CN109666075A

CN109666075A - Glutamine optical probe and its preparation method and application

Info

Publication number: CN109666075A
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: 2019-04-23
Anticipated expiration: 2039-03-01
Also published as: CN109666075B

Abstract

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

Description

Glutamine optical probe and its preparation method and application

Technical field

The present invention relates to optical probe technical field more particularly to a kind of glutamine optical probe and preparation method thereof and Using.

Background technique

Amino acid is the compound after the hydrogen atom on carboxylic acid carbon atom is replaced by amino, is containing basic amine group and acidity The organic compound of carboxyl.The amino acid obtained after protein hydrolyzes is all the a-amino acid that amino is connected on α-carbon, and Only twenties kinds, they are the basic units for constituting protein.Amino acid is played a role in human body by metabolism, such as is closed At tissue protein；It synthesizes acid, hormone, antibody, creatine etc. and contains ammoniacal substance；It is changed into carbohydrate and fat；Or it is oxidized to Carbon dioxide and water and urea generate energy.

Glutamine is one of 20 kinds of primary amino acids, and glutamine can produce another amino acid, itself From this amino acid, i.e. glutamic acid.Glutamine and water, which react, produces glutamic acid and ammonia.The ammonia of production can neutralize Hydrogen ion in renal tubule, this is the principle that this amino acid adjusts acid-base balance in kidney.Glutamine is for muscle right and wrong Often important, it can synthesize myogen, prevent the atrophy of muscle.It is in some diseases for treating muscular atrophy.Paddy ammonia Amide has glucose production effect, can generate glucose, provide energy for our body.It is also found to reconcile Blood glucose level.Normal blood glucose level has having very important significance for our brain, because our brain is direct Utilize glucose.Glutamine participates in citrate cycle as a kind of carbon source.It is extremely important for function of intestinal canal, and enteron aisle is The main place of nutrients is provided.It can promote intestinal villi to bring into normal play function.Paddy ammonia can be found in human leukocytes Amide is largely consumed, and illustrates that it is extremely important for the immune system of human body.It can generate glutathione, paddy in liver The sweet peptide of Guang is critically important for the elimination of harmful free radicals.

It is related with tumour growth and increment that some researches show that the metabolism of glutamine, including glutamine metabolism provides nitrogen source With carbon source (2001,131 (9Suppl): 2449S-2459S of Young VR etc., J Nutr；discussion 2486S- 2487S), Redox homeostasis (Xu Y etc., J Cell Physiol, 1997,170 (2): 192-199) and intracellular letter are maintained Number transduction (Csibi A etc., Cell, 2013,153 (4): 840-854).Tumour cell is to the independence of metabolic regulation and to paddy ammonia The dependence of amide is largely derived from the activation of proto-oncogene and the inactivation of tumor suppressor gene, mainly includes oncogene MYC (Yuneva M etc., J Cell Biol, 2007,178 (1): 93-105；Gao P etc., Nature, 2009,458 (7239): 762- 765；The Cancer such as Qing G Cell, 2012,22 (5): 631-644), oncogene RAS (Pylayeva-Gupta Y etc., Nat Rev Cancer,2011,11(11):761-774；) and tumor suppressor gene P53 (Suzuki S etc., Proc Natl Acad Sci USA,2010,107(16):7461-7466)。

There is above-mentioned important role just because of glutamine, therefore the detection of glutamine content is also particularly important. The common detection method of glutamine be capillary electrophoresis (Li X-t etc., Chem Res Chin Univ 2013,29 (3): 434-438；Meng J etc.), high performance liquid chromatography (S, Gunawan etc., Journal of chromatography, 1990, 503(1):177-187；Wadud S etc., Journal of chromatography B, Analytical technologies In the biomedical and life sciences 2002,767 (2): 369-374) etc..

But these detection methods in the prior art be not particularly suited for living cells research, there are many defects: need through Cross time-consuming sample handling processes, such as clasmatosis, separation and Extraction purifying etc.；It cannot be carried out in living cells and subcellular organelle In situ, real-time, dynamic, high-throughput and high-spatial and temporal resolution detection.This field there is still a need for can in the cell, it is outer it is real-time positioning, The method of quantitative, high-throughput detection glutamine.

Summary of the invention

The purpose of the present invention is to provide in the cell, outer real-time positioning, high-throughput, quantitative detection glutamine probe And method.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

In a first aspect, the present invention provides a kind of glutamine optical probe, comprising glutamine sensitivity polypeptide or its function Energy variant and optical activity polypeptide or its functional variety, wherein optical activity polypeptide or its functional variety are located at glutamine sensitivity In the sequence of polypeptide or its functional variety.Glutamine sensitivity polypeptide or its functional variety are become by optical activity polypeptide or its function Body is divided into first part and second part.In one embodiment, optical probe of the present invention is sensitive to glutamine.

The present invention provides a kind of glutamine optical probe, including glutamine sensitivity polypeptide B and optical activity polypeptide A, Wherein optical activity polypeptide A is located in the sequence of glutamine sensitivity polypeptide B, is first by glutamine sensitivity polypeptide B points Divide B1 and second part B2, forms the probe structure of B1-A-B2 formula.

In one embodiment, glutamine sensitivity polypeptide includes glutamine binding protein and its functional variety.? In one embodiment, glutamine binding protein is originated from Escherichia coli (Escherichia coli) or salmonella (Salmonella spp.).In one embodiment, glutamine binding protein is originated from or GlnH or its functional variety. In one embodiment, glutamine sensitivity polypeptide be with sequence shown in SEQ ID NO:1 or its functional variety or with It has the glutamy of the sequence of 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity Amine binding protein.

In one embodiment, optical activity polypeptide is fluorescin or its functional variety.In one embodiment, Fluorescin is selected from yellow fluorescence protein (cpYFP as shown in SEQ ID NO:2), orange fluorescence albumen (such as SEQ ID CpmOrange shown in NO:3), the red fluorescent protein (mKate as shown in SEQ ID NO:4 or 8, such as SEQ ID NO:5 institute The mcherry shown), green fluorescent protein (cpGFP as shown in SEQ ID NO:6), blue fluorescent protein (such as SEQ ID NO: CpBFP shown in 7), apple DsRed (cpmApple as shown in SEQ ID NO:9).Preferably, optical activity polypeptide It is cpYFP.In one embodiment, fluorescin have SEQ ID NO:2-9 in it is any shown in sequence.

In one embodiment, optical probe also includes the one or more connectors for flanking the optical activity polypeptide. Connector of the present invention can be any amino acid sequence of any length.In one embodiment, optical activity polypeptide side The wing includes no more than the connector of 5 amino acid, such as the connector of 0,1,2,3,4 amino acid.In one embodiment, connector Y is located at the N-terminal and/or C-terminal of optical activity polypeptide.In one embodiment, optical probe of the present invention does not include connector.One In a embodiment, optical probe is as follows: first part's B1- optical activity polypeptide A-paddy ammonia of glutamine sensitivity polypeptide The second part B2 of amide sensitivity polypeptide.

In one embodiment, optical probe of the present invention also includes positioning sequence, for example thin for navigating to probe The specific cells device of born of the same parents.

Optical activity polypeptide of the present invention can be located at any position of glutamine sensitivity polypeptide described herein.One In a embodiment, optical activity polypeptide be located at glutamine sensitivity polypeptide residue 175-185 (such as residue 177-180) it Between, number corresponds to the overall length of glutamine sensitivity polypeptide.In one embodiment, optical activity polypeptide displacement glutamine One or more amino acid between the residue 175-185 (such as residue 177-180) of sensitive polypeptide, number correspond to glutamy The overall length of amine sensitivity polypeptide.

In one embodiment, optical activity polypeptide is located at the site selected from the following of glutamine sensitivity polypeptide: 177/178,177/179,177/180,178/179,178/180 and 179/180.Herein, if indicated in the form of " X/Y " Two numbers in site are continuous integers, then it represents that optical activity polypeptide is located between amino acid described in the number.Example Such as being located at site 177/178 indicates that optical activity polypeptide is located between the amino acid 1 77 and 178 of glutamine sensitivity polypeptide.Such as Two numbers in the site that fruit is indicated in the form of " X/Y " are not continuous integers, then it represents that optical activity polypeptide replaces the number Amino acid between amino acid shown in word.Such as indicate that optical activity polypeptide displacement glutamine is sensitive positioned at site 177/180 The amino acid 1 78-179 of polypeptide.

In the exemplary embodiment, B1-A-B2 formula optical probe of the present invention can be when cpYFP is located at the site of GlnH The probe formed when 177/178,177/179,177/180,178/179 or 178/180, as shown in SEQ ID NO:10-14.? In one embodiment, optical probe of the present invention has sequence shown in SEQ ID NO:10-14 or is made from it.

The present invention also provides the glutamine sensitivity polypeptides described herein with one or more mutation, such as with one Or the glutamine binding protein of multiple mutation, such as polypeptide shown in the SEQ ID NO:1 with one or more mutation.Institute State the modification, substitution, the truncation of missing or sequence that amino acid mutation includes amino acid.The mutation for example selected from D10, R75 and The mutation in the sites such as D157.Illustratively, the mutation is selected from D10N, R75K, R75M, D157N, D10N/D157N and D10N/ R75M/D157N。

Glutamine sensitivity polypeptide (such as glutamine binding protein) in optical probe of the present invention may include one or Multiple amino acid mutations.The amino acid mutation includes modification, substitution, missing or the truncation of amino acid.In an embodiment In, D10, R75 and the D157 of the mutation for example selected from glutamine binding protein (such as polypeptide shown in SEQ ID NO:1) The mutation in equal sites.In one embodiment, it is described mutation be selected from D10N, R75K, R75M, D157N, D10N/D157N and D10N/R75M/D157N.In an exemplary embodiment, optical probe of the present invention has shown in SEQ ID NO:15-20 Sequence is made from it.

In the exemplary embodiment, B1-A-B2 formula optical probe of the present invention can have for the 177/180 site fusion of GlnH CpYFP and the spy that there is D10N, R75K, R75M, D157N, D10N/D157N or D10N/R75M/D157N to be formed when being mutated Needle, as shown in SEQ ID NO:15-20.

Optical probe provided by the invention includes any or its variant in amino acid sequence SEQ ID NO:10-20.One In a embodiment, optical probe provided by the invention include with it is any in amino acid sequence SEQ ID NO:10-20 have 35%, 40%, the sequence of 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity.In preferred embodiment In, optical probe provided by the invention includes to be substantially similar or identical with any in amino acid sequence SEQ ID NO:10-20 Sequence.

The present invention also provides fused polypeptides, include optical probe described herein and other polypeptides.In some embodiments, Optical probe described herein also includes the other polypeptides merged therewith.Other polypeptides described herein do not influence the property of optical probe Matter.In some embodiments, other polypeptides are located at the N-terminal and/or C-terminal of the optical probe.In some embodiments, His polypeptide includes the polypeptide that optical probe is navigated to different organelles or subcellular organelle, the label for being used to purify or is used to exempt from The label of epidemic disease trace.There can be connector between optical probe and other polypeptides in fused polypeptide described herein.

Subcellular organelle described herein includes cytoplasm, mitochondria, nucleus, cell membrane and golgiosome etc..In some realities It applies in mode, the label for purifying or the label for immunoblotting include 6 histidines (6*His), glutathione sulphur turn Move enzyme (GST) and/or Flag.

The present invention also provides the nucleic acid sequences or its complementary series that encode optical probe described herein or fused polypeptide.One In a embodiment, the present invention provides a kind of nucleic acid sequence, encodes any shown amino acid sequence in SEQ ID NO:10-20 Column.In one embodiment, nucleic acid sequence of the present invention includes any in nucleotide sequence SEQ ID NO:21-24 or its change Body.In the preferred embodiment, the present invention provides a kind of nucleic acid sequence, comprising in nucleotide sequence SEQ ID NO:21-24 Any sequence with 99%, 95%, 90%, 80%, the 70% or 50% phase same sex.In another preferred embodiment, this hair It is bright that a kind of nucleic acid sequence is provided, comprising with any core being substantially similar or identical in nucleotide sequence SEQ ID NO:21-24 Nucleotide sequence.

It may include encoding optical probe of the present invention the invention further relates to the complementary series of above-mentioned nucleic acid sequence or its variant Or segment, the nucleic acid sequence or its complementary series of analog, derivative, soluble fragments and variant of fusion protein.

Amino acid sequence and nucleic acid sequence in the present invention are preferably provided with unpack format, are more preferably purified to Matter.

The present invention also provides the nucleic acid sequence of the present invention comprising being operatively connected with expression control sequence or its mutually The expression vector of complementary series, nucleic acid sequence encoding optical probe of the present invention or fused polypeptide.In some embodiments, Expression vector is selected from prokaryotic expression carrier, carrier for expression of eukaryon and viral vectors.In some embodiments, prokaryotic expression carrier Such as it is operatively connected to obtain by plasmid pRSETb and nucleic acid sequence as described herein.In some embodiments, expression control Sequence includes replication orgin, promoter, enhancer, operon, terminator, ribosome bind site.

The present invention also provides the cell comprising expression vector of the present invention, which includes and expression control sequence The nucleic acid sequence of the present invention being operatively connected or its complementary series.The cell is expressed optical probe described herein or is melted Close polypeptide.

The present invention also provides include glutamine optical probe described herein or fused polypeptide or method system as described herein The detection kit of standby glutamine optical probe or fused polypeptide.The kit detects glutamine.

The present invention provides the method for preparing optical probe described herein, comprising: provides and visits comprising expressing optics described herein The cell of needle or the carrier of fused polypeptide cultivates the cell under conditions of cell expression, and separation optical probe or Fused polypeptide.

In one embodiment, 1) method for preparing glutamine optical probe described herein or fused polypeptide includes: The expression vector for encoding glutamine optical probe described herein is transferred in host cell；2) it is being suitble to the expression vector Cultivate the host cell under conditions of expression, 3) separation glutamine optical probe.

The present invention also provides the methods of test sample glutamine, comprising: keeps optical probe described herein or fusion more The optical probe or fused polypeptide of peptide or method as described herein preparation are contacted with sample, and the change of detection optics active peptides Change.The detection can in vivo, in vitro, subcellular or in situ carry out.The sample such as blood.

The method that quantitative sample glutamine is also provided herein, comprising: make optical probe described herein or fused polypeptide Or the optical probe or fused polypeptide of method preparation as described herein are contacted with sample, detect the variation of optics active peptides, and According to the glutamine in the quantitative sample of the variation of optical activity polypeptide.

The present invention also provides the methods of screening compounds (such as drug), comprising: makes optical probe described herein or fusion The optical probe or fused polypeptide of polypeptide or method as described herein preparation are contacted with candidate compound, detect optics active peptides Variation, and variation screening compounds according to optical activity polypeptide.The method can screening compounds with high throughput.

It is prepared the present invention also provides glutamine optical probe described herein or fused polypeptide or method as described herein The application of glutamine optical probe or fused polypeptide in glutamine positions in real time.

Beneficial effects of the present invention: glutamine optical probe provided by the invention is easy to mature, and fluorescence dynamic change is big, Specificity is good, and can be expressed in cell by the method for genetic manipulation, outer in the cell can position in real time, is high-throughput, Quantitative detection amino acid eliminates time-consuming processing sample step.Experiment effect shows glutamine light provided herein Learn 3.5 times or more that probe reaches control to the response of the highest of amino acid, and can be in cytoplasm, mitochondria, nucleus, interior Cell is positioned in the subcellular structures such as matter net, lysosome and golgiosome, qualitative and quantitative analysis, and can be carried out Amino acid quantitative detection in high-throughput screening compound and blood.

Detailed description of the invention

Present invention will be further explained below with reference to the attached drawings and examples.

Fig. 1 is that the SDS-PAGE of exemplary glutamine optical probe described in embodiment 1 schemes；

Fig. 2 is that the illustrative glutamine optics comprising cpYFP and glutamine binding protein as described in example 2 is visited Variation diagram is responded for glutamine；

Fig. 3 is that the illustrative glutamine optics comprising cpGFP and glutamine binding protein described in embodiment 3 is visited Variation diagram is responded for glutamine；

Fig. 4 is that the illustrative glutamine optics comprising cpBFP and glutamine binding protein as described in example 4 is visited Variation diagram is responded for glutamine；

Fig. 5 is the illustrative glutamine light comprising cpmApple and glutamine binding protein described in embodiment 5 It learns probe and variation diagram is responded to glutamine；

Fig. 6 is that the illustrative fusion in the site of glutamine binding protein 177/180 described in embodiment 6 has cpYFP And in the histogram that there is in the site selected from D10, R75 and R75 the glutamine optical probe of mutation to respond to glutamine；

Fig. 7 is the fluorescent spectroscopic properties figure of exemplary glutamine optical probe described in embodiment 7；

Fig. 8 is titration curve of the exemplary glutamine optical probe described in embodiment 7 to various concentration glutamine；

Fig. 9 is that the subcellular organelle of exemplary glutamine optical probe in mammalian cells described in embodiment 8 is fixed Position photo；

Figure 10 is described in embodiment 8 to exemplary glutamine optical probe different subcellulars in mammalian cells The schematic diagram to glutamine detection in device；

Figure 11 is exemplary glutamine optical probe described in embodiment 9 in the high-throughput compound of living cells level progress The point diagram of screening；

Figure 12 is exemplary glutamine optical probe described in embodiment 10 to the glutamine in mouse and human blood Carry out quantitative histogram.

Specific embodiment

When providing several value or ranges, terms used herein " about " refers to the number value or range in given number value or range Within 20%, within 10% and within 5%.

Terms used herein "comprising", " comprising " and its equivalent form include " containing " and " by ... form " contain Justice, such as the composition of "comprising" X can only be made of X or can contain other materials, such as X+Y.

Terms used herein " glutamine sensitivity polypeptide " or " glutamine response polypeptide ", which refer to, generates sound to glutamine The polypeptide answered, the response include the chemistry of polypeptide relevant to the sensitive interaction of polypeptide, biology, electricity or physiology Any response of parameter.The glutamine sensitivity polypeptide is to the amino acid-sensitive including glutamine.Response includes small Variation, for example, the variation in the direction of the amino acid or peptide fragment of polypeptide and, for example, the level-one of polypeptide, second level or tertiary structure Variation, including for example protonate, the variation of electrochemical potential and/or conformation." conformation " is the molecule in molecule comprising side group The three-dimensional arrangement of level-one, second level and tertiary structure；When the three-dimensional structure of molecule changes, conformation changes.Conformation becomes The example of change includes being changed into beta sheet from alpha-helix or being changed into alpha-helix from beta sheet.As long as it is understood that fluorescence The fluorescence of protein part is changed, and detectable change needs not be conformational change.Glutamine sensitivity polypeptide described herein It may also include its functional variety.The functional variety of glutamine sensitivity polypeptide includes but is not limited to that can interact with amino acid To which the variant with the same or similar variation of parent's glutamine sensitivity polypeptide occur.Glutamine sensitivity polypeptide can also be to except paddy The amino acid-sensitive of glutamine.

Glutamine sensitivity polypeptide of the present invention includes but is not limited to glutamine binding protein glutamine- Binding protein, QBP) or have with it the variant of 90% or more homology.Glutamine binding protein of the present invention can From Escherichia coli (Escherichia coli) or salmonella (Salmonella spp.), including with glutamine knot Hop protein has 90% or more homology and any more peptide or proteins sensitive to glutamine.Glutamine binding protein contains week The structure that typical two α/β globular domains are connected by a hinge possessed by matter binding protein can combine glutamine. Glutamine binding protein can incude the variation of pericentral siphon glutamine concentration, in the process of glutamine concentration dynamic change Very big change can also occur for the protein-bonded space conformation of glutamine.Illustratively, glutamine binding protein includes GlnH albumen.

Terms used herein " optical probe " refers to the glutamine sensitivity polypeptide with optical activity peptide fusion.Inventor It was found that glutamine sensitivity polypeptide such as glutamine binding protein in conjunction with the glutamine of physiological concentration after generated structure It can cause the conformation change of optical activity polypeptide (such as fluorescin) as changing, and then lead to the optical of optical activity polypeptide Matter changes.Standard curve is drawn by the fluorescence of the fluorescin measured under different glutamine concentrations, can detecte simultaneously Analyze the presence and/or level of glutamine.Exemplary glutamine binding protein GlnH is as shown in SEQ ID NO:1.When retouching When stating optical probe of the present invention (such as when describing site locating for optical activity polypeptide or mutational site), amino acid residue is referred to Number refers to SEQ ID NO:1, and the alanine (A) after the methionine (M) of sequence starting is calculated as residue 1.But The corresponding residue numbering of other similar glutamine binding protein as known to those skilled in the art.

In optical probe of the invention, optical activity polypeptide (such as fluorescin) is operationally fused to glutamine In sensitive polypeptide." optical activity polypeptide " based on protein is the polypeptide with transmitting fluorescence capability.Fluorescence is optical activity A kind of optical property of polypeptide can be used as the means for detecting the responsiveness of optical probe of the invention.As used herein, term " photoluminescent property " refers to the molar extinction coefficient under appropriate excitation wavelength, fluorescence quantum efficiency, excitation spectrum or emission spectrum Shape, excitation wavelength maximum value and launch wavelength maximum value, the amplitude of two different wave lengths excitation, the transmitting of two different wave lengths Amplitude ratio, lifetime of excited state or fluorescence anisotropy.Any one of these properties between active and inactive state Measurable difference is sufficient to the effectiveness of fluorescent protein substrate of the invention in determination of activity.Measurable difference can pass through The amount of any quantitative fluorescence property is determined to determine, for example, the product of the fluorescence volume or fluorescence of certain wave strong point on emission spectrum Point.Preferably, selection protein substrate is to have the fluorescent characteristic for being easy to distinguish under the conformational state of un-activation and activation.This The text optical activity polypeptide may also include its functional variety.The functional variety of optical activity polypeptide includes but is not limited to that can send out The raw variant with the same or similar photoluminescent property variation of parent's optical activity polypeptide.

" connector " or " bonding pad " refers to the amino acid or core that two parts are connected in polypeptide of the present invention, protein or nucleic acid Nucleotide sequence.Illustratively, the amino of the bonding pad aminoterminal of glutamine sensitivity polypeptide of the present invention and optical activity polypeptide Sour number selection is 0-3, and the amino acid number selection of c-terminus is 0-2；When recombination optical probe conduct is substantially single When member is connect with functional protein, the amino acid or c-terminus in recombination optical probe can be merged.Joint sequence can for one or The short peptide chain of multiple flexible amino acid compositions.

Terms used herein " chromophore ", " fluorogen " are synonymous with " fluorescin ", refer to exciting light irradiation under issue it is glimmering The protein of light.Basic detection means of the fluorescin as bio-science field, such as field of biotechnology are commonly green Fluorescin GFP and the cyclic annular blue fluorescent protein (cpBFP) reset derived by the protein mutation, the cyclic annular green reset Fluorescin (cpGFP), cyclic annular yellow fluorescence protein (cpYFP) reset etc.；There are also the common red fluorescences of the art Albumen RFP, and by the protein derived cyclic annular albumen reset come out, such as cpmApple, cpmOrange, cpmKate etc..Show It is any shown in the sequence such as SEQ ID NO:2-9 of example property fluorescin.

Green fluorescent protein GFP is extracted from Victoria's luminescent jellyfish (Aequorea Victoria) , by 238 Amino acid profiles, molecular weight is about 26kDa.GFP is to form unique barrel-like structure by 12 beta sheet chains, The tripeptides that adds lustre to (Ser65-Tyr66-Gly67) is wrapped in it.When in the presence of oxygen, it is sub- that it can spontaneously form p- hydroxy benzenes Chromophore's structure of methylimidazole quinoline ketone and generate fluorescence.GFP generates fluorescence and does not need co-factor, and fluorescence is highly stable, It is a kind of good imaging tool.GFP is there are two excitation peak, and the main peak of 395nm can produce the transmitting light of 508nm, and acromion The transmitting light for the 503nm that the exciting light irradiation of 475nm can then generate.Exemplary cpGFP is as shown in SEQ ID NO:6

Yellow fluorescence protein YFP is derived from green fluorescent protein GFP, and amino acid sequence and GFP homology are up to 90% More than, YFP is modified in that the 203rd amino acids sport tyrosine (T203Y) by threonine compared to GFP key.Compared to The red shift of wavelength of the main excitation peak of original AvGFP, YFP is to 514nm and launch wavelength then changes into 527nm.On this basis Carrying out rite-directed mutagenesis (S65T) to the 65th amino acids of YFP can get fluorescence enhancement type yellow fluorescence protein EYFP.CpYFP be by The original N-terminal and C-terminal of GFP manufactures a new N by one section of small peptide chain link flexible, in the nearly chromophore position original GFP End and C-terminal, using former 145th~238 amino acids part as the N-terminal of new albumen, former 1st~144 amino acids are as new egg White C-terminal by 5~9 there is small peptide chain link flexible to obtain between two segments.In the present invention, nearly chromophore position is excellent It is selected as at Y144 and N145 amino acids；It is described have short peptide chain flexible be preferably VDGGSGGTG or GGSGG.It is exemplary The sequence of cpYFP is as shown in SEQ ID NO:2.

Red fluorescent protein RFP is extracted from the coral in ocean, and wild RFP is that oligomeric proteins are unfavorable In the amalgamation and expression of organism, the red fluorescent protein of different colours wave band has then further been derived on the basis of RFP, The most commonly used is mCherry and mKate etc..Exemplary cpmKate is as shown in SEQ ID NO:4 or 8.It is exemplary MCherry is as shown in SEQ ID NO:5.

In other embodiments, fluorescin can also be amino acid sequence blue-fluorescence as shown in SEQ ID NO:7 Albumen cpBFP, amino acid sequence orange fluorescence albumen cpmOrange, amino acid sequence such as SEQ as shown in SEQ ID NO:3 One of apple DsRed cpmApple shown in ID NO:9 is a variety of.

Glutamine optical probe of the present invention includes glutamine sensitivity polypeptide B, such as glutamine combination egg White or its variant and optical activity polypeptide A, such as fluorescin.Optical activity polypeptide A is inserted into glutamine sensitivity polypeptide B In, B is divided to for two parts B1 and B2, the probe structure of formation B1-A-B2 formula；Glutamine sensitivity polypeptide B and glutamine Interaction causes the optical signalling of optical activity polypeptide A to become strong.

In optical probe of the invention, optical activity polypeptide can be located at any position of glutamine sensitivity polypeptide. In one embodiment, optical activity polypeptide is located at any position of the glutamine sensitivity polypeptide in the direction N-C with the direction N-C It sets.Specifically, optical activity polypeptide is located at the flexible region of glutamine sensitivity polypeptide, and the flexible region refers to protein Some specific such as loop domain structures present in higher structure, these structural domains compared to protein other are advanced Structure has higher mobility and flexibility, and the region can be after the protein and ligand binding, space structure conformation Dynamic change occurs.Heretofore described flexible region refers mainly to the position of fusion location in glutamine binding protein Domain, such as the region amino acid residue 175-185.In one embodiment, optical activity polypeptide is located at glutamine binding protein Amino acid sequence amino acid residue 175-185 between.Illustratively, optical activity polypeptide is located at glutamine binding protein Amino acid sequence residue 177/178,177/179,177/180,178/179,178/180 and/or 179/180 at, such as SEQ Shown in NO:10~15 ID.

When mentioning certain more peptide or protein, term used herein " variant " or " mutant " include having the polypeptide or egg White identical function but the different variant of sequence.These variants include but is not limited to: lacking in the sequence of more peptide or proteins Losing, be inserted into and/or replace one or more, (usually 1-30, preferably 1-20, more preferably 1-10 is a, most preferably 1-5 It is a) amino acid, and it is one or several (usually within 20, preferably in its carboxyl terminal and/or amino terminal addition More preferably be within 5 within 10) amino acid obtain sequence.It is not intended to be restricted by theory, amino acid residue changes Overall configuration and function without changing polypeptide or protein, i.e. function conservative variants.For example, in the art, with performance phase When close or similar amino acid is replaced, the function of more peptide or proteins is not usually changed.In the art, performance is similar Amino acid often refers to the amino acid residues with similar side chain, has in this field and explicitly defines.These families include having alkali The amino acid (such as lysine, arginine, histidine) of property side chain, amino acid (such as aspartic acid, paddy with acid side-chain Propylhomoserin), amino acid with uncharged polar side chain (such as glycine, asparagine, glutamine, serine, Soviet Union's ammonia Acid, tyrosine, cysteine), amino acid with non-polar sidechain (such as alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), it is (such as threonine, valine, different with β-branched building block amino acid Leucine) and amino acid (such as tyrosine, phenylalanine, tryptophan, histidine) with aromatic side chain.For another example, in ammonia Base end and/or carboxyl terminal, which add one or several amino acid generally also, will not change the function of more peptide or proteins.For being permitted The conserved amino acid of mostly common known nongenetic coding amino acid replaces known in the art.Other undoded amino acids are guarded Substitution can be determined compared with the property of the amino acid genetically encoded based on its physical property.Those skilled in the art are public Know, in gene cloning operation, it is often necessary to design suitable restriction enzyme site, this certainly will be in expressed more peptide or protein ends One or more incoherent residues are introduced, and this has no effect on the activity of desired polypeptides or albumen.For another example melt to construct Hop protein, the expression for promoting recombinant protein obtain the recombinant protein being secreted into outside host cell automatically or conducive to recombinant protein Purifying, it is often necessary to which some amino acid are added to other suitable areas in the end N-, the end C- or the albumen of recombinant protein In domain, it may for example comprise but be not limited to, suitable joint peptide, signal peptide, leader peptide, end extension, glutathione S-transferase (GST), the albumen of maltose E binding protein, the label of albumin A, such as 6His or Flag or Xa factor or fibrin ferment or enterokinase Proteases site.The variant of more peptide or proteins can include: homologous sequence, allelic variant, natural mutation, lures at examples of conservative variations Lead mutant.These variants also may include with the sequence identity of more peptide or proteins be 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% it is more Peptide or protein.

Optical probe of the present invention may include the glutamine sensitivity polypeptide with mutation, such as the glutamine with mutation Binding protein.D10, R75 and/or D157 of the mutation such as glutamine sensitivity polypeptide (such as glutamine binding protein) The mutation in equal sites.Illustratively, the mutation is selected from D10N, R75K, R75M, D157N, D10N/D157N and D10N/R75M/ D157N。

In the exemplary embodiment, B1-A-B2 formula optical probe of the present invention can be in the fusion of 177/180 site of GlnH There are cpYFP and the probe with D10N, R75K, R75M, D157N, D10N/D157N or D10N/R75M/D157N mutation, such as Shown in SEQ ID NO:15-20.

In two or more polypeptides or sequence of nucleic acid molecules, term " the phase same sex " or " phase same sex percentage " refer to and are comparing On window or specified region, using means known in the art such as sequence comparison algorithm, compared by manual alignment and visual inspection When compared with comparing maximum correspondence, two or more sequences or subsequence are identical or in which have certain percentage in specified region Amino acid residue or nucleotide it is identical (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% are identical).For example, being suitble to measurement percent sequence identity Optimization algorithm with sequence similarity percentage is 2.0 algorithm of BLAST and BLAST, respectively reference can be made to Altschul etc. (1977) (1990) J.Mol.Biol.215:403 such as Nucleic Acids Res.25:3389 and Altschul.

Terms used herein " functional variety ", " derivative " and " analog ", which refers to, to be kept substantially and original polypeptide or egg White (such as glutamine binding protein or fluorescin) identical biological function or active albumen.Polypeptide of the invention or The functional variety of albumen (such as glutamine binding protein or fluorescin), derivative or the like, which can be (i), one Or the substituted albumen of multiple conservative or non-conservative amino acid residues (preferably conservative amino acid), and such substitution Amino acid residue can be and may not be by genetic code encoding, or (ii) has in one or more amino acid residues The albumen of substituted group, or (iii) maturation protein and another compound (for example extend the compound of protein half-life, example Such as polyethylene glycol) fusion is formed by albumen, or (iv) additional amino acid sequence is fused to this protein sequence and the egg that is formed White (such as secretion sequence or for purifying the sequence of this albumen or proprotein sequence, or egg is merged with the formation of antigen I gG segment It is white).According to the teaching of this article, these functional varieties, derivative and analogue belong to model well known to those skilled in the art It encloses.

The difference of the analog and original polypeptide or albumen can be the difference on amino acid sequence, be also possible to not shadow The difference on the modified forms of sequence is rung, or is had both at the same time.These albumen include natural or induction genetic variation.Induction becomes Body can be obtained by various technologies, such as generate random mutagenesis by radiating or being exposed to mutagens, can also be lured by fixed point The technology of political reform or other known molecular biology obtains.

The analog further includes the analog with the residue (such as D- amino acid) different from natural L-amino acids, and Analog with non-naturally occurring or synthesis amino acid (such as β, gamma-amino acid).It should be understood that glutamine of the invention Sensitive polypeptide is not limited to the above-mentioned representative albumen enumerated, variant, derivative and analogue.Modification (does not change level-one usually Structure) form include: internal or external albumen chemical derivative form such as acetylation or carboxylated.Modification further includes glycosyl Change, carries out albumen that is glycosylation modified and generating in the synthesis and processing of albumen or in further processing step such as those.This Kind of modification can by albumen is exposed to carry out glycosylated enzyme (glycosylase or deglycosylation enzyme of such as mammal) and It completes.Modified forms further include with phosphorylated amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine) Sequence.It further include being modified to improve its anti-proteolytic properties or optimize the albumen of solubility property.

The present invention also provides the preparation methods of above-mentioned glutamine optical probe, comprising the following steps: 1) by code book The nucleic acid sequence of the text glutamine optical probe is included in expression vector；2) expression vector is transferred in host cell；2) The host cell is cultivated under conditions of being suitble to the expression vector to express, 3) separation glutamine optical probe.

Term used herein " nucleic acid " or " nucleotide " can be DNA form or rna form.DNA form include cDNA, Genomic DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand. When mentioning nucleic acid, terms used herein " variant " can be the variant that the allelic variant naturally occurred or non-natural occur.These Nucleotide variants include degeneracy variant, replace variant, deletion mutants and insertion variant.As known in the art, allelic variant is The alternative forms of one nucleic acid, it may be substitution, missing or the insertion of one or more nucleotide, but not from substantially changing Become the function of the albumen of its coding.It is at least about 70% that nucleic acid of the present invention, which may include with the sequence identity of the nucleic acid sequence, 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% nucleotide sequence.The invention further relates to the nucleic acid fragments hybridized with above-mentioned sequence.As used herein, " nucleic acid piece The length of section " at least contains 15 nucleotide, preferably at least 30 nucleotide, more preferably at least 50 nucleotide, preferably extremely It is more than few 100 nucleotide.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid.

The full length sequence of optical probe or fusion protein of the present invention or its segment can usually use PCR amplification method, artificial conjunction It is obtained at method or recombination method.For PCR amplification method, can disclosed nucleotide sequence design primer according to the present invention, and use city The library cDNA sold or by the library cDNA prepared by conventional method well known by persons skilled in the art as template, amplification and it is related Sequence.When nucleotide sequence is greater than 2500bp, 2~6 PCR amplifications are preferably carried out, then press the segment that each time expands Proper order is stitched together.The present invention is not particularly limited the program and system of the PCR amplification, normal using this field The PCR amplification program and system of rule.Related sequence can also be obtained in large quantity with recombination method.This is usually by its gram It is grand enter carrier, then be transferred to cell, then by conventional method from the host cell after proliferation separation obtained with purifying it is You Guan more Peptide or protein.In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In this hair In bright, when the nucleotide sequence of optical probe is less than 2500bp, artificial synthesis can be used to synthesize.It is described artificial synthesized Method is the artificial synthesis of the DNA of this field routine, without other particular/special requirements.In general, by first synthesizing multiple small fragments, Then it is attached the very long segment of available sequence again.At present, it is already possible to code book be obtained by chemical synthesis completely The DNA sequence dna of invention albumen (or its functional variety, derivative or the like).Then the DNA sequence dna can be introduced into this field In known various existing DNA moleculars (such as carrier) and cell.Mutation can be drawn by mutation the methods of PCR or chemical synthesis Enter in protein sequence of the present invention.

The present invention will encode the nucleotide sequence of the optical probe after the nucleotide sequence for obtaining coded optical probe It is included in expression vector, obtains recombinant expression carrier.The term as used herein " expression vector " and " recombinant vector " are used interchangeably, Refer to protokaryon or eukaryotic vector well known in the art, such as bacterial plasmid, bacteriophage, yeast plasmid, plant cell are viral, lactation is dynamic Object cell virus such as adenovirus, retrovirus or other carriers, these carriers can replicate in host and stablize expression, One important feature of these recombinant vectors is to usually contain expression control sequence.Terms used herein " expression control sequence " refers to Regulate and control target gene transcription, translation and expression the element that can be operatively connected with target gene, can be replication orgin, Promoter, marker gene or translation control element, including enhancer, operon, terminator, ribosome bind site etc., expression The selection of control sequence depends on host cell used.Applicable recombinant vector includes but is not limited to bacterium matter in the present invention Grain.In recombinant expression carrier, " being operatively connected " refers to the nucleotide sequence of purpose and adjusts sequence to allow nucleotides sequence The mode that list reaches connects.Those skilled in the art is known to be used to construct containing fusion protein coded sequence of the present invention and properly Transcription/translation control signal expression vector method.These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to close At.The representative example of these promoters has: lac the or trp promoter of Escherichia coli；λ bacteriophage PL promoter；Eukaryon starting Attached bag includes CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retrovirus The promoter that LTR and some other known controllable gene are expressed in protokaryon or eukaryotic or its virus.Expression vector It further include the ribosome bind site and transcription terminator of translation initiation.In one embodiment, expression vector can be used Commercially available pRSETb carrier, without other particular/special requirements.Illustratively, the coding optics is visited respectively using BamHI and EcoRI The nucleotide sequence and expression vector of needle carry out double digestion, then connect to obtain recombinant expression carrier by the digestion products of the two. The present invention is not particularly limited the specific steps and parameter of digestion and connection, is using the step and parameter of this field routine It can.

After obtaining recombinant expression carrier, which is transformed into host cell, to generate the egg for including fusion protein White or peptide.Such transfer process can be carried out with the routine techniques well known to those skilled in the art such as conversion or transfection.Institute of the present invention The host cell stated be refer to receive and accommodate recombinant DNA molecules cell, be recombination amplification place, preferably by Body cell, which should meet, is easily obtained and is proliferated two conditions." host cell " of the invention may include that prokaryotic cell and eukaryon are thin Born of the same parents specifically include bacterial cell, yeast cells, insect cell and mammalian cell.It specifically can be Escherichia coli, streptomycete Belong to, the bacterial cell of salmonella typhimurium, fungal cell's such as yeast, plant cell, the insect cell of drosophila S2 or Sf9, CHO, COS, HEK293, HeLa cell or the zooblast of Bowes melanoma cells etc., including but not limited to above-mentioned Those host cells.The preferably various cells for being conducive to gene product expression or fermenting and producing of the host cell, such cell is It is well known in the art and commonly uses.Exemplary host cells used are e. coli jm109-DE3 bacterium in embodiments of the present invention Strain.Persons skilled in the art are aware that how to select carrier, promoter, enhancer and host cell appropriate.

The method of the present invention for being transferred to host cell is the method for this field routine, including calcium phosphate or calcium chloride Co-precipitation, DEAE- mannosan-mediation transfection, fat transfection, natural competence, the transfer of chemistry mediation or electroporation.Work as place When master is prokaryotes such as Escherichia coli, the method is preferably CaCl₂Method or MgCl₂Method processing, step used are ability Known in domain.When host cell is eukaryocyte, following DNA transfection method: calcium phosphate precipitation, conventional mechanical can be selected Method such as microinjection, electroporation, liposome packaging etc..

The present invention carries out amplification expression after expression vector is transferred to host cell, to the host cell for being transferred to expression vector Culture, isolated glutamine optical probe.The host cell amplification expression culture is using conventional method.According to Host cell species used, culture medium used in culture can be various conventional mediums.It is being suitable for host cell growth Under conditions of cultivated.

In the present invention, optical probe is expressed or is secreted into the cell, on cell membrane and is extracellular.If desired, can benefit The albumen of recombination is separated by various separation methods or purified with its physics, chemical and other characteristics.The present invention is to separation The method of the amino acid fluorescin is not particularly limited, using the separation method of the fusion protein of this field routine. These methods are well-known to those skilled in the art, including but not limited to: conventional renaturation process, salting-out method, centrifugation, Permeate broken bacterium, ultrasonic treatment, ultracentrifugation, sieve chromatography, adsorption chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) With the combination of various other liquid chromatography technologies and these methods.In one embodiment, the affinity chromatography of His label is utilized The separation of method progress optical probe.

The present invention also provides the glutamine optical probes to position in real time in amino acid, quantitative detection and high throughput Application in screening compound.In one aspect, signal of the glutamine optical probe preferably with cell different parts Peptide connection, is transferred in cell, by the power of fluorescence signal in detection cell, carries out the real-time positioning of amino acid；Pass through ammonia The quantitative detection that curve carries out corresponding amino acid is added dropwise in base acidity scale standard.It is basis that curve, which is added dropwise, in amino acid standard of the present invention Fluorescence signal of glutamine optical probe in the case where various concentration amino acid is drawn.Glutamine of the present invention Optical probe is directly transferred in cell, during amino acid real-time orientational and quantificational detection, does not need time-consuming sample treatment Process, more accurately.Glutamine optical probe of the present invention adds different compounds when carrying out high-throughput screening compound It is added in cell culture fluid, measures the variation of amino acid content, to filter out on the influential chemical combination of amino acid content variation Object.The glutamine optical probe described in the present invention position in real time in amino acid, quantitative detection and high throughput compound Application in screening is non-diagnosing and treating purpose, is not related to the diagnosing and treating of disease.Above-mentioned amino acid can be any ammonia Base acid, such as glutamine.

Herein, the form of concentration, content, percentage and the equal usable range of other numerical value indicates.It will also be appreciated that using This range format is intended merely to convenienct and succinct, it should is flexibly read as including the specifically mentioned number of range bound institute Value should also include all single numbers for including or subrange within the scope of this.

Embodiment

Glutamine optical probe provided by the invention is described in detail below with reference to embodiment, but cannot be They are interpreted as limiting the scope of the present invention.

I. experimental material and reagent

Mainly using conventional genetic engineering molecular biology cloning process and cell culture and imaging side in embodiment Method etc., these methods are well known to those of ordinary skill in the art, such as: " the molecular biology of simple Ross Kai Musi etc. Laboratory reference handbook ", J. Pehanorm Brooker, D.W. Russell writes, and Huang Peitang etc. is translated: " Molecular Cloning:A Laboratory guide " (third edition, In August, 2002, Science Press publish, Beijing)；" animal cell culture: basic fundamental guide " the (the 5th of Fei Leixieni etc. Version), Zhang Jingbo, Xu Cunshuan etc. are translated；J.S. Boneface agriculture, " fine works Cell Biology Experiment guide " of the M. up to rope etc., Zhang Jing Wave etc. is translated.According to the following examples, slightly modified as the case may be of being not difficult succeeds those of ordinary skill in the art with transformation The implementation present invention, these modifications and transformation are all fallen in the range of the claim of this application.

PRSETb-cpYFP is based on used in embodiment, pRSETb-QBP plasmid is by East China University of Science's experimental protein Room building, pRSETb plasmid vector are purchased from Invitrogen company.All primers for PCR are by Shanghai JaRa bioengineering Technology Co., Ltd.'s synthesis is purified and is identified through mass spectrography correct.The expression plasmid constructed in embodiment all passes through sequencing, Sequencing is completed by Huada gene company and Jie Li sequencing company.Taq archaeal dna polymerase used in each embodiment is purchased from Dongsheng Biology, pfu archaeal dna polymerase are purchased from TIANGEN Biotech (Beijing) Co., Ltd., and primeSTAR archaeal dna polymerase is purchased from TaKaRa company, three kinds of polymerases all incidentally give corresponding polymerase buffer and dNTP when buying.BamHI,BglII, The restriction enzymes such as HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, T4 phosphorylase (T4 PNK) are purchased from Fermentas company, when purchase, are accompanied with corresponding buffer etc..Transfection reagent Lip2000Kit is purchased from Invitrogen public affairs Department.The amino acid such as glutamine are purchased from Sigma company.Unless specifically stated otherwise, the chemical reagent such as inorganic salts are purchased from Sigma-Aldrich company.HEPES salt, ampicillin (Amp) and puromycin are purchased from Ameresco company.The detection of 96 holes Blackboard, 384 hole fluorescence detection blackboards are purchased from Grenier company.

DNA purification kit used in embodiment is purchased from BBI company (Canada), and the small pumping kit of ordinary plasmids is purchased from TIANGEN Biotech (Beijing) Co., Ltd..Clone strain Mach1 is purchased from Invitrogen company.Affinity chromatography column and de- Salt column packing is all from GE healthcare company.

The key instrument used in embodiment includes: that (U.S. Bio-Tek is public for 2 multi-function microplate reader of Biotek Synergy Department), X-15R high speed freezing centrifuge (Beckman company, the U.S.), the Microfuge22R table-type high-speed refrigerated centrifuge (U.S. Beckman company), PCR amplification instrument (German Biometra company), Ultrasonic Cell Disruptor (Ningbo Xin Zhi company), nucleic acid electrophoresis apparatus (Shen Neng betting office), sepectrophotofluorometer (Varian company, the U.S.), CO₂Constant temperature cell incubator (SANYO) is inverted glimmering Light microscope (Japanese Nikon company).

II. molecular biology method and cell experiment method

II.1 polymerase chain reaction (PCR):

1. target fragment expands PCR:

This method is mainly used for gene fragment amplification and bacterium colony PCR identification positive colony.The reaction system of the PCR amplification As shown in table 1, amplification program is as shown in table 2.

Table 1.PCR amplification reaction system

Table 2.PCR amplification program

2. long segment (> 2500bp) expands PCR:

The amplification of long segment used in the present invention, mainly Inverse PCR amplification carrier, in the following embodiments for obtaining A kind of technology of rite-directed mutagenesis.Inverse PCR primer is designed at variation position, wherein 5 ' nucleosides of the end comprising variation of a primer Acid sequence.Product after amplification just contains corresponding mutational site.Long segment amplification PCR reaction system is as shown in table 3, expands journey Sequence is as shown in table 4 or table 5.

3. long segment of table (> 2500bp) expands PCR reaction system

4. long segment of table (> 2500bp) expands PCR amplification program

5. long segment of table (> 2500bp) expands PCR amplification program

II.2 endonuclease endonuclease reaction:

The system for carrying out double digestion to plasmid vector is as shown in table 6, wherein n representative make system reach required for total volume plus The sterilizing ultrapure water μ L amount entered.

6. plasmid vector double digestion system of table

II.3 DNA fragmentation 5 ' holds phosphorylation reaction

The plasmid or genome ends extracted from microorganism all contains phosphate group, and PCR product does not have, therefore needs Phosphate group addition reaction is carried out to 5 ' end bases of PCR product, only end is contained phosphate group DNA molecular and could be connected It is reversed to answer.Phosphorylation reaction system is as shown in table 7, and wherein T4 PNK is writing a Chinese character in simplified form for T4 polynueleotide kinase, for DNA points The addition reaction of 5 ' end phosphate groups of son.

7. phosphorylation reaction system of table

The connection of II.4 target fragment and carrier is reacted

Connection method between different segments and carrier difference, is employed herein three kinds of connection methods

1. the blunt end cloning of flat end short-movie section and linearized vector

The principle of this method is that the flat end products that PCR is obtained carry out 5 ' ends of DNA fragmentation under T4 PNK effect After phosphorylation reaction, is connected under the action of PEG4000 and T4 DNA ligase with the carrier of linearisation and obtain recombinant plasmid.Together It is as shown in table 8 that source recombinates linked system.

8. flat terminal fragment coupled reaction system of table

2. the DNA fragmentation containing cohesive end and the connection containing blunt-ended vector segment

Cohesive end outstanding would generally be generated by the DNA fragmentation that restriction enzyme is cut, thus can with contain The blunt-ended vector segment of sequence complementation connects, and forms recombinant plasmid.Coupled reaction system is as shown in table 9, wherein PCR product The mass ratio of segment and carrier double enzyme digestion product is substantially between 2:1-6:1.

9. cohesive end coupled reaction system of table

3. the connection reaction of the DNA fragmentation product recirculation of 5 ' end phosphorylations after inverse PCR introducing rite-directed mutagenesis

The DNA fragmentation of 5 ' end phosphorylations is connected 3 ' ends of linearized vector and 5 ' ends by recirculation connection reaction Reaction obtains recombinant plasmid.Recirculation coupled reaction system is as shown in table 10.

10. recirculation coupled reaction system of table

The preparation and conversion of II.5 competent cell

The preparation of competent cell:

1. picking single colonie (such as Mach1) is inoculated in 5mL LB culture medium, 37 DEG C of shaking tables are stayed overnight.

2. the bacterium solution transferred species for taking 0.5-1mL to be incubated overnight is into 50mL LB culture medium, 37 DEG C, 220rpm culture 3 to 5 is small When, until OD600 reaches 0.5.

3. ice bath is pre-chilled cell 2 hours.

4. 4 DEG C, 4000rpm is centrifuged 10 minutes.

5. abandoning supernatant, cell is resuspended with the buffer of 5mL pre-cooling, resuspension buffer to final volume is added after uniformly is 50mL。

6. ice bath 45 minutes.

7. 4 DEG C of 4000rpm are centrifuged 10 minutes, bacterium is resuspended with the store buffer liquid that 5mL ice is pre-chilled.

8. putting 100 μ L bacterium solutions, -80 DEG C or liquid nitrogen cryopreservation in each EP pipe.

Buffer: CaCl is resuspended₂(100mM)、MgCl₂(70mM)、NaAc(40mM)

Store buffer liquid: 0.5mL DMSO, 1.9mL 80% glycerol, 10 × CaCl of 1mL₂(1M)、1mL 10×MgCl₂ (700mM)、1mL 10×NaAc(400mM)、4.6mL ddH₂O

The conversion of competent cell:

1. taking 100 μ L competent cells in melting on ice bath.

2. the connection product of proper volume is added, gently piping and druming is mixed, ice bath 30 minutes.The connection product being usually added into Volume is less than the 1/10 of competent cell volume.

3. bacterium solution is put into 42 DEG C of water-baths heat shock 90 seconds, it is transferred quickly to place 5 minutes in ice bath.

4. 500 μ L LB are added, cultivated 1 hour in 200rpm on 37 DEG C of constant-temperature tables.

5. bacterium solution 4000rpm is centrifuged 3 minutes, stays 200 μ L supernatants to blow thallus even, be spread evenly across containing appropriate antibiotic Agar plate surface, plate in 37 DEG C of constant incubators be inverted overnight.

The expression of II.6 protein, purifying and fluorescence detection

1. expression vector (such as expression vector of the glutamine optical probe based on pRSETb) is transformed into In JM109 (DE3) cell, it is inverted overnight incubation, picks from the plate and is cloned into 250ml conical flask, be placed in 37 DEG C of shaking tables, 220rpm is cultivated to OD=0.4-0.8, is added the IPTG (1M) of 1/1000 (v/v), 18 DEG C inducing expression 24-36 hours.

2. after the completion of inducing expression, bacterium is received in 4000rpm, centrifugation in 30 minutes, bacterium is resuspended in the phosphate buffer that 50mM is added Body precipitating, ultrasonication to thallus are clarified.9600rpm, 4 DEG C are centrifuged 20 minutes.

Albumen is obtained by the affinity chromatography column purification of self-chambering 3. being centrifuged supernatant, the albumen after affinity chromatography is again The albumen being dissolved in 20mM MOPS buffer (pH 7.4) or phosphate buffer PBS is obtained by the desalting column of self-chambering.

4. purifying albumen after SDS-PAGE is identified, using measurement buffer (100mM HEPES, 100mM NaCl, PH 7.3) or phosphate buffer PBS dilution probe at final concentration of 5-10 μM of protein solution.With measurement buffer Glutamine is configured to the liquid storage of final concentration of 1M by (20mM MOPS, pH 7.4) or phosphate buffer PBS.

5. taking the protein solution of 5 μM of 100 μ l, 37 DEG C are incubated 5 minutes, are separately added into after glutamine mixes to final concentration For 100mM, light absorption of the Multifunction fluorescent microplate reader measurement albumen at 340nm is utilized.

6. taking the protein solution of 1 μM of 100 μ l, 37 DEG C are incubated 5 minutes, and glutamine titration is added, measures albumen The fluorescence intensity that 528nm emits after the excitation of 485nm fluorescence.Multifunction fluorescent enzyme is utilized to fluorescence excitation, the transmitting measurement of sample Instrument is marked to complete.

7. taking the protein solution of 1 μM of 100 μ l, 37 DEG C are incubated 5 minutes, and glutamine is added, measures the absorption spectrum of albumen And fluorescence spectrum.It is complete by spectrophotometer and sepectrophotofluorometer to the measurement of the Absorption and fluorescence spectrum of sample At.

The transfection and fluorescence detection of II.7 mammalian cell

1. the glutamine optical probe plasmid based on pCDNA3.1+ is passed through transfection reagent Lipofectamine2000 (Invitrogen) is transfected into HeLa, is placed in 37 DEG C, 5%CO₂Cell incubator in cultivate. Fluorescence detection is carried out after foreign gene gives full expression to 24~36h.

2. after the completion of inducing expression, adherent HeLa cell is flushed three times with PBS, be placed in HBSS solution respectively into Row fluorescence microscope and microplate reader detection.

Embodiment 1: the plasmid of glutamine binding protein is expressed

By glutamine binding protein QBP (GlnH) gene in PCR amplification soil Agrobacterium gene, PCR product is solidifying BamHI and EcoRI digestion is used after recycling after gel electrophoresis, while corresponding double digestion is carried out to pRSETb carrier.Connected with T4 DNA After connecing enzyme connection, MachI is converted with product, the MachI of conversion is coated on LB plate (ampicillin 100ug/mL), is placed in 37 DEG C overnight incubation.After MachI transformant progress plasmid extraction will be grown, PCR identification is carried out.Positive plasmid is after being sequenced correctly Carry out subsequent plasmid construction.

Embodiment 2: the expression and detection of the cpYFP optical probe of different position of fusion

In the present embodiment, following site fusion cpYFP have been selected based on pRSETb-QBP, have obtained corresponding pRSETb- QBP-cpYFP plasmid: 177/178,177/179,177/180,178/179,178/180 or 179/180.

Using PCR generate cpYFP DNA fragmentation, to the DNA fragmentation using 5 ' ends plus phosphorus operation after inactivate, simultaneously The pRSETb- glutamine binding protein linearized vector for containing different broken sites is generated by Inverse PCR amplification, it will be linear The cpYFP segment of the terminal phosphate of the pRSETb-QBP of change and 5 ' connects production under the action of PEG4000 with T4 DNA ligase Raw recombinant plasmid, by these plates in Kodak multifunctional active imaging system, picking has yellow fluorescence in the case where the channel FITC excites Clone, by Beijing Liuhe Huada Genomics Technology Co., Ltd Shanghai branch company complete be sequenced.

Through sequencing it is correct after, by inducing expression in recombinant plasmid transformed to JM109 (DE3), and protein purification, pass through SDS-PAGE electrophoresis size is near 56Kda.The size meets the purifying containing His-tag that pRSETb-QBP-cpYFP gives expression to and marks The size of the QBP-cpYFP fused protein of label.As a result as shown in Figure 1.

The QBP-cpYFP fused protein of purifying is subjected to glutamine response screening, 100mM glutamine will be contained The detection signal of fluorescence protein is merged divided by the detection signal for merging fluorescence protein of no glutamine.Individual fluorescence egg It is white and fluorescin is fused to the N-terminal of QBP or fusion protein that C-terminal obtains is as control, as shown in Figure 2.

The testing result of Fig. 2 shows that the optical probe for responding glutamine more than 1.5 times has 177/178,177/ The correspondence ammonia in 179,177/180,178/179 and 178/180 sites (as shown in SEQ ID NO 10-14) or its family protein Implement the optical probe of fusion in base acid site.The optical probe and control probe that fluorescin is blended in 179/180 site are almost It does not respond to.

Embodiment 3: the expression and detection of the cpGFP optical probe of different position of fusion

CpYFP is replaced with into cpGFP according to the method in embodiment 2, building glutamine green fluorescent protein fluorescence is visited Needle.As shown in figure 3, testing result shows that the optical probe for responding glutamine more than 1.5 times has 177/178,177/ Implement the optical probe of fusion in 179,177/180,178/179 and 178/180 sites.Fluorescin is blended in 179/180 site Optical probe and control probe almost without response.

Embodiment 4: the expression and detection of the cpBFP optical probe of different position of fusion

CpYFP is replaced with into cpBFP according to the method in embodiment 2, building glutamine blue fluorescent protein fluorescence is visited Needle.As shown in figure 4, testing result shows that the optical probe for responding glutamine more than 1.5 times has 177/178,177/ Implement the optical probe of fusion in 179,177/180,178/179 and 178/180 sites.Fluorescin is blended in 179/180 site Optical probe and control probe almost without response.

Embodiment 5: the expression and detection of the cpmApple optical probe of different position of fusion

CpYFP is replaced with into cpmApple according to the method in embodiment 2, constructs glutamine red fluorescent protein fluorescence Probe.As shown in figure 5, testing result shows that the optical probe for responding glutamine more than 1.5 times has 177/178,177/ Implement the optical probe of fusion in 179,177/180,178/179 and 178/180 sites.Fluorescin is blended in 179/180 site Optical probe and control probe almost without response.

Embodiment 2-5's the result shows that, at 177/178,177/179,177/180,178/179 and 178/180 of QBP Insertion fluorescin can obtain the probe to glutamine response at point, wherein 177/180 and 178/180 of insertion QBP Obtained fusion protein responds glutamine maximum.And by fluorescin be fused to QBP N-terminal or C-terminal it is obtained Fusion protein does not respond to glutamine.So the fusion at the both ends QBP be it is ineffective, only fluorescin is inserted Enter to just generation response inside QBP, and the selection of insertion point is crucial.

Embodiment 6: the expression and detection of the cpYFP optical probe of mutation

Optical probe mutant is constructed on the basis of QBP-177/180-cpYFP.Pass through inverse PCR linearization plasmid PRSETb-QBP-177/180-cpYFP exists to obtained PCR product containing the base sequence for wanting mutational site in primer PNK, T4 DNA ligase add phosphorus to connect under the action of PEG4000, obtain the fixed point saturation in this 3 sites D10, R75 and R75 Mutant plasmid, and complete to be sequenced by Beijing Liuhe Huada Genomics Technology Co., Ltd Shanghai branch company.

As a result as shown in Figure 6.Fluorescence detection, which as the result is shown responds glutamine, has D10N, R75K more than 1.5 times, R75M and D157N mutant.And double prominent body body D10N/D157N and Trimutant D10N/R75M/D157N do not have glutamine Response, therefore prominent body body D10N/D157N and D10N/R75M/D157N can be used as the pH correction matter of glutamine fluorescence probe Grain.

The performance of 7. optical probe mutant of embodiment

The processing of 0mM and 100mM glutamine is carried out respectively after ten minutes to the glutamine optical probe of purifying, use is glimmering The detection of light spectrophotometer progress fluorescence Spectra.

Measurement to excitation spectrum: recording excitation spectrum with the excites scope of 360nm to 510nm and the launch wavelength of 530nm, Every 3nm reads primary.The result shows that there are two excitation peak is as shown in Figure 7 at~420 and 490nm for probe.

The glutamine detection of concentration gradient is carried out to the glutamine optical probe of purifying.The probe of purifying is handled 10 After minute, fluorescence intensity and 485nm excite the change of fluorescence intensity ratio at 528nm transmitting at detection 420nm excitation 528nm transmitting Change.As a result as shown in figure 8, the K of 5 kinds of glutamine fluorescence probes_d(binding constant) be followed successively by 2.3 μM, 8.5mM, 0.5mM, 12.7mM and 4.5mM, amplitude of variation are followed successively by 3.5 times, 1.6 times, 2.5 times, 2.1 times and 1.6 times.Therefore, sample can be directed to Glutamine contents level selects more suitable glutamine probe to carry out quantitative detection.The preferable inspection of QBP-177/180 Surveying range can are as follows: 0.1-20 μM；The preferable detection range of QBP-177/180-D10N can are as follows: 1.3mM-55mM；QBP-177/ The preferable detection range of 180-R75K can are as follows: 0.05-5.5mM；The preferable detection range of QBP-177/180-R75M can are as follows: 1.0- 100mM；The preferable detection range of QBP-177/180-D157N can are as follows: 0.5mM-25mM.

Embodiment 8: the subcellular organelle positioning of optical probe and performance of the optical probe in subcellular organelle

In the present embodiment, merged using different localization signal peptides with optical probe glutamine optical probe, by optics Probe navigates in different organelles.

After optical probe plasmid transfection HeLa cell 36 hours that merge different localization signal peptides, is rinsed, set using PBS It carries out carrying out fluorescence detection under the channel FITC using inverted fluorescence microscope in HBSS solution.As a result as shown in Figure 9.Glutamy Amine optical probe from different special localization signal peptide fusions by that can navigate to including cytoplasm, mitochondria, nucleus, thin In the subcellular organelles such as after birth and golgiosome.All show fluorescence in different subcellular structures, and the distribution of fluorescence and strong It spends different.

It is detected in cell cytosol and mitochondria respectively using the glutamine optical probe being mutated containing R75K.It will be through The HeLa cell for crossing transfection glutamine probe, after being rinsed using PBS, is placed in HBSS solution, 10mM 0mM paddy is added Glutamine is strong using fluorescence at fluorescence intensity at microplate reader detection 420nm excitation 528nm transmitting and 485nm excitation 528nm transmitting Spend ratio.The results are shown in Figure 10.It is detected 30 minutes after addition 10mM glutamine, 485/420 gradually increases, in endochylema and line Highest can reach 2.4 times in plastochondria, and control group does not add glutamine 485/420 and remains unchanged for 1.

Embodiment 9: high-throughput screening compound is carried out based on optical probe in living cells

In the present embodiment, we carry out high-throughput compound using the HeLa cell of endochylema expression glutamine optical probe Screening.

HeLa cell through transfecting is rinsed using PBS, is placed in HBSS solution (no glutamine) processing 1 hour, then It is handled 1 hour using 10 μM of compounds.Glutamine is added dropwise in each sample respectively.It is excited using microplate reader record 420nm Fluorescence intensity and 485nm excite fluorescence intensity ratio variation at 528nm transmitting at 528nm transmitting.At unused any compound The sample of reason is standardized as control.As a result as shown in figure 11.In the 2000 kinds of compounds used, most changes It is minimum that conjunction object enters impact cell to glutamine.The intake ability for thering are 12 kinds of compounds to can be improved cell to glutamine, In addition there are 10 kinds of compounds that can be substantially reduced intake of the cell to glutamine.

Embodiment 10: the glutamine in optical probe quantitative detection blood

In this embodiment, using the glutamine optical probe of purifying to the glutamine in the blood supernatant of mouse and people It is analyzed.

After ten minutes by glutamine optical probe and diluted blood supernatant mixed processing, it is detected using microplate reader 420nm excites fluorescence intensity and 485nm at 528nm transmitting to excite fluorescence intensity ratio at 528nm transmitting.As a result such as Figure 12 institute Show, the glutamine content in mouse blood is at 466 μM or so, and the glutamine content in human blood is at 560 μM or so.

As seen from the above embodiment, glutamine optical probe provided by the invention, molecular weight of albumen are relatively small and easy In maturation, fluorescence dynamic change is big, and specificity is good, and can be expressed in cell by the method for genetic manipulation, can be thin It is intracellular it is outer it is real-time positioning, quantitative detection amino acid；And it is able to carry out high-throughput screening compound.

Other embodiment

Present specification describes many embodiments.It should be understood, however, that those skilled in the art are by reading this specification The various improvement without departing substantially from the spirit and scope of the present invention known, should also be included within the scope of the following claims.

Sequence table

<110>East China University of Science

<120>glutamine optical probe and its preparation method and application

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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His Lys Leu Glu Tyr Asn

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

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

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

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

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

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

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

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

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

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

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Glu Ala

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

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

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

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

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

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

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

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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

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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

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

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

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

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

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Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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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

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

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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

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

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

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

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Asn

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

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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

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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

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

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

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

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

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Glu Tyr Asn

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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

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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

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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

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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. a kind of optical probe becomes comprising glutamine sensitivity polypeptide or its functional variety and optical activity polypeptide or its function Body, wherein optical activity polypeptide or its functional variety are located in the sequence of glutamine sensitivity polypeptide or its functional variety.

2. optical probe as described in claim 1, wherein glutamine sensitivity polypeptide has sequence shown in SEQ ID NO:1 It arranges, or has the sequence of 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity with it.

3. optical probe as described in any one of the preceding claims, wherein optical activity polypeptide is located at glutamine sensitivity Between the residue 175-185 of polypeptide, number corresponds to the overall length of glutamine sensitivity polypeptide.

4. optical probe as described in any one of the preceding claims, the optical probe includes prominent at following sites Become: D10, R75 and D157, number correspond to the overall length of glutamine sensitivity polypeptide.

5. a kind of nucleic acid sequence encodes optical probe of any of claims 1-4.

6. a kind of expression vector, it includes the nucleic acid sequences as claimed in claim 5 being operatively connected with expression control sequence.

7. a kind of host cell, it includes described in claim 5 nucleic acid sequence or expression vector as claimed in claim 6.

8. a kind of method for preparing optical probe of any of claims 1-4, comprising the following steps:

(1) expression vector described in claim 5 is transferred in host cell,

(2) host cell is cultivated under conditions of being suitble to the expression vector to express, and

(3) optical probe is separated by the host cell.

9. optical probe of any of claims 1-4 or the optical probe of claim 8 the method preparation are being examined The application in glutamine in sample.

10. a kind of detection kit, it includes described in optical probe of any of claims 1-4 or claim 8 The optical probe of method preparation.