
<211>63
<212>PRT
<213> Escherichia coli
<400>1
Gln Ala Lys Lys Val Gln Glu His Leu Asn Lys Thr Gly Ile Pro Asp
1 5 10 15
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Gly Lys Ala Thr Val Thr
20 25 30
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
35 40 45
Gly Asn Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr
50 55 60
<210>2
<211>50
<212>PRT
<213> Escherichia coli
<400>2
Gln Phe Tyr Thr Val Lys Ser Gly Asp Thr Leu Ser Ala Ile Ser Lys
1 5 10 15
Gln Val Tyr Gly Asn Ala Asn Leu Tyr Asn Lys Ile Phe Glu Ala Asn
20 25 30
Lys Pro Met Leu Lys Ser Pro Asp Lys Ile Tyr Pro Gly Gln Val Leu
35 40 45
Arg Ile
50
<210>3
<211>149
<212>PRT
<213> Escherichia coli
<400>3
Met Gly Leu Phe Asn Phe Val Lys Asp Ala Gly Glu Lys Leu Trp Asp
1 5 10 15
Ala Val Thr Gly Gln His Asp Lys Asp Asp Gln Ala Lys Lys Val Gln
20 25 30
Glu His Leu Asn Lys Thr Gly Ile Pro Asp Ala Asp Lys Val Asn Ile
35 40 45
Gln Ile Ala Asp Gly Lys Ala Thr Val Thr Gly Asp Gly Leu Ser Gln
50 55 60
Glu Ala Lys Glu Lys Ile Leu Val Ala Val Gly Asn Ile Ser Gly Ile
65 70 75 80
Ala Ser Val Asp Asp Gln Val Lys Thr Ala Thr Pro Ala Thr Ala Ser
85 90 95
Gln Phe Tyr Thr Val Lys Ser Gly Asp Thr Leu Ser Ala Ile Ser Lys
100 105 110
Gln Val Tyr Gly Asn Ala Asn Leu Tyr Asn Lys Ile Phe Glu Ala Asn
115 120 125
Lys Pro Met Leu Lys Ser Pro Asp Lys Ile Tyr Pro Gly Gln Val Leu
130 135 140
Arg Ile Pro Glu Glu
145
<210>4
<211>50
<212>PRT
<213> Artificial sequence
<220>
<223>LysM D104N,E125Q,D135N
<400>4
Gln Phe Tyr Thr Val Lys Ser Gly Asn Thr Leu Ser Ala Ile Ser Lys
1 5 10 15
Gln Val Tyr Gly Asn Ala Asn Leu Tyr Asn Lys Ile Phe Gln Ala Asn
20 25 30
Lys Pro Met Leu Lys Ser Pro Asn Lys Ile Tyr Pro Gly Gln Val Leu
35 40 45
Arg Ile
50
<210>5
<211>63
<212>PRT
<213> Artificial sequence
<220>
<223>BON Q26R,N35Q, N75Q, G52D
<400>5
Gln Ala Lys Lys Val Gln Glu His Leu Gln Lys Thr Gly Ile Pro Asp
1 5 10 15
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Asp Lys Ala Thr Val Thr
20 25 30
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
35 40 45
Gly Gln Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr
50 55 60
<210>6
<211>228
<212>PRT
<213> Artificial sequence
<220>
<223>mseCFP
<400>6
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 1015
Val Glu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Trp Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala
225
<210>7
<211>245
<212>PRT
<213> Artificial sequence
<220>
<223>cpV
<400>7
Met Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
1 5 10 15
Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr
20 25 30
Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val
35 40 45
Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
50 55 60
Leu Tyr Lys Gly Gly Ser Gly Gly Met Val Ser Lys Gly Glu Glu Leu
65 70 75 80
Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
85 90 95
Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr
100 105 110
Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val
115 120 125
Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Gln Cys Phe
130 135 140
Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala
145 150 155 160
Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp
165 170 175
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu
180 185 190
Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn
195 200 205
Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr
210 215 220
Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile
225 230 235 240
Arg His Asn Ile Glu
245
<210>8
<211>251
<212>PRT
<213> Artificial sequence
<220>
<223>Glover
<400>8
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Phe Gly Tyr Gly Val Ala Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser His Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Ser
225 230 235 240
Arg Gly Pro Tyr Ser Ile Val Ser Pro Lys Cys
245 250
<210>9
<211>237
<212>PRT
<213> Artificial sequence
<220>
<223>mRuby2
<400>9
Met Val Ser Lys Gly Glu Glu Leu Ile Lys Glu Asn Met Arg Met Lys
1 5 10 15
Val Val Met Glu Gly Ser Val Asn Gly His Gln Phe Lys Cys Thr Gly
20 25 30
Glu Gly Glu Gly Asn Pro Tyr Met Gly Thr Gln Thr Met Arg Ile Lys
35 40 45
Val Ile Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr
50 55 60
Ser Phe Met Tyr Gly Ser Arg Thr Phe Ile Lys Tyr Pro Lys Gly Ile
65 70 75 80
Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg
85 90 95
Val Thr Arg Tyr Glu Asp Gly Gly Val Val Thr Val Met Gln Asp Thr
100 105 110
Ser Leu Glu Asp Gly Cys Leu Val Tyr His Val Gln Val Arg Gly Val
115 120 125
Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Lys Gly Trp
130 135 140
Glu Pro Asn Thr Glu Met Met Tyr Pro Ala Asp Gly Gly Leu Arg Gly
145 150 155 160
Tyr Thr His Met Ala Leu Lys Val Asp Gly Gly Gly His Leu Ser Cys
165 170 175
Ser Phe Val Thr Thr Tyr Arg Ser Lys Lys Thr Val Gly Asn Ile Lys
180 185 190
Met Pro Gly Ile His Ala Val Asp His Arg Leu Glu Arg Leu Glu Glu
195 200 205
Ser Asp Asn Glu Met Phe Val Val Gln Arg Glu His Ala Val Ala Lys
210 215 220
Phe Ala Gly Leu Gly Gly Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210>10
<211>189
<212>DNA
<213> Escherichia coli
<400>10
caggcgaaga aggtgcagga gcatctgaac aaaaccggta taccggatgc cgataaagtg 60
aatattcaaa ttgccgacgg caaagcgacg gtcactggtg acggcctgag tcaggaggcg 120
aaggagaaaa tccttgttgc ggtggggaat atttccggta ttgccagtgt cgatgatcag 180
gtgaaaacg 189
<210>11
<211>150
<212>DNA
<213> Escherichia coli
<400>11
cagttttata ccgttaagtc tggcgacact ctgagtgcca tttccaaaca ggtctacggt 60
aacgctaatc tgtacaataa aatcttcgaa gcgaataaac cgatgctaaa aagcccggat 120
aaaatttatc cggggcaagt gttgcgtatt 150
<210>12
<211>447
<212>DNA
<213> Escherichia coli
<400>12
atgggtctgt tcaattttgt gaaagatgcc ggagaaaaac tctgggacgc ggttacaggt 60
cagcacgata aagacgatca ggcgaagaag gtgcaggagc atctgaacaa aaccggtata 120
ccggatgccg ataaagtgaa tattcaaatt gccgacggca aagcgacggt cactggtgac 180
ggcctgagtc aggaggcgaa ggagaaaatc cttgttgcgg tggggaatat ttccggtatt 240
gccagtgtcg atgatcaggt gaaaacggcg acaccagcca ctgccagcca gttttatacc 300
gttaagtctg gcgacactct gagtgccatt tccaaacagg tctacggtaa cgctaatctg 360
tacaataaaa tcttcgaagc gaataaaccg atgctaaaaa gcccggataa aatttatccg 420
gggcaagtgt tgcgtattcc ggaagag 447
<210>13
<211>626
<212>PRT
<213> Artificial sequence
<220>
<223>GEP II 1.0
<400>13
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Trp Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Ile Asp Met Gly Leu Phe Asn Phe Val Lys Asp Ala
225 230 235 240
Gly Glu Lys Leu Trp Asp Ala Val Thr Gly Gln His Asp Lys Asp Asp
245 250 255
Gln Ala Lys Lys Val Gln Glu His Leu Asn Lys Thr Gly Ile Pro Asp
260 265 270
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Gly Lys Ala Thr Val Thr
275 280 285
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
290 295 300
Gly Asn Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr Ala
305 310 315 320
Thr Pro Ala Thr Ala Ser Gln Phe Tyr Thr Val Lys Ser Gly Asp Thr
325 330 335
Leu Ser Ala Ile Ser Lys Gln Val Tyr Gly Asn Ala Asn Leu Tyr Asn
340 345 350
Lys Ile Phe Glu Ala Asn Lys Pro Met Leu Lys Ser Pro Asp Lys Ile
355 360 365
Tyr Pro Gly Gln Val Leu Arg Ile Pro Glu Glu Glu Phe Met Asp Gly
370 375 380
Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
385 390 395 400
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Lys
405 410 415
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
420 425 430
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
435 440 445
Gly Gly Ser Gly Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly
450 455 460
Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys
465 470 475 480
Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
485 490 495
Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
500 505 510
Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr
515 520 525
Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu
530 535 540
Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
545 550 555 560
Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
565 570 575
Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
580 585 590
His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala
595 600 605
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
610 615 620
Ile Glu
625
<210>14
<211>127
<212>PRT
<213> Artificial sequence
<220>
<223>GEPII 2.7
<400>14
Gln Ala Lys Lys Val Gln Glu His Leu Asn Lys Thr Gly Ile Pro Asp
1 5 10 15
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Gly Lys Ala Thr Val Thr
20 25 30
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
35 40 45
Gly Asn Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr Ala
50 55 60
Thr Pro Ala Thr Ala Ser Gly Gly Gly Gly Ser Gly Gly Gln Phe Tyr
65 70 75 80
Thr Val Lys Ser Gly Asn Thr Leu Ser Ala Ile Ser Lys Gln Val Tyr
85 90 95
Gly Asn Ala Asn Leu Tyr Asn Lys Ile Phe Gln Ala Asn Lys Pro Met
100 105 110
Leu Lys Ser Pro Asn Lys Ile Tyr Pro Gly Gln Val Leu Arg Ile
115 120 125
<210>15
<211>130
<212>PRT
<213> Artificial sequence
<220>
<223>GEPII 2.10
<400>15
Gln Ala Lys Lys Val Gln Glu His Leu Asn Lys Thr Gly Ile Pro Asp
1 5 10 15
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Gly Lys Ala Thr Val Thr
20 25 30
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
35 40 45
Gly Asn Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr Ala
50 55 60
Thr Pro Ala Thr Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
65 70 75 80
Gln Phe Tyr Thr Val Lys Ser Gly Asn Thr Leu Ser Ala Ile Ser Lys
85 90 95
Gln Val Tyr Gly Asn Ala Asn Leu Tyr Asn Lys Ile Phe Gln Ala Asn
100 105 110
Lys Pro Met Leu Lys Ser Pro Asn Lys Ile Tyr Pro Gly Gln Val Leu
115 120 125
Arg Ile
130
<210>16
<211>135
<212>PRT
<213> Artificial sequence
<220>
<223>GEPII 2.15
<400>16
Gln Ala Lys Lys Val Gln Glu His Leu Asn Lys Thr Gly Ile Pro Asp
1 5 10 15
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Gly Lys Ala Thr Val Thr
20 25 30
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
35 40 45
Gly Asn Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr Ala
50 55 60
Thr Pro Ala Thr Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
65 70 75 80
Gly Gly Gly Gly Ser Gln Phe Tyr Thr Val Lys Ser Gly Asn Thr Leu
85 90 95
Ser Ala Ile Ser Lys Gln Val Tyr Gly Asn Ala Asn Leu Tyr Asn Lys
100 105 110
Ile Phe Gln Ala Asn Lys Pro Met Leu Lys Ser Pro Asn Lys Ile Tyr
115 120 125
Pro Gly Gln Val Leu Arg Ile
130 135
<210>17
<211>142
<212>PRT
<213> Artificial sequence
<220>
<223>GEPII 2.22
<400>17
Gln Ala Lys Lys Val Gln Glu His Leu Asn Lys Thr Gly Ile Pro Asp
1 5 10 15
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Gly Lys Ala Thr Val Thr
20 25 30
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
35 40 45
Gly Asn Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr Ala
50 55 60
Thr Pro Ala Thr Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gln Phe Tyr Thr
85 90 95
Val Lys Ser Gly Asn Thr Leu Ser Ala Ile Ser Lys Gln Val Tyr Gly
100 105 110
Asn Ala Asn Leu Tyr Asn Lys Ile Phe Gln Ala Asn Lys Pro Met Leu
115 120 125
Lys Ser Pro Asn Lys Ile Tyr Pro Gly Gln Val Leu Arg Ile
130 135 140
<210>18
<211>148
<212>PRT
<213> Artificial sequence
<220>
<223>GEP II 2.28
<400>18
Gln Ala Lys Lys Val Gln Glu His Leu Asn Lys Thr Gly Ile Pro Asp
1 5 10 15
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Gly Lys Ala Thr Val Thr
20 25 30
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
35 40 45
Gly Asn Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr Ala
50 55 60
Thr Pro Ala Thr Ala Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
65 70 75 80
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
85 90 95
Gly Ser Gln Phe Tyr Thr Val Lys Ser Gly Asn Thr Leu Ser Ala Ile
100 105 110
Ser Lys Gln Val Tyr Gly Asn Ala Asn Leu Tyr Asn Lys Ile Phe Gln
115 120 125
Ala Asn Lys Pro Met Leu Lys Ser Pro Asn Lys Ile Tyr Pro Gly Gln
130 135 140
Val Leu Arg Ile
145
<210>19
<211>124
<212>PRT
<213> Artificial sequence
<220>
<223>GEPII 2.4
<400>19
Gln Ala Lys Lys Val Gln Glu His Leu Asn Lys Thr Gly Ile Pro Asp
1 5 10 15
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Gly Lys Ala Thr Val Thr
20 25 30
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
35 40 45
Gly Asn Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr Ala
50 55 60
Thr Pro Ala Thr Ala Ser Gly Gly Gly Gly Gln Phe Tyr Thr Val Lys
65 70 75 80
Ser Gly Asn Thr Leu Ser Ala Ile Ser Lys Gln Val Tyr Gly Asn Ala
85 9095
Asn Leu Tyr Asn Lys Ile Phe Gln Ala Asn Lys Pro Met Leu Lys Ser
100 105 110
Pro Asn Lys Ile Tyr Pro Gly Gln Val Leu Arg Ile
115 120
<210>20
<211>127
<212>PRT
<213> Artificial sequence
<220>
<223>GEPII 2.7
<400>20
Gln Ala Lys Lys Val Gln Glu His Leu Asn Lys Thr Gly Ile Pro Asp
1 5 10 15
Ala Asp Lys Val Asn Ile Gln Ile Ala Asp Gly Lys Ala Thr Val Thr
20 25 30
Gly Asp Gly Leu Ser Gln Glu Ala Lys Glu Lys Ile Leu Val Ala Val
35 40 45
Gly Asn Ile Ser Gly Ile Ala Ser Val Asp Asp Gln Val Lys Thr Ala
50 55 60
Thr Pro Ala Thr Ala Ser Gly Gly Gly Gly Ser Gly Gly Gln Phe Tyr
65 70 75 80
Thr Val Lys Ser Gly Asn Thr Leu Ser Ala Ile Ser Lys Gln Val Tyr
85 90 95
Gly Asn Ala Asn Leu Tyr Asn Lys Ile Phe Gln Ala Asn Lys Pro Met
100 105 110
Leu Lys Ser Pro Asn Lys Ile Tyr Pro Gly Gln Val Leu Arg Ile
115 120 125
<210>21
<211>641
<212>PRT
<213> Artificial sequence
<220>
<223>R-GEPII 1.0
<400>21
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Phe Gly Tyr Gly Val Ala Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser His Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Ser
225 230 235 240
Arg Gly Pro Tyr Ser Ile Val Ser Pro Lys Cys Ile Asp Met Gly Leu
245 250 255
Phe Asn Phe Val Lys Asp Ala Gly Glu Lys Leu Trp Asp Ala Val Thr
260 265 270
Gly Gln His Asp Lys Asp Asp Gln Ala Lys Lys Val Gln Glu His Leu
275 280 285
Asn Lys Thr Gly Ile Pro Asp Ala Asp Lys Val Asn Ile Gln Ile Ala
290 295 300
Asp Gly Lys Ala Thr Val Thr Gly Asp Gly Leu Ser Gln Glu Ala Lys
305 310 315 320
Glu Lys Ile Leu Val Ala Val Gly Asn Ile Ser Gly Ile Ala Ser Val
325 330 335
Asp Asp Gln Val Lys Thr Ala Thr Pro Ala Thr Ala Ser Gln Phe Tyr
340 345 350
Thr Val Lys Ser Gly Asp Thr Leu Ser Ala Ile Ser Lys Gln Val Tyr
355 360 365
Gly Asn Ala Asn Leu Tyr Asn Lys Ile Phe Glu Ala Asn Lys Pro Met
370 375 380
Leu Lys Ser Pro Asp Lys Ile Tyr Pro Gly Gln Val Leu Arg Ile Pro
385 390 395 400
Glu Glu Glu Phe Met Val Ser Lys Gly Glu Glu Leu Ile Lys Glu Asn
405 410 415
Met Arg Met Lys Val Val Met Glu Gly Ser Val Asn Gly His Gln Phe
420 425 430
Lys Cys Thr Gly Glu Gly Glu Gly Asn Pro Tyr Met Gly Thr Gln Thr
435 440 445
Met Arg Ile Lys Val Ile Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp
450 455 460
Ile Leu Ala Thr Ser Phe Met Tyr Gly Ser Arg Thr Phe Ile Lys Tyr
465 470 475 480
Pro Lys Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe
485 490 495
Thr Trp Glu Arg Val Thr Arg Tyr Glu Asp Gly Gly Val Val Thr Val
500 505 510
Met Gln Asp Thr Ser Leu Glu Asp Gly Cys Leu Val Tyr His Val Gln
515 520 525
Val Arg Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys
530 535 540
Thr Lys Gly Trp Glu Pro Asn Thr Glu Met Met Tyr Pro Ala Asp Gly
545 550 555 560
Gly Leu Arg Gly Tyr Thr His Met Ala Leu Lys Val Asp Gly Gly Gly
565 570 575
His Leu Ser Cys Ser Phe Val Thr Thr Tyr Arg Ser Lys Lys Thr Val
580 585 590
Gly Asn Ile Lys Met Pro Gly Ile His Ala Val Asp His Arg Leu Glu
595 600 605
Arg Leu Glu Glu Ser Asp Asn Glu Met Phe Val Val Gln Arg Glu His
610 615 620
Ala Val Ala Lys Phe Ala Gly Leu Gly Gly Gly Met Asp Glu Leu Tyr
625 630 635 640
Lys
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