CN109666075B

CN109666075B - Glutamine optical probe and preparation method and application thereof

Info

Publication number: CN109666075B
Application number: CN201910156124.1A
Authority: CN
Inventors: 杨弋; 赵玉政; 李写; 顾燕芳; 胡晗阳; 陈念
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2019-03-01
Filing date: 2019-03-01
Publication date: 2022-05-31
Anticipated expiration: 2039-03-01
Also published as: CN109666075A

Abstract

The invention relates to a glutamine optical probe and a preparation method and application thereof. In one aspect, the invention relates to an optical probe comprising a glutamine-sensitive polypeptide or functional variant thereof and an optically active polypeptide or functional variant thereof, wherein the optically active polypeptide or functional variant thereof is within the sequence of the glutamine-sensitive polypeptide or functional variant thereof. The invention also relates to a preparation method of the probe and application of the probe in detecting amino acid. The glutamine optical probe provided by the invention has relatively small molecular weight, is easy to mature, has large fluorescence dynamic change and good specificity, can be expressed in different subcellular organelles of cells, and can detect amino acid in a high-flux and quantitative manner inside and outside the cells.

Description

Glutamine optical probe and preparation method and application thereof

Technical Field

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

Background

The amino acid is a compound in which a hydrogen atom on a carbon atom of a carboxylic acid is substituted with an amino group, and is an organic compound containing a basic amino group and an acidic carboxyl group. The amino acids obtained by proteolysis are all alpha-amino acids with amino groups attached to the alpha-carbon, and only twenty are those which are the basic units constituting proteins. Amino acids play a role in the human body through metabolism, such as synthesis of tissue proteins; synthetic acids, hormones, antibodies, creatine and other ammonia-containing substances; conversion to carbohydrates and fats; or to carbon dioxide and water and urea, producing energy.

Glutamine is one of the 20 basic amino acids, and glutamine can produce another amino acid, which itself is also derived from this amino acid, i.e., glutamic acid. Glutamine reacts with water to produce glutamic acid and ammonia. The produced ammonia can neutralize the hydrogen ions in the kidney tubules, which is the principle of this amino acid in regulating the acid-base balance in the kidney. Glutamine is very important for muscle, and it can synthesize muscle protein to prevent muscle atrophy. It is used in some diseases to treat muscle atrophy. Glutamine has a glucose-producing effect and is capable of producing glucose and providing energy to our body. It has also been found to regulate blood glucose levels. Normal blood glucose levels are of great importance to our brain, since our brain uses glucose directly. Glutamine is a carbon source involved in the citric acid cycle. It is very important for the function of the intestine, which is the main site for providing nutrients. It can promote the normal function of intestinal villus. Glutamine can be found to be consumed in large amounts in human leukocytes, suggesting that it is important for the immune system of the human body. It can produce glutathione in the liver, which is important for the elimination of harmful free radicals.

There are studies that indicate that glutamine metabolism is involved in tumor growth and proliferation, including glutamine metabolism providing both a nitrogen and carbon source (Young VR et al, J Nutr 2001,131(9Suppl): 2449S-2459S; disuccinon 2486S-2487S), maintenance of redox homeostasis (Xu Y et al, J Cell Physiol,1997,170(2): 192-. The autonomy of tumor cells in the regulation of metabolism and the dependence on glutamine are largely due to the activation of protooncogenes and the inactivation of tumor suppressor genes, mainly including the oncogene MYC (Yuneva M et al, J Cell Biol, 2007,178 (1): 93-105; Gao P et al, Nature, 2009,458 (7239): 762-.

It is precisely because glutamine has the above-mentioned important role, and therefore the detection of the glutamine content is also particularly important. Common detection methods for glutamine are capillary electrophoresis (Li X-t et al, Chem Res Chin Univ 2013,29(3): 434-.

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

Disclosure of Invention

The invention aims to provide a probe and a method for detecting glutamine in real time and in real time in a cell with high flux.

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

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

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

In one embodiment, the glutamine sensitive polypeptide includes glutamine binding proteins and functional variants thereof. In one embodiment, the glutamine binding protein is derived from Escherichia coli (Escherichia coli) or Salmonella (Salmonella spp.). In one embodiment, the glutamine binding protein is derived from or is GlnH or a functional variant thereof. In one embodiment, the glutamine sensitive polypeptide is a glutamine binding protein having the sequence shown in SEQ ID NO. 1 or a functional variant thereof, or a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity thereto.

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

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

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

The optically active polypeptide of the present invention can be located at any position of the glutamine sensitive polypeptide described herein. In one embodiment, the optically active polypeptide is located between residues 175-185 (e.g., residues 177-180) of the glutamine-sensitive polypeptide, the numbering corresponding to the full length of the glutamine-sensitive polypeptide. In one embodiment, the optically active polypeptide replaces one or more amino acids between residues 175-185 (e.g., residues 177-180) of the glutamine-sensitive polypeptide, the numbering corresponding to the full length of the glutamine-sensitive polypeptide.

In one embodiment, the optically active polypeptide is located at a site of the glutamine sensitive polypeptide selected from the group consisting of: 177/178, 177/179, 177/180, 178/179, 178/180, and 179/180. Herein, if two numbers in the site expressed in the form of "X/Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids described in the numbers. For example, a position at position 177/178 indicates that the optically active polypeptide is located between amino acids 177 and 178 of the glutamine sensitive polypeptide. If two numbers in the position indicated in the form "X/Y" are not consecutive integers, this indicates that the optically active polypeptide replaces an amino acid between the amino acids indicated by the numbers. For example, position 177/180 indicates that the optically active polypeptide replaces amino acids 178-179 of the glutamine sensitive polypeptide.

In an exemplary embodiment, the optical probe of the present invention, type B1-A-B2, can be a probe formed when cpYFP is located at position 177/178, 177/179, 177/180, 178/179 or 178/180 of GlnH, as shown in SEQ ID NO: 10-14. In one embodiment, the optical probe of the invention has or consists of the sequence shown in SEQ ID NO 10-14.

The invention also provides a glutamine sensitive polypeptide described herein having one or more mutations, e.g., a glutamine binding protein having one or more mutations, e.g., a polypeptide set forth in SEQ ID No. 1 having one or more mutations. The amino acid mutation comprises modification, substitution, deletion or truncation of the sequence of the amino acid. The mutation is selected from, for example, mutations at positions D10, R75, and D157. Illustratively, the mutation is selected from the group consisting of D10N, R75K, R75M, D157N, D10N/D157N and D10N/R75M/D157N.

The glutamine-sensitive polypeptide (e.g., glutamine-binding protein) in the optical probe of the present invention may comprise one or more amino acid mutations. The amino acid mutation includes modification, substitution, deletion or truncation of an amino acid. In one embodiment, the mutation is, for example, a mutation at a position selected from the group consisting of D10, R75, and D157 of a glutamine binding protein (e.g., a polypeptide set forth in SEQ ID NO: 1). In one embodiment, the mutation is selected from the group consisting of D10N, R75K, R75M, D157N, D10N/D157N and D10N/R75M/D157N. In an exemplary embodiment, the optical probe of the invention has or consists of the sequence shown in SEQ ID NO 15-20.

In an exemplary embodiment, the optical probe of the invention B1-A-B2 can be a probe formed when GlnH is fused with cpYFP at position 177/180 and has mutations D10N, R75K, R75M, D157N, D10N/D157N or D10N/R75M/D157N, as shown in SEQ ID NO: 15-20.

The optical probe provided by the invention comprises any one of amino acid sequences SEQ ID NO 10-20 or a variant thereof. In one embodiment, the present invention provides an optical probe comprising a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of amino acid sequences SEQ ID NO 10-20. In a preferred embodiment, the present invention provides an optical probe comprising a sequence substantially similar or identical to any of the amino acid sequences SEQ ID NO 10-20.

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

Subcellular organelles described herein include cytoplasm, mitochondria, nucleus, cell membrane, golgi apparatus, and the like. In some embodiments, the tag for purification or the tag for immunoblotting comprises 6 histidine (6 × His), glutathione S-transferase (GST), and/or Flag.

The invention also provides nucleic acid sequences encoding the optical probes or fusion polypeptides described herein, or complements thereof. In one embodiment, the invention provides a nucleic acid sequence encoding an amino acid sequence set forth in any one of SEQ ID NOs 10-20. In one embodiment, the nucleic acid sequence of the invention comprises any of the nucleotide sequences SEQ ID NO 21-24 or a variant thereof. In a preferred embodiment, the invention provides a nucleic acid sequence comprising a sequence having 99%, 95%, 90%, 80%, 70% or 50% identity to any one of the nucleotide sequences SEQ ID NO 21-24. In another preferred embodiment, the present invention provides a nucleic acid sequence comprising a nucleotide sequence substantially similar or identical to any of the nucleotide sequences SEQ ID NO 21-24.

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

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

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

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

The invention also provides a detection kit comprising a glutamine optical probe or fusion polypeptide described herein or a glutamine optical probe or fusion polypeptide prepared by a method described herein. The kit detects glutamine.

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

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

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

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

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

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

The invention has the beneficial effects that: the glutamine optical probe provided by the invention is easy to mature, has large fluorescence dynamic change and good specificity, can be expressed in cells by a gene operation method, can be used for real-time positioning, high-flux and quantitative detection of amino acid inside and outside the cells, and saves the time-consuming step of sample processing. The experimental effect shows that the highest response of the glutamine optical probe provided by the application to amino acid reaches more than 3.5 times of that of a control, and the glutamine optical probe can be used for positioning, qualitatively and quantitatively detecting cells in subcellular structures such as cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosome, Golgi body and the like, and can be used for high-flux compound screening and quantitative detection of amino acid in blood.

Drawings

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

FIG. 1 is an SDS-PAGE pattern of exemplary glutamine optical probes described in example 1;

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

FIG. 3 is a graph of the change in glutamine response of an exemplary glutamine optical probe comprising a cPGFP and a glutamine binding protein described in example 3;

FIG. 4 is a graph of the change in glutamine response of an exemplary glutamine optical probe comprising a cPBF and a glutamine binding protein according to example 4;

FIG. 5 is a graph of the change in glutamine response of an exemplary glutamine optical probe comprising cpmApple and a glutamine binding protein according to example 5;

FIG. 6 is a bar graph of the response of an exemplary optical probe described in example 6 to glutamine of a glutamine fusion protein having cpYFP at position 177/180 and having a mutation at a position selected from the group consisting of D10, R75, and R75;

FIG. 7 is a graph of the fluorescence spectrum properties of an exemplary glutamine optical probe described in example 7;

FIG. 8 is a titration curve of an exemplary optical glutamine probe described in example 7 for varying concentrations of glutamine;

FIG. 9 is a photograph of subcellular organelle localization in a mammalian cell of an exemplary glutamine optical probe described in example 8;

FIG. 10 is a schematic illustration of the detection of glutamine within different subcellular organelles in mammalian cells for an exemplary glutamine optical probe described in example 8;

FIG. 11 is a dot plot of high throughput compound screening at the viable cell level using the exemplary glutamine optical probe described in example 9;

FIG. 12 is a bar graph of the quantification of glutamine in mouse and human blood by the exemplary glutamine optical probe described in example 10.

Detailed Description

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

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

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

The glutamine sensitive polypeptide of the present invention includes, but is not limited to, glutamine-binding protein, QBP) or variants thereof having more than 90% homology. The glutamine-binding protein of the present invention can be derived from Escherichia coli (Escherichia coli) or Salmonella (Salmonella spp.), and includes any polypeptide or protein having more than 90% homology with glutamine-binding protein and sensitive to glutamine. Glutamine-binding proteins have a structure in which two α/β globular domains, which are typical of periplasmic binding proteins, are connected by a hinge, and can bind glutamine. Glutamine-binding proteins can sense the change of glutamine concentration in periplasm, and the spatial conformation of the glutamine-binding proteins can be greatly changed in the process of dynamic change of the glutamine concentration. Illustratively, glutamine binding proteins include GlnH proteins.

The term "optical probe" as used herein refers to a glutamine-sensitive polypeptide fused to an optically active polypeptide. The inventors have found that the conformational change of a glutamine-sensitive polypeptide, such as a glutamine-binding protein, upon binding to a physiological concentration of glutamine, results in a conformational change in an optically active polypeptide, such as a fluorescent protein, which in turn results in a change in the optical properties of the optically active polypeptide. The presence and/or level of glutamine can be detected and analyzed by plotting a standard curve with the aid of the fluorescence of the fluorescent protein measured at different glutamine concentrations. Exemplary glutamine binding protein GlnH is shown in SEQ ID NO 1. When describing the optical probe of the present invention (e.g., when describing the site or mutation site where the optically active polypeptide is located), reference to the amino acid residue numbering is made to SEQ ID NO:1, and the alanine (A) after the methionine (M) at the beginning of the sequence is counted as residue 1. However, those skilled in the art know the corresponding residue numbering of other similar glutamine binding proteins.

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

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

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

The green fluorescent protein GFP was originally extracted from Victoria luminifera (Aequorea Victoria), and was composed of 238 amino acids and had a molecular weight of approximately 26 kDa. GFP is a unique barrel-shaped structure formed by 12 beta-folded strands, and a chromogenic tripeptide (Ser65-Tyr66-Gly67) is wrapped in the GFP. When in the presence of oxygen, it spontaneously forms a chromophore structure of p-hydroxybenzylideneimidazolidinone to generate fluorescence. GFP produces fluorescence without the need for cofactors, and fluorescence is very stable and a good imaging tool. GFP has two excitation peaks, the main peak at 395nm can generate 508nm emission, and the excitation light irradiation at the shoulder 475nm can generate 503nm emission. Exemplary cpGFP is shown in SEQ ID NO 6

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

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

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

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

In the optical probe of the present invention, the optically active polypeptide may be located at any position of the glutamine sensitive polypeptide. In one embodiment, the optically active polypeptide is positioned anywhere in the N-C direction of the glutamine sensitive polypeptide that is in the N-C direction. Specifically, the optically active polypeptide is located in a flexible region of the glutamine sensitive polypeptide, wherein the flexible region refers to some specific structures such as a ring-shaped domain existing in a higher-order structure of the protein, the domains have higher mobility and flexibility compared with other higher-order structures of the protein, and the region can dynamically change a spatial structure conformation after the protein is combined with a ligand. The flexible region in the present invention mainly refers to the region where the fusion site is located in the glutamine binding protein, such as the region of amino acid residue 175-185. In one embodiment, the optically active polypeptide is located between amino acid residues 175-185 of the amino acid sequence of the glutamine binding protein. Illustratively, the optically active polypeptide is located at residues 177/178, 177/179, 177/180, 178/179, 178/180 and/or 179/180 of the amino acid sequence of the glutamine binding protein, as shown in SEQ ID NO 10-15.

The term "variant" or "mutant" as used herein in reference to a polypeptide or protein includes variants having the same function as the polypeptide or protein, but differing in sequence. These variants include, but are not limited to: a sequence obtained by deleting, inserting and/or substituting one or more (usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 5) amino acids in the sequence of the polypeptide or protein, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the carboxyl terminal and/or the amino terminal thereof. Without wishing to be bound by theory, amino acid residues are changed without changing the overall configuration and function of the polypeptide or protein, i.e., function conservative mutations. For example, in the art, substitutions with amino acids having similar or analogous properties will not generally alter the function of the polypeptide or protein. Amino acids with similar properties are often referred to in the art as families of amino acids with similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Also, for example, the addition of one or more amino acids at the amino-and/or carboxy-terminus will not generally alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of genetically coded amino acids. It is well known to those skilled in the art that in gene cloning procedures, it is often necessary to design appropriate cleavage sites, which may introduce one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. Also for example, to construct a fusion protein, to facilitate expression of a recombinant protein, to obtain a recombinant protein that is automatically secreted outside of a host cell, or to facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, for example, including, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, glutathione S-transferase (GST), maltose E binding protein, protein a, a tag such as 6His or Flag, or a proteolytic enzyme site for factor Xa or thrombin or enterokinase. Variants of a polypeptide or protein may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants may further comprise a polypeptide or protein having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the polypeptide or protein.

The optical probe of the present invention may comprise a glutamine-sensitive polypeptide having a mutation, for example a glutamine-binding protein having a mutation. Such as mutations at positions D10, R75, and/or D157 of a glutamine-sensitive polypeptide (e.g., a glutamine-binding protein). Illustratively, the mutation is selected from the group consisting of D10N, R75K, R75M, D157N, D10N/D157N and D10N/R75M/D157N.

In an exemplary embodiment, the optical probe of the invention, B1-A-B2, can be a probe fused with cpYFP at position 177/180 of GlnH and having mutations D10N, R75K, R75M, D157N, D10N/D157N or D10N/R75M/D157N, as shown in SEQ ID NO: 15-20.

The term "identity" or "percent identity" in two or more polypeptide or nucleic acid molecule sequences refers to two or more sequences or subsequences that are the same or wherein a percentage of amino acid residues or nucleotides are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) over a window of comparison or designated region, when compared and aligned for maximum correspondence by manual alignment and visual inspection using methods known in the art, e.g., sequence comparison algorithms. For example, preferred algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al (1977) Nucleic Acids Res.25:3389 and Altschul et al (1990) J.Mol.biol.215:403, respectively.

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

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

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

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

The term "nucleic acid" or "nucleotide" as used herein may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The term "variant" as used herein in reference to a nucleic acid may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substituted variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the protein encoded thereby. A nucleic acid of the invention can comprise a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the nucleic acid sequence. The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR).

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

After obtaining the nucleotide sequence for coding the optical probe, the invention brings the nucleotide sequence for coding the optical probe into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" are used interchangeably herein and refer to prokaryotic or eukaryotic vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors, which are capable of replication and stable expression in a host, and one important feature of these recombinant vectors is that they typically contain expression control sequences. The term "expression control sequence" as used herein refers to an element which can be operably linked to a gene of interest to control transcription, translation and expression of the gene of interest, and may be an origin of replication, a promoter, a marker gene or a translation control element, including enhancers, operators, terminators, ribosome binding sites, and the like, and the choice of expression control sequence depends on the host cell used. Recombinant vectors suitable for use in the present invention include, but are not limited to, bacterial plasmids. In a recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. Those skilled in the art are familiar with methods which can be used to construct expression vectors containing the coding sequences of the fusion proteins of the present invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTR of retrovirus, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector may be a commercially available pRSETb vector without any other special requirement. Illustratively, the nucleotide sequence encoding the optical probe and the expression vector are subjected to double enzyme digestion by BamHI and EcoRI respectively, and then enzyme digestion products of the nucleotide sequence and the expression vector are connected to obtain the recombinant expression vector. The invention has no special restriction on the specific steps and parameters of enzyme digestion and connection, and the conventional steps and parameters in the field can be adopted.

After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including the fusion protein. Such transfer procedures may be carried out by conventional techniques known to those skilled in the art, such as transformation or transfection. The host cell of the invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, and is a place for recombinant gene amplification, and an ideal receptor cell should meet two conditions of easy acquisition and proliferation. The "host cells" of the present invention may include prokaryotic and eukaryotic cells, including in particular bacterial cells, yeast cells, insect cells and mammalian cells. Specific examples thereof include bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, and the like, including but not limited to those host cells described above. The host cell is preferably a variety of cells that facilitate expression or fermentative production of the gene product, such cells being well known and commonly used in the art. An exemplary host cell used in the examples of the present invention is Escherichia coli JM109-DE3 strain. It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

The methods of transfer to host cells described herein are conventional in the art and include calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemically mediated transfer, or electroporation. When the host is a prokaryote such as E.coli, the method is preferably CaCl₂Method or MgCl₂Methods, the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

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

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

The invention also provides application of the glutamine optical probe in real-time amino acid positioning, quantitative detection and high-throughput compound screening. In one aspect, the glutamine optical probe is preferably connected with signal peptides at different parts of a cell, transferred into the cell, and used for carrying out real-time positioning on amino acid by detecting the strength of a fluorescence signal in the cell; and (4) carrying out quantitative detection on the corresponding amino acid through an amino acid standard dripping curve. The amino acid standard dripping curve is drawn according to fluorescence signals of the glutamine optical probe under the condition of different concentrations of amino acid. The glutamine optical probe is directly transferred into cells, and in the real-time positioning and quantitative detection process of amino acid, a time-consuming sample processing process is not needed, so that the glutamine optical probe is more accurate. When the glutamine optical probe is used for screening high-flux compounds, different compounds are added into a cell culture solution, and the change of the amino acid content is measured, so that the compounds influencing the amino acid content change are screened. The application of the glutamine optical probe in real-time amino acid positioning, quantitative detection and high-throughput compound screening is non-diagnosis and treatment purposes and does not relate to diagnosis and treatment of diseases. The amino acid may be any amino acid, such as glutamine.

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

Examples

The following examples are provided to illustrate the glutamine optical probe of the present invention in detail, but they should not be construed as limiting the scope of the present invention.

I. Test materials and reagents

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

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

The DNA purification kit used in the examples was purchased from BBI (Canada) and the general plasmid minipump kit was purchased from Tiangen Biochemical technology (Beijing) Ltd. The cloning strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and the desalting column packing are both from GE healthcare.

The main instruments used in the examples include: biotek Synergy 2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R desktop high-speed refrigerated centrifuge (Beckman, USA), PCR amplificationZener (Biometra, Germany), sonicator (Ningbo Co.), nucleic acid electrophoresis (Shenneng Bo Co.), fluorescence spectrophotometer (Varian, USA), CO₂Isothermal cell culture chamber (SANYO), inverted fluorescence microscope (japan nikon).

Methods of molecular biology and cell experiments

II.1 Polymerase Chain Reaction (PCR):

1. and (3) target fragment amplification PCR:

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

TABLE 1 PCR amplification reaction System

TABLE 2 PCR amplification procedure

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

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

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

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

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

II.2 endonuclease cleavage reaction:

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

TABLE 6 plasmid vector Dual enzyme digestion System

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

The ends of plasmids or genomes extracted from microorganisms contain phosphate groups, and PCR products do not contain phosphate groups, so that the 5' end base of the PCR product needs to be subjected to phosphate group addition reaction, and only DNA molecules with the phosphate groups at the ends can be subjected to ligation reaction. The phosphorylation reaction system is shown in table 7, wherein T4 PNK is abbreviated as T4 polynucleotide kinase, and is used for addition reaction to the 5' phosphate group of DNA molecule.

TABLE 7 phosphorylation reaction System

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

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

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

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

TABLE 8 blunt-ended fragment ligation reaction System

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

DNA fragments cut by restriction endonucleases will generally produce overhanging sticky ends and can therefore be ligated with sticky end vector fragments containing sequence complementarity to form recombinant plasmids. The ligation reaction system is shown in Table 9, wherein the mass ratio of the PCR product fragment to the vector double-enzyme digestion product is approximately between 2:1 and 6: 1. .

TABLE 9 cohesive end-joining reaction System

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

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

TABLE 10 self-cyclizing ligation reaction System

II.5 preparation and transformation of competent cells

Preparation of competent cells:

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

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

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

Centrifuge at 4000rpm for 10 minutes at 4.4 ℃.

5. Discard the supernatant, resuspend the cells with 5mL of pre-cooled buffer, add resuspension buffer until uniform to a final volume of 50 mL.

6. Ice-bath for 45 min.

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

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

Resuspension buffer CaCl₂(100mM)、MgCl₂(70mM)、NaAc(40mM)

Storage buffer 0.5mL DMSO, 1.9mL 80% glycerol, 1mL 10 × CaCl₂(1M)、1mL 10×MgCl₂(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH₂O

Transformation of competent cells:

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

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

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

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

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

II.6 expression, purification and fluorescence detection of proteins

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

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

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

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

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

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

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

II.7 transfection and fluorescence detection of mammalian cells

1. The pCDNA3.1+ -based glutamine optical probe plasmid was transfected into HeLa by the transfection reagent Lipofectamine2000(Invitrogen) and placed at 37 ℃ with 5% CO₂Cultured in a cell culture box. And carrying out fluorescence detection after the exogenous gene is fully expressed for 24-36 h.

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

Example 1: plasmid for expressing glutamine-binding protein

The glutamine binding protein QBP (GlnH) gene in the agrobacterium tumefaciens gene is amplified by PCR, a PCR product is subjected to gel electrophoresis, then is recovered and is cut by BamHI and EcoRI, and simultaneously, the pRSETb vector is correspondingly cut by double enzyme. After ligation with T4 DNA ligase, MachI was transformed with the product, and the transformed MachI was plated on LB plates (ampicillin 100ug/mL) and incubated overnight at 37 ℃. After plasmid extraction of the MachI transformant, PCR identification is carried out. And (4) carrying out subsequent plasmid construction after the positive plasmid is sequenced correctly.

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

In this example, the following site fusion cpYFP was selected based on pRSETb-QBP to obtain the corresponding pRSETb-QBP-cpYFP plasmid: 177/178, 177/179, 177/180, 178/179, 178/180, or 179/180.

Generating a DNA fragment of cpYFP by utilizing PCR, inactivating the DNA fragment after using a 5 'terminal phosphorization operation, generating a pRSETb-glutamine binding protein linearized vector containing different fracture sites by reverse PCR amplification, connecting the linearized pRSETb-QBP and the cpYFP fragment phosphorylated at the 5' terminal under the action of PEG4000 and T4 DNA ligase to generate recombinant plasmids, placing the plates in a Kodak multifunctional living body imaging system, selecting a clone with yellow fluorescence under the excitation of a FITC channel, and completing sequencing by Shanghai Branch of great Gene science and technology Limited in Heixiang Hua.

After the sequencing was correct, the recombinant plasmid was transformed into JM109(DE3) to induce expression, and the protein was purified and electrophoresed to have a size of around 56Kda by SDS-PAGE. The size of the fusion protein is consistent with the size of the expressed QBP-cpYFP fusion protein containing His-tag purification label of pRSETb-QBP-cpYFP. The results are shown in FIG. 1.

The purified QBP-cpYFP fusion protein was subjected to glutamine response screening, and the detection signal of the fusion fluorescent protein containing 100mM glutamine was divided by the detection signal of the fusion fluorescent protein without glutamine. The fluorescent protein alone and the fusion protein obtained by fusing the fluorescent protein to the N-terminus or C-terminus of QBP were used as controls, respectively, as shown in fig. 2.

The results of the assay in FIG. 2 show that the optical probes responding to glutamine by more than 1.5 times have optical probes fused at positions 177/178, 177/179, 177/180, 178/179 and 178/180 (shown as SEQ ID NO 10-14) or corresponding amino acid positions of the family proteins thereof. The optical probe and the control probe, in which the fluorescent protein was fused at position 179/180, showed little response.

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

The fluorescent probe for glutamine green fluorescent protein was constructed by replacing cpYFP with cpGFP according to the method in example 2. As shown in FIG. 3, the results of the examination revealed that there were optical probes which responded to glutamine by more than 1.5 times, including those fused at positions 177/178, 177/179, 177/180, 178/179 and 178/180. The optical probe and the control probe, in which the fluorescent protein was fused at position 179/180, showed little response.

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

A glutamine blue fluorescent protein fluorescent probe was constructed by replacing cpYFP with cppBFP according to the method in example 2. As shown in FIG. 4, the results of the examination revealed that there were optical probes which responded to glutamine by more than 1.5 times, including those fused at the 177/178, 177/179, 177/180, 178/179 and 178/180 positions. The optical probe and the control probe, in which the fluorescent protein was fused at position 179/180, showed little response.

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

The fluorescent probe for glutamine red fluorescent protein was constructed by replacing cpYFP with cpmpample as in example 2. As shown in FIG. 5, the results of the examination revealed that there were optical probes which responded to glutamine by more than 1.5 times, including those fused at the 177/178, 177/179, 177/180, 178/179 and 178/180 positions. The optical probe and the control probe, in which the fluorescent protein was fused at position 179/180, showed little response.

The results of examples 2-5 show that probes responsive to glutamine were obtained by inserting fluorescent proteins at positions 177/178, 177/179, 177/180, 178/179 and 178/180 of QBP, wherein fusion proteins obtained by inserting QBP at positions 177/180 and 178/180 were most responsive to glutamine. And fusion proteins obtained by fusing fluorescent proteins to the N-terminus or C-terminus of QBP do not respond to glutamine. Therefore, fusion at both ends of the QBP is not effective, and only insertion of fluorescent proteins inside the QBP produces a response, and the choice of the insertion site is critical.

Example 6: expression and detection of mutated cpYFP optical probes

An optical probe mutant is constructed on the basis of QBP-177/180-cpYFP. The plasmid pRSETb-QBP-177/180-cpYFP is linearized by inverse PCR, a primer contains a base sequence of a site to be mutated, an obtained PCR product is added with phosphorus under the action of PNK, T4 DNA ligase and PEG4000 for connection to obtain the site-specific saturated mutation plasmids of the 3 sites D10, R75 and R75, and sequencing is completed by Shanghai division company of the Beijing Liuhe Huada Gene science and technology Limited.

The results are shown in FIG. 6. Fluorescence detection results showed that there were D10N, R75K, R75M and D157N mutants that responded more than 1.5-fold to glutamine. And the double-bulge D10N/D157N and the triple-bulge D10N/R75M/D157N do not respond to glutamine, so the bulge D10N/D157N and the bulge D10N/R75M/D157N can be used as pH correction plasmids of a glutamine fluorescent probe.

EXAMPLE 7 Performance of optical Probe mutants

The purified glutamine optical probe was treated with 0mM glutamine and 100mM glutamine for 10 minutes, respectively, and then fluorescence spectrum was detected using a fluorescence spectrophotometer.

Measurement of excitation spectra: excitation spectra were recorded with an excitation range of 360nm to 510nm and an emission wavelength of 530nm, read every 3 nm. The results show that the probe has two excitation peaks at 420 and 490nm as shown in FIG. 7.

The purified glutamine optical probe is subjected to concentration gradient glutamine detection. After 10 minutes of treatment of the purified probe, the change in the ratio of the fluorescence intensity at 528nm excitation at 420nm to the fluorescence intensity at 528nm excitation at 485nm was detected. The results are shown in FIG. 8, K for 5 glutamine fluorescent probes_d(binding constant) was 2.3. mu.M, 8.5mM, 0.5mM, 12.7mM and 4.5mM in this order, and the range of change was 3.5-fold, 1.6-fold, 2.5-fold, 2.1-fold and 1.6-fold in this order. Therefore, a more appropriate glutamine probe can be selected for quantitative detection according to the glutamine content level in the sample. Preferred detection ranges for QBP-177/180 may be: 0.1-20 μ M; the preferred detection range for QBP-177/180-D10N may be: 1.3mM-55 mM; preferred detection ranges for QBP-177/180-R75K may be: 0.05-5.5 mM; the preferred detection range for QBP-177/180-R75M may be: 1.0-100 mM; preferred detection ranges for QBP-177/180-D157N may be: 0.5mM-25 mM.

Example 8: subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

In this example, different localization signal peptides were used to fuse with the optical probe glutamine optical probe to localize the optical probe to different organelles.

After HeLa cells were transfected with optical probe plasmids fused with different localization signal peptides for 36 hours, they were washed with PBS and placed in HBSS solution for fluorescence detection under FITC channel using an inverted fluorescence microscope. The results are shown in FIG. 9. The glutamine optical probe can be localized to subcellular organelles including cytoplasm, mitochondria, nucleus, cell membrane and Golgi body by fusing with different specific localization signal peptides. Fluorescence is shown in different subcellular structures and the distribution and intensity of fluorescence varies.

Detection was performed in the cytosol and in the mitochondria, respectively, using optical glutamine probes containing the R75K mutation. The HeLa cells transfected with the glutamine probe are washed by PBS, placed in HBSS solution, added with 10mM or 0mM glutamine, and detected by a microplate reader to obtain the ratio of the fluorescence intensity at 528nm excitation of 420nm to the fluorescence intensity at 528nm excitation of 485 nm. The results are shown in FIG. 10. The increase in 485/420 was gradual 30 minutes after the addition of 10mM glutamine, reaching a maximum of 2.4-fold in both cytosol and mitochondria, whereas 485/420 for the control group without glutamine remained unchanged at 1.

Example 9: high throughput compound screening in living cells based on optical probes

In this example, we used HeLa cells expressing cytoplasmic glutamine optical probes for high-throughput compound screening.

Transfected HeLa cells were washed with PBS, treated in HBSS solution (without glutamine) for 1 hour, and then treated with 10. mu.M of the compound for 1 hour. Glutamine was added dropwise to each sample. The change of the ratio of the fluorescence intensity at 528nm excitation of 420nm to the fluorescence intensity at 528nm excitation of 485nm was recorded by a microplate reader. Samples not treated with any compound were used as controls for normalization. The results are shown in FIG. 11. Of the 2000 compounds used, the vast majority of compounds had minimal effect on glutamine entry into cells. 12 compounds can improve the glutamine uptake capability of cells, and 10 compounds can obviously reduce the glutamine uptake of the cells.

Example 10: quantitative detection of glutamine in blood by optical probe

In this example, glutamine in mouse and human blood supernatants was analyzed using a purified glutamine optical probe.

And mixing the glutamine optical probe and the diluted blood supernatant for 10 minutes, and detecting the ratio of the fluorescence intensity at 528nm excitation of 420nm to the fluorescence intensity at 528nm excitation of 485nm by using a microplate reader. As shown in FIG. 12, the glutamine content in the blood of the mouse was about 466 μ M, and the glutamine content in the blood of the human was about 560 μ M.

The above embodiments show that the glutamine optical probe provided by the invention has relatively small protein molecular weight, easy maturation, large fluorescence dynamic change and good specificity, can be expressed in cells by a gene operation method, and can be used for real-time positioning and quantitative detection of amino acid inside and outside the cells; and enables high throughput screening of compounds.

Other embodiments

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

SEQUENCE LISTING

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

<120> glutamine optical probe, preparation method and application thereof

<130> 191302 1CNCN

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> GlnH

<400> 1

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asp Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys Trp Phe Gly Thr

210 215 220

Glu Pro Lys

225

<210> 2

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<223> cpYFP

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

His Lys Leu Glu Tyr Asn

245

<210> 3

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> cpmOrange

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Glu Ala

<210> 4

<211> 250

<212> PRT

<213> Artificial sequence

<220>

<223> cpmKate

<400> 4

Met Gly Gly Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly

1 5 10 15

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

20 25 30

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

35 40 45

Leu Pro Ser Lys Leu Gly His Lys Leu Asn Gly Gly Thr Gly Gly Ser

50 55 60

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

65 70 75 80

Leu Tyr Met Glu Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly

210 215 220

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

225 230 235 240

Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys

245 250

<210> 5

<211> 236

<212> PRT

<213> Artificial sequence

<220>

<223> mCherry

<400> 5

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

1 5 10 15

Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe

20 25 30

Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly

210 215 220

Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 6

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<223> cpGFP

<400> 6

Asn Val Tyr Ile Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn

1 5 10 15

Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Asn

<210> 7

<211> 243

<212> PRT

<213> Artificial sequence

<220>

<223> cpBFP

<400> 7

Asn Val Tyr Ile Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Glu Tyr Asn

<210> 8

<211> 233

<212> PRT

<213> Artificial sequence

<220>

<223> mKate

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Ser Lys Leu Gly His Lys Leu Asn

225 230

<210> 9

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> cpmApple

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Glu Ala

<210> 10

<211> 473

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-177/178-cpYFP

<400> 10

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asp Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Gly Ile Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn

435 440 445

Gly Ala Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr

450 455 460

Lys Lys Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 11

<211> 472

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-177/179-cpYFP

<400> 11

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asp Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Leu Gly His Lys Leu Glu Tyr Asn Ser Leu Glu Ala Gln Gln Tyr Gly

420 425 430

Ile Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn Gly

435 440 445

Ala Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys

450 455 460

Lys Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 12

<211> 471

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-177/180-cpYFP

<400> 12

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asp Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Leu Gly His Lys Leu Glu Tyr Asn Leu Glu Ala Gln Gln Tyr Gly Ile

420 425 430

Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn Gly Ala

435 440 445

Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys

450 455 460

Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 13

<211> 473

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-178/179-cpYFP

<400> 13

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asp Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Ile Leu Gly His Lys Leu Glu Tyr Asn Ser Leu Glu Ala Gln Gln Tyr

420 425 430

Gly Ile Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn

435 440 445

Gly Ala Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr

450 455 460

Lys Lys Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 14

<211> 472

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-178/180-cpYFP

<400> 14

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asp Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Ile Leu Gly His Lys Leu Glu Tyr Asn Leu Glu Ala Gln Gln Tyr Gly

420 425 430

Ile Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn Gly

435 440 445

Ala Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys

450 455 460

Lys Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 15

<211> 471

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-177/180-D10N-cpYFP

<400> 15

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asn Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asp Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Leu Gly His Lys Leu Glu Tyr Asn Leu Glu Ala Gln Gln Tyr Gly Ile

420 425 430

Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn Gly Ala

435 440 445

Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys

450 455 460

Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 16

<211> 471

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-177/180-R75K-cpYFP

<400> 16

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asp Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Leu Gly His Lys Leu Glu Tyr Asn Leu Glu Ala Gln Gln Tyr Gly Ile

420 425 430

Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn Gly Ala

435 440 445

Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys

450 455 460

Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 17

<211> 471

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-177/180-R75M-cpYFP

<400> 17

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

Leu Ala Leu Ala Gly Ile Thr Ile Thr Asp Glu Met Lys Lys Ala Ile

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asp Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Leu Gly His Lys Leu Glu Tyr Asn Leu Glu Ala Gln Gln Tyr Gly Ile

420 425 430

Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn Gly Ala

435 440 445

Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys

450 455 460

Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 18

<211> 471

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-177/180-D157N-cpYFP

<400> 18

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asn Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Leu Gly His Lys Leu Glu Tyr Asn Leu Glu Ala Gln Gln Tyr Gly Ile

420 425 430

Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn Gly Ala

435 440 445

Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys

450 455 460

Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 19

<211> 471

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-177/180-D10N/D157N-cpYFP

<400> 19

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asn Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asn Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Leu Gly His Lys Leu Glu Tyr Asn Leu Glu Ala Gln Gln Tyr Gly Ile

420 425 430

Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn Gly Ala

435 440 445

Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys

450 455 460

Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 20

<211> 471

<212> PRT

<213> Artificial sequence

<220>

<223> GlnH-177/180-D10N/R75M/D157N-cpYFP

<400> 20

Met Ala Asp Lys Lys Leu Val Val Ala Thr Asn Thr Ala Phe Val Pro

1 5 10 15

Phe Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu

20 25 30

Trp Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro

35 40 45

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

50 55 60

Leu Ala Leu Ala Gly Ile Thr Ile Thr Asp Glu Met Lys Lys Ala Ile

65 70 75 80

Asp Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys

85 90 95

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

100 105 110

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

115 120 125

Ile Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr

130 135 140

Met Glu Leu Gly Thr Asn Arg Ala Asp Ala Val Leu His Asn Thr Pro

145 150 155 160

Asn Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Leu Gly His Lys Leu Glu Tyr Asn Leu Glu Ala Gln Gln Tyr Gly Ile

420 425 430

Ala Phe Pro Lys Gly Ser Asp Glu Leu Arg Asp Lys Val Asn Gly Ala

435 440 445

Leu Lys Thr Leu Arg Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys

450 455 460

Trp Phe Gly Thr Glu Pro Lys

465 470

<210> 21

<211> 1416

<212> DNA

<213> Artificial sequence

<220>

<223> GlnH-177/180-cpYFP nucleic acid

<400> 21

atggcggata aaaaattagt tgtcgcgacg gataccgcct tcgttccgtt tgaatttaaa 60

cagggcgata aatatgtggg ctttgacgtt gatctgtggg ctgccatcgc taaagagctg 120

aagctggatt acgaactgaa gccgatggat ttcagtggga tcattccggc actgcaaacc 180

aaaaacgtcg atctggcgct ggcgggcatt accatcaccg acgagcgtaa aaaagcgatc 240

gatttctctg acggctacta caaaagcggc ctgttagtga tggtgaaagc taacaataac 300

gatgtgaaaa gcgtgaaaga tctcgacggg aaagtggttg ctgtgaagag cggtactggc 360

tccgttgatt acgcgaaagc aaacatcaaa actaaagatc tgcgtcagtt cccgaacatc 420

gataacgcct atatggaact gggcaccaac cgcgcagacg ccgttctgca cgatacgcca 480

aacattctgt acttcatcaa aaccgccggt aacggtcagt tcaaagcggt aggttacaac 540

agcgacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc caacttcaag 600

atccgccaca acgtcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 660

cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagctt ccagtccgtc 720

ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 780

gccgggatca ctctcggcat ggacgagctg tacaacgtgg atggcggtag cggtggcacc 840

ggcagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 900

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 960

aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 1020

gtgaccaccc tcggctacgg cctgaagtgc ttcgcccgct accccgacca catgaagcag 1080

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 1140

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1200

aaccgcatcg agctgaaggg catcggcttc aaggaggacg gcaacatcct ggggcacaag 1260

ctggagtaca acctggaagc gcagcaatac ggtattgcgt tcccgaaagg tagcgacgag 1320

ctgcgtgaca aagtcaacgg cgcgttgaaa accctgcgcg agaacggaac ttacaacgaa 1380

atctacaaaa aatggttcgg tactgaaccg aaataa 1416

<210> 22

<211> 1416

<212> DNA

<213> Artificial sequence

<220>

<223> GlnH-177/180-R75K-cpYFP nucleic acid

<400> 22

atggcggata aaaaattagt tgtcgcgacg gataccgcct tcgttccgtt tgaatttaaa 60

cagggcgata aatatgtggg ctttgacgtt gatctgtggg ctgccatcgc taaagagctg 120

aagctggatt acgaactgaa gccgatggat ttcagtggga tcattccggc actgcaaacc 180

aaaaacgtcg atctggcgct ggcgggcatt accatcaccg acgagaaaaa aaaagcgatc 240

gatttctctg acggctacta caaaagcggc ctgttagtga tggtgaaagc taacaataac 300

gatgtgaaaa gcgtgaaaga tctcgacggg aaagtggttg ctgtgaagag cggtactggc 360

tccgttgatt acgcgaaagc aaacatcaaa actaaagatc tgcgtcagtt cccgaacatc 420

gataacgcct atatggaact gggcaccaac cgcgcagacg ccgttctgca cgatacgcca 480

aacattctgt acttcatcaa aaccgccggt aacggtcagt tcaaagcggt aggttacaac 540

agcgacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc caacttcaag 600

atccgccaca acgtcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 660

cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagctt ccagtccgtc 720

ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 780

gccgggatca ctctcggcat ggacgagctg tacaacgtgg atggcggtag cggtggcacc 840

ggcagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 900

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 960

aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 1020

gtgaccaccc tcggctacgg cctgaagtgc ttcgcccgct accccgacca catgaagcag 1080

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 1140

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1200

aaccgcatcg agctgaaggg catcggcttc aaggaggacg gcaacatcct ggggcacaag 1260

ctggagtaca acctggaagc gcagcaatac ggtattgcgt tcccgaaagg tagcgacgag 1320

ctgcgtgaca aagtcaacgg cgcgttgaaa accctgcgcg agaacggaac ttacaacgaa 1380

atctacaaaa aatggttcgg tactgaaccg aaataa 1416

<210> 23

<211> 1416

<212> DNA

<213> Artificial sequence

<220>

<223> GlnH-177/180-R75M-cpYFP nucleic acid

<400> 23

atggcggata aaaaattagt tgtcgcgacg gataccgcct tcgttccgtt tgaatttaaa 60

cagggcgata aatatgtggg ctttgacgtt gatctgtggg ctgccatcgc taaagagctg 120

aagctggatt acgaactgaa gccgatggat ttcagtggga tcattccggc actgcaaacc 180

aaaaacgtcg atctggcgct ggcgggcatt accatcaccg acgagatgaa aaaagcgatc 240

gatttctctg acggctacta caaaagcggc ctgttagtga tggtgaaagc taacaataac 300

gatgtgaaaa gcgtgaaaga tctcgacggg aaagtggttg ctgtgaagag cggtactggc 360

tccgttgatt acgcgaaagc aaacatcaaa actaaagatc tgcgtcagtt cccgaacatc 420

gataacgcct atatggaact gggcaccaac cgcgcagacg ccgttctgca cgatacgcca 480

aacattctgt acttcatcaa aaccgccggt aacggtcagt tcaaagcggt aggttacaac 540

agcgacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc caacttcaag 600

atccgccaca acgtcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 660

cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagctt ccagtccgtc 720

ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 780

gccgggatca ctctcggcat ggacgagctg tacaacgtgg atggcggtag cggtggcacc 840

ggcagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 900

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 960

aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 1020

gtgaccaccc tcggctacgg cctgaagtgc ttcgcccgct accccgacca catgaagcag 1080

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 1140

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1200

aaccgcatcg agctgaaggg catcggcttc aaggaggacg gcaacatcct ggggcacaag 1260

ctggagtaca acctggaagc gcagcaatac ggtattgcgt tcccgaaagg tagcgacgag 1320

ctgcgtgaca aagtcaacgg cgcgttgaaa accctgcgcg agaacggaac ttacaacgaa 1380

atctacaaaa aatggttcgg tactgaaccg aaataa 1416

<210> 24

<211> 1416

<212> DNA

<213> Artificial sequence

<220>

<223> GlnH-177/180-D157N-cpYFP nucleic acid

<400> 24

atggcggata aaaaattagt tgtcgcgacg gataccgcct tcgttccgtt tgaatttaaa 60

cagggcgata aatatgtggg ctttgacgtt gatctgtggg ctgccatcgc taaagagctg 120

aagctggatt acgaactgaa gccgatggat ttcagtggga tcattccggc actgcaaacc 180

aaaaacgtcg atctggcgct ggcgggcatt accatcaccg acgagcgtaa aaaagcgatc 240

gatttctctg acggctacta caaaagcggc ctgttagtga tggtgaaagc taacaataac 300

gatgtgaaaa gcgtgaaaga tctcgacggg aaagtggttg ctgtgaagag cggtactggc 360

tccgttgatt acgcgaaagc aaacatcaaa actaaagatc tgcgtcagtt cccgaacatc 420

gataacgcct atatggaact gggcaccaac cgcgcagacg ccgttctgca caacacgcca 480

aacattctgt acttcatcaa aaccgccggt aacggtcagt tcaaagcggt aggttacaac 540

agcgacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc caacttcaag 600

atccgccaca acgtcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 660

cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagctt ccagtccgtc 720

ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 780

gccgggatca ctctcggcat ggacgagctg tacaacgtgg atggcggtag cggtggcacc 840

ggcagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 900

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 960

aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 1020

gtgaccaccc tcggctacgg cctgaagtgc ttcgcccgct accccgacca catgaagcag 1080

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 1140

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1200

aaccgcatcg agctgaaggg catcggcttc aaggaggacg gcaacatcct ggggcacaag 1260

ctggagtaca acctggaagc gcagcaatac ggtattgcgt tcccgaaagg tagcgacgag 1320

ctgcgtgaca aagtcaacgg cgcgttgaaa accctgcgcg agaacggaac ttacaacgaa 1380

atctacaaaa aatggttcgg tactgaaccg aaataa 1416

Claims

1. An optical probe comprising a glutamine sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the glutamine sensitive polypeptide, wherein,

the glutamine sensitive polypeptide is shown as SEQ ID NO:1, and

the optically active polypeptide is located at a site of the glutamine sensitive polypeptide selected from the group consisting of: 177/178, 177/179, 177/180, 178/179 and 178/180, and

the optically active polypeptide is a fluorescent protein.

2. An optical probe comprising a functional variant of a glutamine-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the functional variant of a glutamine-sensitive polypeptide, wherein,

the glutamine sensitive polypeptide is shown as SEQ ID NO. 1, and the mutation of the functional variant is as follows: D10N, R75K, R75M or D157N, the numbering corresponding to the full length of the glutamine-sensitive polypeptide,

the optically active polypeptide is located at a site of a functional variant of a glutamine sensitive polypeptide selected from the group consisting of: 177/178, 177/179, 177/180, 178/179 and 178/180, and

the optically active polypeptide is a fluorescent protein.

3. A nucleic acid molecule encoding the optical probe of any one of claims 1-2.

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

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4, which is not a plant cell.

6. A method of making the optical probe of any one of claims 1-2, comprising the steps of:

(1) transferring the expression vector of claim 4 into a host cell,

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

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

7. Use of an optical probe according to any one of claims 1-2 or an optical probe prepared by the method of claim 6 for the preparation of a kit for detecting glutamine in a sample.

8. A detection kit comprising the optical probe of any one of claims 1-2 or an optical probe prepared by the method of claim 6.