CN101473031A - Permuted and nonpermuted luciferase biosensors - Google Patents

Abstract

A modified luciferase protein which is a sensor for molecules including cAMP, cGMP, calcium, chelators thereof, kinases, or phosphatases is provided. Also provided is a circularly permuted anthozoan luciferase protein and a decapod crustacean luciferase protein, optionally containing one or more heterologous amino acid sequences, at least one of which directly or indirectly interacts with a molecule of interest. Further provided is a modified anthozoan luciferase protein and a decapod crustacean luciferase protein containing an insertion of one or more heterologous amino acid sequences, at least one of which directly or indirectly interacts with a molecule of interest.

Description

Conversion and unmapped luciferase biosensor

Cross reference with related application

The application requires in the U. S. application sequence number 60/788 of submission on April 3rd, 2006 according to 35 U.S.C. § 119 (e), 608, the U. S. application sequence number of submitting on January 10th, 2,007 60/879,771, the U. S. application sequence number of submitting on February 14th, 2,007 60/901,133 interests, the disclosure integral body of described patent is integrated with this paper as a reference.

Invention field

The present invention relates to biochemical measurement and reagent field.More specifically, the present invention relates to modified luciferase and about the method for its use.

Background

Luciferase is the enzyme of catalytic substrate (for example, luciferin) oxidation, follows the photon of light to discharge.Luciferase separates from numerous species comprise Coleoptera arthropods and many marine organisms.Because it is to detect easily, and its activity can pinpoint accuracy carry out quantitatively, studies genetic expression and protein positioning so luciferase has been widely used in.To be up to the green fluorescent protein (GFP) that formed chromophore in 30 minutes different with needs, and the product of luciferase can detect (if substrate and oxygen also exist) after finishing immediately in that polypeptide chain is synthetic.In addition, do not need posttranslational modification to be used for enzymic activity, and this enzyme does not comprise prothetic group, bonded cofactor or disulfide linkage.Luciferase is a useful reporter in many species and extensively various cell.

Luciferase has makes it as the useful especially other feature of reporter molecule that is used for bio-sensing, the i.e. molecule of the molecular property of exposing system.Biosensor (that is the transmitter that, comprises biological components) generally works by means of 2 step processes: the signal by biological components mediation produces and signal conduction and/or amplification by electric component.Signal produces generally and reaches by combination, energy transfer or catalysis.Because the inherent validity and the specificity of these chemical processes, it can be useful especially producing by enzymatic signal.Most of catalyzed reactions produce the hydrolysis energy that is less than two molecules that are used for ATP, or about 70kJ/ mole.Yet, luminously have a much higher intrinsic energy by what luciferase caused.For example, send the energy of 214kJ/ mole by the catalytic reaction of Lampyridea luciferase (560nm).In addition, luciferase also is highly effective in that chemical energy is converted to aspect the photon, and promptly they have high quantum production rate.Therefore luciferase is very effective for producing detectable signal.

The luciferase biosensor has obtained describing.For example, people (1991) such as Sala-Newby discloses North America Lampyridea (Photinus pyralis) luciferase cDNA and has modified to produce ring-type AMP deopendent protein kinase phosphorylation site.Especially, the Xie Ansuan on the 217th is mutated into arginine producing site RRFS (SEQ ID NO:117), and with seven peptides, i.e. kemptide (kemptide), and promptly the phosphorylation site of pig pyruvate kinase adds on the N or C-terminal of luciferase.People such as Sala-Newby set forth the protein carry phosphorylation site with regard to its specific activity, pI, pH to the influence of the color of the light that sends and in the presence of ATP the effect of the catalytic subunit of protein kinase A characterize.They find the unique a kind of (RRFS in the recombinant protein; SEQ ID NO:117) significantly is different from the wild-type luciferase, and RRFS (SEQ ID NO:117) mutant has lower specific activity, lower optimal pH, sends greener light under low pH, and when phosphorylation, make its active minimizing be up to 80%.The effect that discloses the back is reversed by Phosphoric acid esterase.

People such as Waud (1996) arrive protein kinase recognition sequence and protease site through engineering approaches in the Lampyridea luciferin enzyme dna of North America.Two structural domains of luciferase are modified by people such as Waud; 1 between amino acid 209 and 227 and another is on C-terminal, between the amino acid 537 and 550.The amino acid mutation that people such as Waud disclose between the residue 209 and 227 is reduced to less than 1% of wild-type recombinant chou the noclilucence activity, and the through engineering approaches peptide sequence causes the specific activity of wild-type reorganization luciferase 0.06%-120% on C-terminal.People such as Waud also disclose and have added ring-type AMP deopendent protein kinase catalytic subunit for the variant luciferase, described luciferase mixes kinases recognition sequence LRRASLG (SEQ ID NO:1), follow the Serine on amino acid position 543, cause reducing 30% activity.It is active that alkaline phosphatase treatment is recovered.When people such as Waud further disclose the cleavage site that has between amino acid 542 and 543, the noclilucence activity that comprises the variant luciferase of zymoplasm recognition sequence LVPRES (SEQ ID NO:2) reduces 50% when carrying out incubation in the presence of zymoplasm.

People such as Ozawa (2001) have described based on the segmental protein splicing of Lampyridea luciferase of appropriate design and have induced the complementary biosensor.Protein splicing is to cut from the precursor fusion rotein by its intein (internal protein), and side joint extein (outer egg white matter) is connected to post translational protein modification in the polypeptide.Disclose from the N of synechocystis species (Synechocystis sp.) PCC6803 and C-terminal intein DnaE and merged with the N and the C-terminal fragment of luciferase respectively separately.Protein-protein interaction triggers the folding of DnaE intein, cause protein splicing, and the extein of the luciferase that is thus connected recovers its enzymic activity.People such as Ozawa disclose known binding partners---and the interaction between phosphorylation substrate 1 (IRS-1) and the kinase whose target N-terminal of its PI3-SH2 structural domain, this is to use the division luciferase to monitor in the presence of Regular Insulin.

People such as Paulmurugan (2002) use the mensuration based on division Lampyridea luciferase, to use 2 kind the interactions that protein be MyoD and Id of reconstruction strategy monitoring in cell culture and mouse of complementary strategy and intein mediation.In order to keep the reporter activity, in complementary strategy, fusion rotein needs protein interaction, promptly, interaction via protein mating partner MyoD and Id, and in reconstruction strategy, the new complete beetle luciferase that the montage that mediates via intein forms is kept its activity, even also is like this under the lasting interactional situation that does not exist between the protein mating partner.

Protein fragments is complementary to be measured among the people (U.S. Patent number 6,270,964,6,294,330 and 6,428,951) such as being disclosed in Michnick.Particularly, Michnick has described the mensuration based on division mouse dihydrofolate reductase (DHFR) gene, and wherein the C-terminal fragment of the N-terminal fragment of DHFR and DHFR merges with GCN4 leucine zipper sequence separately.The DHFR activity detects in the cell of expressing 2 kinds of fusion roteins.People such as Michnick have also described another kind of complementarity method, wherein will in glucosaminide kinases (AK) gene, the nested groups of the disappearance of S1 nuclease generation introduce in the leucine zipper construct, and screen with resulting construct group introducing cell and with regard to the AK activity.

Need be as biosensor, for example for example be used as biosensor in protein-protein interaction, intracellular signal transduction or the physiology conversion, have the reorganization luciferase of the improvement of high degree of specificity and high signal sensitivity in detection cell incident.

Summary of the invention

The invention provides the gene product of improvement, for example, the beetle luciferase that modified luciferase is for example modified, for example Lampyridea or Pleonomus luciferase, coral polyp (anthozoan) luciferase is sea pansy (Renilla) luciferase for example, or the Crustacean luciferase, it has the activity of one or more changes in the presence of one or more molecules of interest, and described molecules of interest is cAMP, cGMP, kinases, Phosphoric acid esterase or calcium for example.In one embodiment, the aminoacid sequence of modified luciferase be different from corresponding unmodified (natural, wild-type or parent's, the mutant luciferase that for example has one or more replacements) luciferase, this is because to modifying tolerance, for example the site (residue) of insertion, disappearance, circular permutation or its any combination tolerance is gone up or the zone in one or more modifications.In one embodiment, the zone of modifying tolerance is comprised secondary structure, for example βZhe Die or the ring of on natural, wild-type luciferase, finding of the surface between the α spiral.One or more modifications can be inner with respect to the N or the C-terminal of the luciferase of unmodified, and/or can be on the N and/or C-terminal of the luciferase of unmodified, the for example insertion of the disappearance of luciferase sequence and/or one or more amino-acid residues, the optional luciferase sequence that is included on the decorating site, thus modified luciferase produced.Disappearance within the scope of the present invention is included on the site of the luciferase sequence of disappearance tolerance or the disappearance of the one or more amino-acid residues in the zone.Modification can comprise the circular permutation and the introducing (insertion) of one or more discrete (isolating) allogeneic amino acid sequences, in the described allogeneic amino acid sequence at least one and molecules of interest directly or indirectly interact, and described modification is optional can to comprise one or more amino acid whose disappearances, for example on to the site of modifying tolerance or in the zone, the N and/or the C-terminal that comprise the luciferase of unmodified, as long as resulting modified luciferase has the noclilucence activity before or after the interaction of molecules of interest, the biological example luminescence activity is changing behind the molecules of interest incubation.In one embodiment, modification can be the shortage of peptide bond between two amino acid that connect via the peptide bond in the luciferase of corresponding unmodified in modified luciferase, in described peptide bond and the modified luciferase on the N-terminal of the luciferase of corresponding unmodified and the C-terminal residue or near peptide bond between the residue of discovery combine, described shortage produces the circular permutation luciferase, it is chosen wantonly and comprises one or more isolating allogeneic amino acid sequences, and at least one in the described allogeneic amino acid sequence and molecules of interest directly or indirectly interact.In one embodiment, with molecules of interest directly or indirectly interactional one or more allogeneic amino acid sequences in the circular permutation luciferase corresponding on the sequence of the N-terminal of the luciferase of corresponding unmodified and/or C-terminal residue or near.In another embodiment, with molecules of interest directly or indirectly interactional one or more allogeneic amino acid sequences on the N-terminal of circular permutation or non-annularity conversion luciferase and/or the C-terminal residue or near.In one embodiment, in the circular permutation luciferase with molecules of interest directly or indirectly interactional one or more allogeneic amino acid sequences on to the site of modifying tolerance or in the zone, its not on the N-terminal of circular permutation luciferase and/or the C-terminal residue or near, promptly heterologous sequence is inner for N and C-terminal.In one embodiment, the circular permutation luciferase is modified to comprise two or more allogeneic amino acid sequences, described allogeneic amino acid sequence independently on corresponding to the sequence of the N-terminal of the luciferase of corresponding unmodified and/or C-terminal residue or near, on the N-terminal of circular permutation luciferase and/or the C-terminal residue or near, on to the site of modifying tolerance or in the zone, described site or zone not on the N-terminal of circular permutation or non-annularity conversion luciferase and/or the C-terminal residue or near, or its any combination.In one embodiment, the allogeneic amino acid sequence directly or indirectly interacts with different molecules of interest separately.In a further embodiment, the circular permutation luciferase comprises at least two allogeneic amino acid sequences, and it is in the existence of specific exogenous agent or interact with each other not.Two allogeneic amino acid sequences can comprise identical or different sequence.In addition, modified luciferase can be included in the N of luciferase of corresponding unmodified or 1-about 10 or about 30 residues of C-terminal, or any integer between the two, for example N of 15 residues and the disappearance on the C-terminal.Rely on concrete luciferase, the length of disappearance can be to surpass 30 residues, and the length of required disappearance can be determined by conventional deletion analysis.Modified luciferase can be used to detect reversible interaction, the for example combination of two or more molecules, the formation of disulfide linkage or other conformational change, the for example hydrophobic variation of pH, temperature or solvent of condition, or via the irreversible interaction of the activity change of modified luciferase, for example change of light intensity, color or dynamic curve.Modified luciferase can also be used to detect the interaction of the structural modification that causes modified luciferase.Modified luciferase can also be used to detect the interaction of the structural modification that causes modified luciferase, for example via kinase whose phosphorylation or via the bond rupture of proteolytic enzyme.

As described below, be inserted on the residue 21,25,117,358,376,379,398,399,400,401,402,403,405,406,407,409 or 490 of Pleonomus luciferase but cause having in the frame of modified Pleonomus luciferase of detection of active, i.e. near zone amino those residues of modifying tolerance and/or those.Also as described below, be inserted on the residue 7,121,233,267,294,303,361,540 or 541 of Lampyridea luciferase but cause having in the frame of modified Lampyridea luciferase of detection of active, i.e. near zone amino those residues of modifying tolerance and/or those.Other residue or the zone of modifying tolerance also hereinafter obtained describing in this article.

Therefore, the beetle luciferase can be modified on residue, the residue 21 of Pleonomus luciferase for example, 25,117,358,376,379,398,399,400,401,402,403,405,406,407,409 or 490, or the residue 15-30 of corresponding Pleonomus luciferase, residue 21 or 25 for example, residue 112-122, for example residue 117, residue 352-362, for example residue 358, residue 371-384, for example residue 379, residue 393-414, or in the zone of residue 485-495, or at the residue 7 of Lampyridea luciferase, 37,47,75,83,107,121,144,160,174,188,198,205,225,233,242,255,268,308,316,358,377,403,435, on 490 or 540, or the residue 2-12 of corresponding Lampyridea luciferase, residue 32-53, for example residue 32-43 or residue 42-52, residue 70-88, for example residue 70-80 or residue 78-88, residue 102-126, for example residue 102-112 or residue 116-126, residue 139-165, residue 183-203, residue 220-247, for example residue 228-238, residue 262-273, residue 303-313, residue 353-408, residue 485-495, or in the zone of residue 535-546.Correspondence position can be identified by comparison luciferase sequence by for example using the sequence alignment program.Can be used as the site to residue or the zone of modifying tolerance in the luciferase,, be used for inserting, or make luciferase " division " become two molecules that can in complementary action of protein or protein splicing mensuration, use with the circular permutation luciferase.

The present invention further comprises modified coral polyp luciferase, it has on to the site of modifying tolerance or at least one modification in the zone, described site or zone include but not limited to the residue 2 at corresponding sea pansy luciferase (Genbank ID AF025843), 30,31,42,45,46,68,69,90,91,92,110,111,150,151,168,169,193,207,208,223,224,251,259, on 274 or 311 the residue, or at the residue 2-12 of corresponding sea pansy luciferase (Genbank ID AF025843), residue 26-36, residue 37-47, residue 64-74, residue 86-97, residue 90 or 91 for example, residue 96-116, residue 147-157, residue 218-234, for example residue 223,234,228,229 or 230, or in the zone of residue 301-311.Correspondence position can be identified by comparison luciferase sequence by for example using the sequence alignment program.Can be used as the site to residue or the zone of modifying tolerance in the luciferase,, be used for inserting, or make luciferase " division " become two molecules that can in complementary action of protein or protein splicing mensuration, use with the circular permutation luciferase.

What further comprise is modified Crustacean luciferase, for example, oar foot animal (copepod) luciferase, it has on to the site of modifying tolerance or at least one modification in the zone, include but not limited to residue 43-53 at the bent firefly of correspondence (Gaussia) luciferase, residue 63-73, residue 79-89, residue 95-105, residue 105-115, residue 109-119, in the zone of residue 121-131 or residue 157-168, for example referring to Figure 41, or in the zone of the residue 45-55 of corresponding sophisticated thorn shrimp (Oplophorus) luciferase or residue 79-89.Correspondence position can be identified by comparison luciferase sequence by for example using the sequence alignment program.Can be used as the site to residue or the zone of modifying tolerance in the luciferase,, be used for inserting, or make luciferase " division " become two molecules that can in complementary action of protein or protein splicing mensuration, use with the circular permutation luciferase.

In one embodiment, modified luciferase has detectable activity, and the luciferase with respect to corresponding unmodified is included in modifying on the site that tolerates or the one or more amino acid whose insertion in the zone, described insertion comprises such aminoacid sequence, itself and molecules of interest direct interaction, for example comprise the insertion of the recognition sequence of molecules of interest, or interact indirectly, for example interact via another kind of molecule with molecules of interest.In one embodiment, modified luciferase comprises two or more, and for example 3,4,5,10,20,50,100,200,300 or more, but less than about 1000, or the insertion of any integer amino-acid residue between the two.For example, the insertion of IP3 sequence can comprise about 700 amino-acid residues.In one embodiment, modified luciferase with insertion further comprises the disappearance of luciferase sequence, and for example 1 or more, but less than about 100, for example less than 50,40,30,20,10 or 5, or the disappearance of any integer residue between the two.

In one embodiment, the invention provides the circular permutation luciferase of further modification with the insertion that comprises aminoacid sequence, described aminoacid sequence and molecules of interest direct interaction, for example comprise the insertion of the recognition sequence of molecules of interest, or interact indirectly with molecules of interest, for example via another kind of interaction of molecules.For example, as mentioned below, have luciferase to the N that modifies tolerance and/or C-terminal and inner residue or zone on tolerance residue or zone with on different tolerance residues or zone, carry out circular permutation, and insert one or more allogeneic amino acid sequences, at least one in the described allogeneic amino acid sequence and molecules of interest directly or indirectly interact.But resulting modified luciferase shows the change with detection of active in the presence of molecules of interest.

In one embodiment, circular permutation beetle luciferase, circular permutation Decapoda Crustacean luciferase (for example stinging the shrimp luciferase) or the circular permutation sea pansy luciferase with cAMP or cGMP binding site the ring nucleus thuja acid for example cAMP or cGMP in the presence of show to have the luciferase activity of change.Useful ring-type nucleotide binding site can have (L/K/S/I) (A/I/C/G) (L/I) (A/S) (V/T/S/N/W) (SEQ ID NO:118) of X (P/V/T/R/E) R (A/T/H/S) of G (E/Q/K) in luciferase of the present invention, and wherein X is a 2-6 amino acid.Useful cAMP binding site (structural domain) includes but not limited to by direct (people such as Bos, 2003 of the cAMP binding site in the activated exchanger matter of cAMP (Epac) in circular permutation luciferase of the present invention; And referring to for example, NCBI registration number AF115480), comprise Epac 2B, Epac 1 and Epac IIA, the ionic channel of ring nucleus thuja acid gate is the passage regulated of hyperpolarization activatory ring nucleus thuja acid (people such as Zagotta for example, 2003), neuropathy target esterase (people such as Dremier, 2003), PKA modulability II β type subunit (referring to for example NCBI registration number M124921), for example PKA II β A and PKA II β B, PKA modulability I α type subunit, for example PKA I α A and PKA I α B, PKG IIA, PKG IIB and katabolic product activation of protein.As described herein, non-annularity conversion sea pansy luciferase and non-annularity conversion Decapoda Crustacean luciferase with cAMP binding site have the luciferase activity of change in the presence of cAMP.Useful cGMP binding site includes but not limited to the cGMP binding site in the cGMP deopendent protein kinase (GK) in circular permutation luciferase of the present invention, for example, and GK I, or the GAF regulatory region in the phosphodiesterase (PDEs), for example PDE2 or PDE5, adenylate cyclase, or FnlA.In one embodiment, the ring nucleus thuja acid binding domains that comprises luciferase of the present invention further comprises the Subcellular Localization signal, and it is used to detect the Subcellular Localization and/or the concentration of ring-type Nucleotide.

As mentioned below, prepare the luciferase biosensor of the insertion of various sequences with the folding classification of at least 4 kinds of different structures of representative.Especially, one of folding classification interacts by different small molecules and participates in the adjusting of numerous enzymes.In addition, the allosteric structural domain inserts and can be used to detect conformational change for example phosphorylation or proteolytic enzyme cutting in the luciferase of the present invention, and described allosteric structural domain promptly changes the structural domain of structure conformation after in conjunction with another kind of molecule.

Therefore, in one embodiment, the modified luciferase of the present invention comprises such aminoacid sequence, it is with respect to the aminoacid sequence of corresponding luciferase, for example the wild-type luciferase of unmodified is a circular permutation, obtain new N and C-terminal in the circular permutation luciferase, wherein at least one is on to the site of modifying tolerance or in the zone, and be engineered to and have functionally by introducing the allogeneic amino acid sequence, described allogeneic amino acid sequence directly or indirectly interacts with for example ring nucleus thuja acid.In another embodiment, the circular permutation luciferase comprises other modifications, include but not limited to the N of circular permutation luciferase or the insertion and/or the disappearance of C-terminal inside, for example on the N of the luciferase of corresponding unmodified and the C-terminal or near another insertion and/or disappearance, for example at the residue 1-about 10 or about 30 of corresponding N-terminal, or on the residue of any integer between the two, and/or corresponding to last 1 residue of the C-terminal of the luciferase of corresponding unmodified or last approximately 30, for example last 15, or on the residue of 1-30 any integer between the two.

In one embodiment, under the situation that does not have molecules of interest, the activity of the luciferase that the present invention is modified is less than the activity of the luciferase of corresponding unmodified, the reporter activity of for example modified luciferase be corresponding unmodified luciferase activity about 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 50%, 70% or more, but less than 100%, the activity of described modified luciferase is optional to be detectable.In another embodiment, under the situation that does not have molecules of interest, the activity of the luciferase that the present invention is modified is active substantially the same or bigger with the luciferase of corresponding unmodified, for example the reporter activity of the modified luciferase of the present invention is about 1.5 times of luciferase activity of corresponding unmodified, for example at least 2,3 or 5 times or more.In the presence of molecules of interest, the activity of the luciferase that the present invention is modified can change with detecting.For example, activity with respect to luciferase modified under the situation that does not have molecules of interest, modified luciferase active detects that to change be at least 0.001%, 0.01%, 0.1%, 1%, 10% or 100% in the presence of molecules of interest, with be up to 2 times, 4 times, 10 times, 100 times, 1,000 times, 10,000 times or more the change.Therefore, approaching with the physics of the interactional molecules of interest of modification that exists in the luciferase of modified luciferase rather than corresponding unmodified, can change, for example reduce, eliminate or increase the activity of modified luciferase.For example, modified beetle, coral polyp luciferase or Decapoda Crustacean can be circular permutation beetle, coral polyp or the Decapoda Crustacean luciferases with cAMP binding site.Luminous signal with respect to luciferase modified under the situation that does not have cAMP, cAMP exist or non-existent condition under the luminous signal of beetle, coral polyp or Decapoda Crustacean luciferase of corresponding unmodified, the luminous signal of the modified luciferase of this kind can be to reduce, eliminate or increase in the presence of cAMP.

Therefore, the modified luciferase of the present invention can be used as biosensor.

The present invention also provides isolated nucleic acid molecule (polynucleotide), and it comprises the nucleotide sequence of the modified luciferase of code book invention.Further provide the isolated nucleic acid molecule of the nucleotide sequence that comprises encoding fusion protein, described fusion rotein comprises modified luciferase and at the N-terminal (N-terminal fusion partner) of modified luciferase and/or the one or more amino-acid residues on the C-terminal (C-terminal fusion partner).Therefore, as used herein, " fusion rotein " is to be included in the N-terminal of the modified luciferase of the present invention and/or the one or more amino acid whose polypeptide on the C-terminal.Preferably, with respect to corresponding modified luciferase, but the existence of the one or more fusion partners in the fusion rotein does not significantly change the detection of active of fusion rotein.N or C-terminal fusion partner can be the sequences that is used for purifying, for example glutathione S-transferase (GST) or poly-His sequence, wish the sequence of the character of the luciferase that change is modified, for example protein removes critical sequences, protein or nucleic acid interaction sequence are (for example, binding sequence), the Subcellular Localization sequence, or have can with the sequence of the differentiable character of one or more character of luciferase in the fusion rotein.In one embodiment, fusion rotein comprises modified luciferase and fusion partner, described fusion partner is the report protein that is different from luciferase, and contrast is useful to described report protein as intramolecularly, for example fluorescence protein or another kind of luciferase.In another embodiment, the present invention includes the carrier of the nucleotide sequence that comprises encoding fusion protein, described fusion rotein comprises modified luciferase of the present invention and the proteinic nucleic acid fragment of coding report, and described report protein is different from the luciferase in the modified luciferase.Randomly, at least the encode modified luciferase of luciferase and optimized encoding or comprise the nucleotide sequence of optimization of the fusion rotein of modified luciferase, for example the codon optimized sequence of people is used in nucleic acid molecule of the present invention, because those majorizing sequences can increase the intensity of the signal of luciferase.The optimization of nucleotide sequence is known in the art, referring to for example WO 02/16944.

The present invention also comprises the expression modified luciferase of the present invention or the stable cell lines of fusion rotein, and the expression cassette that comprises the nucleic acid molecule of coding modified luciferase of the present invention or fusion rotein, with the carrier that in host cell, can express nucleic acid molecule of the present invention (for example, plasmid, virus or defective virus particle).Preferably, expression cassette comprises the promotor that is operably connected with nucleotide sequence, for example composing type or adjustment type promotor.In one embodiment, expression cassette comprises inducible promoter.Also provide the host cell that comprises expression cassette of the present invention or carrier, for example plant or vertebrate cells of prokaryotic cell prokaryocyte or eukaryotic cell for example, mammalian cell for example, (for example include but not limited to people, non-human primate, dog, cat, ox, horse, sheep or rodent, rabbit, rat, ferret or mouse) cell and comprise nucleic acid molecule of the present invention, expression cassette, carrier, host cell or the modified luciferase or the test kit of fusion rotein.

Use in the application that the modified luciferase of the present invention can be such, promptly wherein the luciferase of unmodified can not be for example as functional reporter to measure or to detect various conditioned disjunction molecules of interest, for example via inserting for example steroid of oestrogenic hormon binding domains, calcium binding domains of hormone receptor binding site, via the proteolytic enzyme that inserts the proteolytic enzyme recognition site, or via the ring nucleus thuja acid that inserts the ring-type nucleotide binding site.For example, coding comprises the carrier of modified luciferase of the insertion of cAMP binding site, or comprise the modified luciferase and the sample mix of the insertion of cAMP binding site, described sample is cell for example, cell lysate, in-vitro transcription/translation mixture, or supernatant liquor, and the active following of the modified luciferase in the sample detects or measures, for example choose wantonly on one or more time points, and optional luciferase with respect to corresponding unmodified, or with respect to having the luciferase (for example further modifying to reduce the binding affinity with cAMP) of the interactional similar modification of minimizing, or with respect to not containing cAMP or having the control sample of different amount cAMP by being mutated into specific amino acids with cAMP.For example in time and/or with respect to the change of luminescence activity of contrast (cell that for example has specified amount cAMP), show existence or the amount of cAMP in the sample in the sample, or with respect to the change of the cAMP amount of experiment condition.In one embodiment, make cell with comprise promotor for example the nucleotide sequence of the modified luciferase of carrier and the code book invention of adjustment type or constitutive promoter contact, described modified luciferase comprises and the interactional insertion of ring nucleus thuja acid.In one embodiment, transfectional cell promotor is therein induced under the condition of transient expression of modified luciferase and is cultivated, and measures luminous existence or amount.In another embodiment, will comprise with the modified luciferase of the present invention of the interactional insertion of ring nucleus thuja acid and suspect to have the sample mix of ring nucleus thuja acid.Measure luminous amount subsequently.Therefore the present invention provides the method that detects the amount of ring-type Nucleotide.

In one embodiment, modified luciferase is modified coral polyp luciferase, for example modified sea pansy luciferase.In one embodiment, modified coral polyp luciferase is circular permutation coral polyp luciferase, for example circular permutation sea pansy luciferase.In another embodiment, modified coral polyp luciferase is not a circular permutation.Modified coral polyp luciferase has one or more allogeneic amino acid sequences, comprises and direct or indirect interactional at least one the allogeneic amino acid sequence of molecules of interest.In one embodiment, aminoacid sequence is during interacting with molecules of interest or experiences the aminoacid sequence of conformational change afterwards, described conformational change changes the activity of luciferase subsequently, for example, the modified sea pansy luciferase with this seed amino acid sequence is useful for detecting allosteric interaction.

In one embodiment, modified luciferase is modified Decapoda Crustacean luciferase, for example modified thorn shrimp luciferase.In one embodiment, modified Decapoda Crustacean luciferase is a circular permutation Decapoda Crustacean luciferase, for example circular permutation thorn shrimp luciferase.In another embodiment, modified Decapoda Crustacean luciferase is not a circular permutation.Modified Decapoda Crustacean luciferase has one or more allogeneic amino acid sequences, comprises and direct or indirect interactional at least one the allogeneic amino acid sequence of molecules of interest.In one embodiment, aminoacid sequence is during interacting with molecules of interest or experiences the aminoacid sequence of conformational change afterwards, described conformational change changes the activity of luciferase successively, for example, the modified thorn shrimp luciferase with this seed amino acid sequence is useful for detecting allosteric interaction.

The aminoacid sequence of the illustrative purpose that merges with modified coral polyp luciferase of the present invention or modified Decapoda Crustacean luciferase includes but not limited to the enteropeptidase site, proteolytic enzyme cutting site, for example about the site of Caspase, for example Caspase 3 cleavage sites, Caspase 8 cleavage sites, PSA, or virus protease for example rhinovirus proteolytic enzyme cutting site, SARS proteolytic enzyme cutting site or TEV proteolytic enzyme cutting site (NLYFQG; SEQ ID NO:119), the ring-type nucleotide binding site, hormone binding site, calcium binding domains are for example by EGTA and CaCl ₂The calmodulin of regulating, or have and dual fusions interact with each other and optional sequence of regulating by exogenous agent, for example FKBP and FRB, wherein rapamycin induce in conjunction with and FK506 promote that bonded dissociates; From the structural domain of PKA-R with from the structural domain of PKA-C, it can be regulated by cAMP; From the structural domain of SH2 and structural domain that can phosphorylation, it can be regulated by for example Tyrosylprotein kinase or Phosphoric acid esterase; From the structural domain of 14-3-3t and structural domain that can phosphorylation, it can be regulated by for example cAMP-PKA; From the structural domain of WW and structural domain that can phosphorylation, it can be regulated by for example Ser-Thr kinases; From the structural domain of Tetrahydrofolate dehydrogenase (DHFR), it can be regulated by methotrexate (MTX) or BisMTX; From the structural domain of gyrase B (GyrB), it can be regulated by Notomycin or Vulkamycin. PA-93; Or has a dual fusions from the sequence in same structure territory.Therefore, in one embodiment, circular permutation coral polyp luciferase or modified Decapoda Crustacean luciferase are modified to comprise two or more heterologous sequences, described heterologous sequence independently on corresponding to the sequence of the N-terminal of the luciferase of corresponding unmodified and/or C-terminal residue or near, on the N-terminal of circular permutation luciferase and/or the C-terminal residue or near, on to the site of modifying tolerance or in the zone, described site or zone not on the sequence of the N-terminal of circular permutation luciferase and/or C-terminal residue or near, or its any combination, wherein two allogeneic amino acid sequences can with the various objectives interaction of molecules.

Further provide and identify one or more compositions and methods of directly or indirectly regulating molecules of interest.

In one embodiment, the invention provides the active method of molecules of interest that detects or measure in the cell.This method comprises provides the luminous reaction mixture that comprises cell and have the carrier of nucleotide sequence, and described nucleotide sequence comprises modified luciferase, the open reading-frame (ORF) of for example modified beetle luciferase.Modified luciferase has insertion with respect to the luciferase of corresponding unmodified, described being inserted in the luciferase sequence on the residue of modifying tolerance or in the zone.With respect to the luciferase of corresponding unmodified, insertion comprises and the direct or indirect interactional aminoacid sequence of molecules of interest.About 47 ℃ in mixture at about 20-, for example about 37 ℃-Yue 45 ℃.Luminous in the mixture detects subsequently or measures, thereby detects or measure existence, amount or the activity of the molecule in the cell.As mentioned below, before adding test agent, make the luminous reaction mixture with the cell of coding luciferase in physiological temp and/or condition, for example about 37 ℃ and/or about 5% CO ₂Following incubation for some time can provide reaction and bigger dynamicrange faster, and described luciferase is the biosensor of cAMP.

Also providing biosensor of the present invention is used in for example the live purposes of Mammals imaging of cell or multicellular organism.

The accompanying drawing summary

Fig. 1. the position that the Tn5 in Pleonomus luciferase (the corresponding SEQ ID of aminoacid sequence NO:3) inserts (runic).

Fig. 2. the aminoacid sequence (luc+) (SEQID NO:210) of parent's (unmodified) Lampyridea luciferase.

Fig. 3. the luminous cAMP that uses the circular permutation luciferase is in conjunction with the synoptic diagram of measuring.

Fig. 4 .PKA modulability II β type subunit (RII β B).The x-ray crystal structure of rat RII β B amino acid 264-412 (PDB 1CX4).RII β B is rendered as red zone; CAMP is rendered as ball and rod.Primary sequence similarity between rat (amino acid 264-412) and the people RII β B (amino acid 266-414) be 96.6% (program Megallign, DNAStar).

Fig. 5 A. circular permutation Lampyridea luciferase (CPM-FF Luc) expression plasmid.HSV-TK or T7 promotor are respectively applied for and express circular permutation Lampyridea luciferase in mammalian cell or lysates.The amino acid 544 of Lampyridea luciferase is connected by 4 two amino acid whose peptides that are rich in Gly/Ser (SEQ ID NO:196) with 4.

The expression plasmid of the CPM-FF Luc fusions of Fig. 5 B. and RII β B (CPM-FF Luc/RII β B).The unique combination of restriction enzyme allows the DNA of coding RII β B to connect in frame, has plasmid (the corresponding SEQ ID of the GSTG NO:122 of the coding CPM-FF Luc/RII β B fusion rotein of various X/Y peptide linker length with generation; The corresponding SEQ ID of GSSG NO:197; The corresponding SEQ ID of GSSGGSGGSG NO:198, the corresponding SEQ ID of GSGGSGGSSG NO:199; The corresponding SEQ ID of GSSGGSGGSGGGSGGSGGSG NO:200; With the corresponding SEQ ID of GSGGSGGSGGTSGGSGGSSG NO:201).

Fig. 5 C. modifies and is used for the Epac dna sequence dna (SEQID NO:15) that intestinal bacteria (E.coli) are expressed.

Fig. 5 D. expresses circular permutation sea pansy luciferase (CPM-hRL) expression plasmid and the construct of the fusions (CPM-hRL/RII β B) of CPM-hRL and RII β B.The unique combination of restriction enzyme allows the DNA of coding RII β B to connect in frame, has plasmid (the corresponding SEQ IDNO:122 of GSTG of the coding CPM-hRL/RII β B fusion rotein of various X/Y peptide linker length with generation; The corresponding SEQ ID of GSSG NO:197; The corresponding SEQ IDNO:198 of GSSGGSGGSG; The corresponding SEQ ID of GSGGSGGSGGTSGGSGGSSG NO:201).Be rich in 4 two the corresponding SEQ ID of amino acid whose peptide NO:196 of Gly/Ser.

Fig. 6. the SDS-PAGE of in-vitro transcription/translation product with circular permutation beetle luciferase of cAMP binding site analyzes.Have (X=4, Y=4), (X=10, Y=10) and (X=20, Y=20) the CPM-FF Luc/RII β B Expression of Fusion Protein of the X/Y joint length of individual amino-acid residue.

Fig. 7. have (X=4, Y=4), (X=10, Y=10) and (X=20, Y=20) function based on the cAMP transmitter of CPM-FF Luc/RII β B of the X/Y joint length of individual amino-acid residue characterizes.

Fig. 8 A. use and to have (X=4, Y=4), (X=10, Y=10) and (X=20, Y=20) dose response experiments based on the cAMP transmitter of CPM-FF Luc/RII β B of the X/Y joint length of individual amino-acid residue.

Fig. 8 B. has (X=10, Y=10) the cAMP Selectivity of Sensor based on CPM-FF Luc/RII β B of the X/Y joint length of individual amino-acid residue.

Fig. 9. from have (X=10, the homogeneity cAMP determination data of the reaction of the CPM-FFLuc/RII β B cAMP transmitter of X/Y joint length Y=10).

Figure 10 A-B. is about the active comparison of RLU of the circular permutation sea pansy luciferase that comprises the cAMP binding site.

Figure 11 A-B. uses the measurement of 2 kinds of different CPM-FF Luc/RII β B cAMP biosensors cAMP concentration in the lysate of the HEK293 cell that Fu Sikelin handles.

Figure 11 C. in time RLU in the HEK293 cell of DNA transient transfection, described dna encoding has (X=10, the cAMP luciferase biosensor based on CPM-FFLuc/RII β B of X/Y joint length Y=10).

Figure 12. the function of CPM-FF Luc/RII β B cAMP biosensor with X/Y joint length of the total number of atnino acid in the set [2x (x=0-5), 2y (y=0-5)] characterizes.In the existence of 100 μ M cAMP with the luciferase activity not.Splice combinations (10,2) and (10,6) do not show.

Figure 13. the function of CPM-FF Luc/RII β B cAMP transmitter with X/Y joint length of the total number of atnino acid in the set [2x (x=0-5), 2y (y=0-5)] characterizes.Induce multiple in the luciferase activity in the presence of 100 μ M cAMP.Splice combinations (10,2) and (10,6) do not show.

Figure 14. the function of CPM-FFLuc/RII β B cAMP transmitter with X/Y joint length of the total number of atnino acid in set [10 ,-2n (n=1-7)], [10,2n (n=1-5)] and [10+2n (n=1-5), 0] characterizes.In the existence of 100 μ M cAMP with the luciferase activity not.

Figure 15. the function of CPM-FFLuc/RII β B cAMP transmitter with X/Y joint length of the total number of atnino acid in set [10 ,-2n (n=1-7)], [10,2n (n=1-5)] and [10+2n (n=1-5), 0] characterizes.Induce multiple in the luciferase activity in the presence of 100 μ M cAMP.

Figure 16 A-B. use and to have (X=4, Y=4) and (X=10, Y=4) comparison of the dose response experiments of the CPM-Pleonomus Luc/RII β B cAMP transmitter of the X/Y joint length of individual amino-acid residue and corresponding C PM-FF luciferase.

Figure 17 A-B. use and to have (X=4, Y=4) and (X=20, Y=20) comparison of the dose response experiments of the CPM-FF Luc/RI α B cAMP transmitter of the X/Y joint length of individual amino-acid residue and corresponding C PM-FF Luc/RII β B.

Figure 18 A-B. use and to have (X=4, Y=4) and (X=20, Y=20) comparison of the dose response experiments of the heat-resisting Luc/RII β of the CPM-B cAMP transmitter of the X/Y joint length of individual amino-acid residue and corresponding C PM-FF luciferase.

Figure 19. use (X=4, the variation of the cAMP concentration in the CPM-hRL/RII β B cAMP bio-sensor monitors HEK293 cell of X/Y joint length Y=20).

The sequence of Figure 20 .CPM-FF Luc (SEQ ID NO:16).

The synoptic diagram of Figure 21 .CPM-FF Luc GAF construct.The corresponding SEQ ID of GSTG NO:122; The corresponding SEQ ID of GSSG NO:197; The corresponding SEQ ID of GSSGGSGGSG NO:198; The corresponding SEQ ID of GSGGSGGSSG NO:199; The corresponding SEQ ID of GSSGGSGGSGGGSGGSGGSG NO:200; The corresponding SEQ ID of GSGGSGGSGGTSGGSGGSSG NO:201; With the corresponding SEQ ID of 42RT control peptide NO:196.

Figure 22. the RLU of various CPM-FF Luc GAF constructs in the existence of cGMP and not.

Figure 23. follow the multiple of inducing of the cGMP that increases concentration or cAMP for CPM-FF Luc GAF construct.

The synoptic diagram of Figure 24 .CPM-FF Luc calcium biosensor.The corresponding SEQ IDNO:122 of GSTG; The corresponding SEQ ID of GSSG NO:197; The corresponding SEQ IDNO:198 of GSSGGSGGSG, the corresponding SEQ ID of GSGGSGGSSG NO:199; The corresponding SEQ ID of GSSGGSGGSGGGSGGSGGSG NO:200; With the corresponding SEQ ID of GSGGSGGSGGTSGGSGGSSG NO:201; The corresponding SEQ ID of 42RT control peptide NO:196; The corresponding SEQ ID of LEGSGGGG NO:202; With the corresponding SEQ ID of GGGGSGPW NO:203.

Figure 25. at CaCl ₂Or the RLU of various CPM-FF Luc calcium biosensors under the existence of EDTA and EGTA.

Figure 26. the other site of the modification of Lampyridea luciferase.

The external RLU of Figure 27 .CPM-FF Luc cAMP biosensor on each site and induce multiple.

The external RLU of Figure 28 .CPM-FF Luc cAMP biosensor on each site and induce multiple.

Figure 29. have the construct of the insertion of the RII β B in the sea pansy luciferase of non-annularity conversion.

Figure 30. the RLU of the construct shown in Figure 29 and induce multiple.

Figure 31. have the RII β B in the sea pansy luciferase of non-annularity conversion and the construct of various terminal length.

Figure 32. the RLU of the construct shown in Figure 31 and induce multiple.

Figure 33. have the construct of the RI α B in the sea pansy luciferase of circular permutation.

Figure 34. the RLU of the construct among Figure 33 and induce multiple.

Figure 35. the external activity test.Construct pBFB287 is used for 91 sites.After using TnTT7 link coupled rabbit reticulocyte split product system (Coupled Rabbit ReticulocyteLysate System) to express, make 8.5 μ L TNT reaction and be supplemented with 1.7 μ L1mM cAMP stoste or dH ₂The 8.5 μ L 300mM HEPES/200mM thiocarbamides (pH about 7.5) of O mix; Permission was reacted at room temperature incubation about 10 minutes.Every kind of sample of 5 μ L is added in 96 orifice bores in triplicate, and use 100 μ L sea pansy luciferase assay reagent on the Glomax photometer, to measure luminous.

Figure 36. the external activity test.Construct 201325.44.H6 is used for 91 sites.After using TnT T7 link coupled malt extract system expression, make 5 μ L TNT reaction replenish 1.5 μ L 1mM cAMP stoste or dH ₂O; Permission was reacted at room temperature incubation about 10 minutes.This mixture of 15 μ L is added in the 75ul 1X sea pansy lysis buffer subsequently, and every kind of sample of 20 μ L is added in 96 orifice bores in triplicate, and use 100 μ L sea pansy luciferase assay reagent on the Glomax photometer, to measure 91 and 223 kind of construct luminous.For 229 kinds of constructs, cAMP induces as shown in Figure 35 the measurement.

The transient transfection data of Figure 37 .CPM RLuc cAMP biosensor.

The synoptic diagram that Figure 38 A. uses the single step of the GPCR of CPM FF Luc cAMP biosensor to measure.

Figure 38 B. is the Fu Sikelin concentration mapping of RLU to increasing in CPM FF Luc cAMP measures.

Figure 39. from the data of using CPM FF Luc cAMP biosensor SCREENED COMPOUND library.

Figure 40. use the dose response of the particular compound of CPM FF Luc cAMP biosensor.

Figure 41. aminoacid sequence (the SEQ ID NO:204 of exemplary oar foot animal luciferase; Genbank ID AAG54095).

In time the comparison of relative response under room temperature and 37 ℃ of Figure 42 .CPM-FF Luc/RII β cAMP biosensor.

Figure 43 A-B. in the presence of various agonists (A) or antagonist (B) at the RLU of room temperature and 37 ℃ of following CRE reporter and CPM-FF Luc/RII β cAMP biosensor.

Figure 44. with stable transfection CPM-FF Luc/RII β and the cell of Dopamine HCLs that is exposed to different amounts under 37 ℃ in time induce multiple.

Figure 45. at 37 ℃ of following RLU log M Dopamine HCL is mapped.

Figure 46. the effectiveness about various agonists under 37 ℃ sorts.

Figure 47. the effectiveness about various antagonists under 37 ℃ sorts.

Figure 48. the effectiveness ordering of the agonist of the beta 2-adrenergic receptor of use HEK293/CPM-FF Luc/RII β.The HEK293 cell of stably express CPM-FF Luc/RIIB stimulates with the agonist of endogenous beta 2-adrenergic receptor.Luminous incubation was at room temperature measured after 26 minutes.

Figure 49. the effectiveness ordering of the agonist of the beta 2-adrenergic receptor of use HEK293.The HEK293 cell of stably express CPM-FF Luc/RIIB in the presence of 0.033 μ M Racemic isoproterenol with the antagonist incubation.Luminous incubation was at room temperature measured after 31 minutes.

Figure 50. use the comparison of the noclilucence GPCR mensuration of various agonists.

Figure 51. use the comparison of the noclilucence GPCR mensuration of various antagonists.

Figure 52. use the detection that changes in the cell of CPM RLuc/RII β B cAMP biosensor to cAMP.A) comparison of the detection of use different promoters.B) Fu Sikelin induces.C) SK38393 induces.D) Induced by Dopamine.

Figure 53. the detection that changes in the cell of the cAMP in cell with CPM RLuc/RII β B cAMP biosensor.

Figure 54. have the RLU of the FLuc construct of RII β B and various terminal length.

Figure 55. the nucleotide sequence (SEQ ID NOs:205,206,207,208,209) of thorn shrimp luciferase and fusion constructs thereof.

Figure 56. measure the RLU that stings shrimp luciferase fusions in (PCA) in external complementary action of protein.After background rejection, measure and induce multiple.

Figure 57. the SDS-PAGE of thorn shrimp luciferase (OpLuc) fusions analyzes.Swimming lane 1) total length OpLuc; Swimming lane 2) 50-FRB of coexpression and FKBP-51; Swimming lane 3) 50-FRB; Swimming lane 4) FKBP-51; With swimming lane 5) no DNA contrast.

Figure 58. the RLU of thorn shrimp luciferase fusions in external PCA.After background rejection, measure and induce multiple.

Figure 59. the SDS-PAGE of thorn shrimp luciferase fusions analyzes.Swimming lane 1) total length OpLuc; Swimming lane 2) 84-FRB of coexpression and FKBP-85; Swimming lane 3) 84-FRB; Swimming lane 4) FKBP-85; With swimming lane 5) no DNA contrast.

Figure 60. the RLU of thorn shrimp luciferase fusions in based on the PCA of cell.N＝3。Ss=divides the site.After background rejection, measure and induce multiple.

Figure 61. the RLU of thorn shrimp luciferase circular permutation sample fusions in external PCA.After background rejection, measure and induce multiple.

Figure 62. the SDS-PAGE of thorn shrimp luciferase circular permutation sample fusions analyzes.Swimming lane 1) total length OpLuc; Swimming lane 2) 51-FKBP of coexpression and FRB-50; Swimming lane 3) 85-FKBP of coexpression and FRB-84; Swimming lane 4) FRB-50; Swimming lane 5) FRB-84; Swimming lane 5) 51-FKBP; Swimming lane 7) 85-FKBP; With swimming lane 8) no DNA contrast.84-FRB; Swimming lane 4) FKBP-85; With swimming lane 5) no DNA contrast.

Figure 63. based on the carrier of CP thorn shrimp luciferase.

Figure 64. use result based on the carrier of RII β B CP thorn shrimp luciferase.The post on the left side is pointed out the activity of complete luciferase (contrast).The twoth post that begins from the left side: correspondence has the construct of 4aa joint.The 3rd post that begins from the left side: have the 4aa joint-"-.The 4th post that begins from the left side: have the 20aa joint-"-.

Figure 65. serine/threonine kinase/Phosphoric acid esterase construct.The concrete peptide sequence of identifying is in this table: EIYGEFGSSG (SEQ ID NO:267), EIYGEFGSSGGSGGSG (SEQ ID NO:268), EIYGEFGSSGGSGGSGGGSGGSGGSG (SEQ IDNO:269), GSSG (SEQ ID NO:270), GSTSGSGKPGSGEGSEIYGEFGSSG (SEQ ID NO:271), GSTSGSGKPGSGEGSEIYGEFGSGGSGGSSG (SEQ ID NO:272), GSTSGSGKPGSGEGSEIYGEFGSGGSGGSGGGSGGSGGSSG (SEQ IDNO:273), GSTSGSGKPGSGEGSEIYGEFGSGSGGSGGSSG (SEQ IDNO:274), GSTG (SEQ ID NO:275), GSSGGSGGSG (SEQ ID NO:276), GSSGGSGGSGGGSGGSGGSG (SEQ ID NO:277), GSGGSGGSGGTSGGSGGSSG (SEQ ID NO:278), GSSGRKRDRLGTLGIGGSSGGGSGGGGSGG (SEQ ID NO:279), GGSGGSGSSGRKRDRLGTLGIGGSSGGGSGGGGSGG (SEQ ID NO:280), GSGGSGGSGG (SEQ ID NO:281), GSSGGSGGSGGGSGGSGSSGRKRDRLGTLGIGGSSGGGSGGGGSGG (SEQ ID NO:282), RKRDRLGTLGIGGSSGGGSGGGGSGG (SEQ IDNO:283), GGSSGRKRDRLGTLGIGGSSG (SEQ ID NO:284), GGSSGRKRDRLGTLGIGSSGSGGSGG (SEQ ID NO:285), GGSSGRKRDRLGTLGIGSGGSGGSGGTSGGSGGSSG (SEQ ID NO:287), GSSGGSGGSGGGSGGSG (SEQ ID NO:288), GGSSGRKRDRLGTLGIGSSGSGGSGGTSGGSGGSSG (SEQ ID NO:289), GSSGGSGGSGGGRKRDRLGTLGIGGSSGGGSGGGGSGG (SEQID NO:290), GSGGSGGSSG (SEQ ID NO:291), GSSGGSGGSGGGSGGSGGSGRKRDRLGTLGIGGSSGGGSGGGGSGG (SEQ ID NO:292), GSGG (SEQ ID NO:293), and GGSGGGGSGG (SEQ ID NO:294).

Figure 66. external Fluc serine/threonine kinase is measured.

Figure 67 .A-DDDDDDDDDDDD.Representative nucleotide sequence.

Detailed Description Of The Invention

Definition

As used herein, term " nucleic acid molecules ", " polynucleotides " or " nucleotide sequence " refer to nucleic acid, DNA or RNA, and it comprises polypeptide or protein precursor is produced necessary coded sequence. The polypeptide of coding can be the fusions of full-length polypeptide, its fragment (less than total length) or full-length polypeptide or its fragment and another kind of polypeptide, produces fused polypeptide.

As used herein, " nucleic acid " is covalently bound nucleotide sequence, wherein 3 ' position of the pentose of 1 nucleotides is connected by the 5 ' position of phosphodiester group with the pentose of next nucleotides, and wherein nucleotide residue (base) namely connects in the linear precedence of nucleotides at particular sequence. As used herein, " polynucleotides " are to comprise the nucleic acid that length surpasses the sequence of about 100 nucleotides. As used herein, " oligonucleotides " or " primer " is the part of short polynucleotides or polynucleotides. Oligonucleotides generally comprises the sequence of about 100 bases of about 2-. Word " oligonucleotides (oligo) " replaces word " oligonucleotides (oligonucleotide) " to use sometimes.

Nucleic acid molecules is said to be and has " 5 '-end " (5 ' end) and " 3-is terminal " (3 ' end), because the phosphatase nucleic acid diester linkage appears on the 5 ' carbon and 3 ' carbon of pentose ring of replacement mononucleotide. New key will be that the end of the polynucleotides of 5 ' carbon is its 5 ' terminal nucleotide thereon. New key will be that the end of the polynucleotides of 3 ' carbon is its 3 ' terminal nucleotide thereon. As used herein, terminal nucleotide be 3 '-or 5 '-nucleotides on the terminal terminal position.

Dna molecular is said to be has " 5 ' end " and " 3 end " because mononucleotide reacts to form oligonucleotides in the following manner: namely so that 5 ' phosphoric acid of 1 mononucleotide pentose ring be connected with 3 ' oxygen of its neighbours via phosphodiester bond in one direction. Therefore, if 5 ' phosphoric acid of oligonucleotides is not connected with 3 ' oxygen of mononucleotide pentose ring, then the end of oligonucleotides is called " 5 ' end ", if its 3 ' oxygen is not connected with 5 ' phosphoric acid of subsequently mononucleotide pentose ring, then is called " 3 ' end ".

As used herein, even nucleotide sequence is inner for larger oligonucleotides or polynucleotides, also can be said to be and has 5 ' and 3 ' end. In linearity or ring-shaped DNA molecule, discrete elements be called " upstream " or 5 of " downstream " or 3 ' element '. The reflection of this term transcribe along the DNA chain with 5 ' to 3 ' mode carry out. The promoter of transcribing of the gene (for example, open read frame or code area) that usually, instruct to connect and enhancer element generally are positioned at 5 of code area ' or upstream. Yet even when the 3 ' time that is positioned at promoter element and code area, enhancer element also can be brought into play its effect. Tanscription termination and polyadenylation signal are positioned at 3 of code area ' or downstream.

The basic genetic coding units as used herein, that term " codon " is comprised of the sequence of 3 nucleotides, its appointment are impregnated in specific amino acids or the initial or termination signal in the polypeptide chain. Term " code area " refers to be coded in the amino acid whose nucleotide sequence of finding in the newborn polypeptide owing to the translation of mRNA molecule when using with regard to structural gene. Usually, the code area is border by the nucleotide triplet " ATG " of the initial sub-methionine of coding and in 3 ' side by terminator codon (for example, TAA, TAG, TGA) in 5 ' side. In some cases, the code area is also known initial by nucleotide triplet " TTG ".

Term " gene " refers to comprise coded sequence and optional comprising for the dna sequence dna of being produced the necessary control sequence of polypeptide by dna sequence dna.

As used herein, term " allos " nucleotide sequence or protein refer to have separate sources with respect to canonical sequence, for example come from the sequence of alien species, if or from same species, it can significantly be modified by primitive form so.

Nucleic acid is known to comprise dissimilar sudden changes. " point " sudden change refers in nucleotide sequence in the locational change of single base from wild-type sequence. Sudden change can also refer to insertion or the disappearance of one or more bases, thereby so that nucleotide sequence is different from reference, wild-type sequence for example.

As used herein, term " hybridization " refers to the annealing of complementary series and target nucleic acid, namely comprises the ability that two nucleic acid polymers (polynucleotides) of complementary series are annealed by base pairing. Term " annealing " and " hybridization " are used interchangeably from start to finish, and are intended to comprise any specific and reproducible interaction between complementary series and the target nucleic acid, comprise the combination in the zone that only has the part complementarity. Usually undiscovered some base can be included in the nucleic acid of the present invention in natural acid, and comprises for example inosine and 7-deazaguanine. The technical staff in nucleic acid field can consider numerous variablees, determines by rule of thumb duplex stability, and described variable comprises for example right incidence of length, base composition and oligonucleotide sequence, ionic strength and base mismatch of complementary series. The stability of nucleic acid duplex is by melting temperature or " T_m" measure. The T of the specific nucleic acid duplex under specified requirements_mIt is the temperature that average half base-pair dissociates.

Term " recombinant DNA molecules " means to be included in the hybrid dna sequence that occurring in nature is not found at least two nucleotide sequences together usually.

Term " carrier " is used in reference to such nucleic acid molecules, and namely dna fragmentation can insert or be cloned in it and can be used for the DNA sector transfer in cell and can copy at cell. Carrier can derive from plasmid, bacteriophage, virus, clay etc.

As used herein, term " recombinant vector " and " expression vector " refer to such DNA or RNA sequence, and it comprises required coded sequence and expresses necessary suitable DNA or RNA sequence for the coded sequence that is operably connected in the specific host biology. Prokaryotic expression carrier comprises promoter, ribosome bind site, is used at the origin of replication of host cell self-replicating and comprises possibly other sequences, the operon sequence of for example choosing wantonly, optional restriction enzyme sites. Promoter is defined as the dna sequence dna that the guide RNA polymer is combined with DNA and initial RNA is synthetic. Carrier for expression of eukaryon comprises promoter, randomly comprises that polyadenylation signal also randomly comprises enhancer sequence.

Polynucleotides with nucleotide sequence of coded protein or polypeptide mean to comprise the nucleotide sequence of the code area of gene, or nucleic acid sequence encoding gene outcome in other words. The code area can exist with cDNA, genomic DNA or rna form. When existing with dna form, oligonucleotides can be strand (that is, sense strand) or double-stranded. When needing, suitable control element such as enhancers/promoters, montage junction surface, polyadenylation signal etc. can place the code area close proximity with gene, with suitable transcription initiation and/or the correct processing that allows elementary rna transcription thing. Alternately, code area. Other regulating elements include but not limited to transcription factor binding site point, splicing signal, polyadenylation signal, termination signal and enhancer element.

The control signal of transcribing in the eucaryote comprises " promoter " and " enhancer " element. Promoter and enhancer are comprised of the short array of dna sequence dna, its with relate to the cell protein specificity of transcribing and interact. Promoter is separated from various eucaryotes source with enhancer element, and described source comprises the gene in yeast, insect and the mammalian cell. Promoter is also separated from virus with enhancer element, and similarly control element for example promoter also in prokaryotes, find. The selective dependency of specific promoter and enhancer is used for expressing the cell type of destination protein matter. Some eukaryotic promoter and enhancer have widely host range, and other is useful in limited cell type subset. For example, SV40 early gene enhancer has activity very much in the extensive various cell type from many mammalian species, and has been widely used in marking protein in mammalian cell. Two of activated promoter/enhancer element other examples are from those of people's EF-1 gene of Rous sarcoma virus and human cytomegalovirus and LTR in the mammalian cell types of broad range.

Term " promoter/enhancer " refers to comprise the DNA section of the sequence that promoter and enhancer function (that is the function that, is provided by aforesaid promoter element and enhancer element) can be provided. For example, retroviral LTR comprises promoter and enhancer function. Enhancers/promoters can be " endogenous " or " external source " or " allos ". " endogenous " enhancers/promoters be in genome with natural be connected the sort of of given gene. " external source " or " allos " enhancers/promoters is the enhancers/promoters of placing side by side by means of genetic manipulation (that is, Protocols in Molecular Biology) and gene, thereby so that transcribing by the enhancers/promoters that connects of gene instruct.

At the existence of " splicing signal " on the expression vector transcript higher expression in eukaryotic host cell that usually causes recombinating. The splicing signal mediation forms from the removal of the introne of one-level rna transcription thing and by donor splicing site and acceptor site. Normally used donor splicing site and acceptor site are the montage joints from the 16S RNA of SV40.

The effective expression of recombinant DNA sequence in eukaryotic cells need to instruct the expression of the signal of effective termination of resulting transcript and polyadenylation. Transcription terminator generally finds in the downstream of polyadenylation signal and length is hundreds of nucleotides. As used herein, the termination of nascent RNA transcript and the dna sequence dna of polyadenylation are instructed in term " poly-(A) site " or " poly-(A) sequence " expression. Effective polyadenylation of restructuring transcript is wished, is unstable and fast degradation in default of the transcript that gathers (A) tail. Poly-(A) signal that uses in expression vector can be " allos " or " endogenous ". Natural discovery is the sort of on 3 ' end that endogenous poly-(A) signal is the code area of given gene in genome. Poly-(A) signal of allos be from a kind of gene, separated and place another kind of gene 3 ' the sort of. Poly-(A) signal of normally used allos is poly-(A) signal of SV40. Poly-(A) signal of SV40 is included on the 237bp BamH I/Bcl I restricted fragment and instructs and stops and polyadenylation.

Carrier for expression of eukaryon can also comprise " virus replication " or " virus replication starting point ". Virus replication is the viral DNA sequence that allows carrier extrachromosomal replication in the host cell of expressing suitable replicator. The carrier that comprises SV40 or polyomavirus origin of replication is copied to high copy number (being up to 104 copy/cells) in the cell of expressing suitable viral T antigen. By contrast, comprise carrier from the replicon of bovine papilloma virus or Epstein-Barr virus with low copy number (about 100 copy/cells) extrachromosomal replication.

Process or reaction that term " external " refers to artificial environment and refers to occur in artificial environment. External environment includes but not limited to test tube and cell lysate. Process or reaction that term " in the body " refers to natural surroundings (for example, animal or cell) and refers to occur in natural surroundings.

Term " expression system " refers to be used for measuring any mensuration or the system of (for example, detecting) destination gene expression.Technician in the biology field is to be understood that and can uses any in extensively various expression system.The suitable mammalian cell of broad range can (for example, American type culture collection, Rockland, MD) acquisition from extensively various source.The vectorial selection of conversion or transfection method and expression will rely on selected host system.Conversion and transfection method are well-known in the art.Expression system comprises outer-gene expression mensuration, and wherein goal gene (for example, reporter gene) is connected with the adjusting sequence, and expression of gene is monitored after the agent treated with inhibition or inducible gene expression.The detection of genetic expression can be by any suitable manner, include but not limited to the mRNA that expresses or protein (for example, the detected product of reporter gene) detection, but or by the change detected in the cell phenotype of expressing goal gene.Expression system can also comprise the mensuration that wherein detects cutting incident or other nucleic acid or cellular change.

As used herein, term " wild-type " refers to have the gene or the gene product of the feature of from naturally occurring source isolating the sort of gene or gene product.Therefore wild type gene is the most frequent observed and be arbitrarily designated as the sort of of gene " wild-type " form in colony.By contrast, term " mutant " refers to when comparing with wild type gene or gene product, the gene or the gene product of the modification in display sequence and/or the functional property (that is the feature of change).Should be understood that the mutant that can separating natural exists; These are identified by the fact that has the feature of change when with wild type gene or gene product comparison.

Term " isolating " when with regard to nucleic acid, using, when in " isolating oligonucleotide " or " isolating polynucleotide ", using, refer to be identified and with its source in the nucleotide sequence that at least a pollutent of pass separates that associates usually with it.Therefore, isolating nucleic acid is with the form that is different from it and finds at occurring in nature or be provided with and exist.By contrast, the status discovery that exists at occurring in nature with them of non-isolating nucleic acid (for example, DNA and RNA).For example, given dna sequence dna (for example, gene) approaches the contiguous gene discovery on host cell chromosome; RNA sequence (for example, the specific mRNA sequence of encode specific protein matter) conduct in cell is found with the mixture of numerous other mRNAs of coding numerous protein.Yet isolating nucleic acid for example comprises this nucleic acid in the cell of expressing described nucleic acid usually, and its amplifying nucleic acid is in being different from the chromosome position of n cell, or originally by the different nucleotide sequence side joint of finding with occurring in nature of nucleotide sequence.Isolating nucleic acid or oligonucleotide can exist with strand or double chain form.When isolating nucleic acid or oligonucleotide will be used for marking protein, the oligonucleotide bottom line included justice or coding strand (that is, oligonucleotide can be a strand), but can include justice and antisense strand (that is, oligonucleotide can be double-stranded).

" peptide ", " protein " and " polypeptide " mean any amino acid chain, and be irrelevant with length or posttranslational modification (for example, glycosylation or phosphorylation).Can also the encode variant of naturally occurring protein or its polypeptide fragment of nucleic acid molecule of the present invention, it has naturally occurring (natural or wild-type) the proteinic aminoacid sequence at least 85%, 90%, 95% or 99% aminoacid sequence that is equal to of originating with it.Term " fusion polypeptide " or " fusion rotein " refer to be included in the chimeric protein with reference to protein (for example, luciferase) that connects with one or more heterologous sequences (for example, non-luciferase polypeptide) on N and/or the C-terminal.In certain embodiments, the part of modified polypeptide, fusion polypeptide or the full-length polypeptide of the present invention can keep some activity at least of corresponding total length functional (non-chimeric) polypeptide.In other embodiments, with respect to corresponding total length functional polypeptide, under the situation that does not have exogenous agent or molecules of interest, the part of polypeptide, fusion polypeptide or total length functional polypeptide that the present invention is modified can lack activity.In other embodiments, with respect to corresponding total length functional polypeptide, in the presence of exogenous agent, the part of polypeptide, fusion polypeptide or total length functional polypeptide that the present invention is modified can keep some activity at least, or have substantially the same activity, or alternately lack active.

Peptide molecule is said to be has " N-terminal " (N-terminal) and " C-terminal " (C-terminal), because peptide bond is present between the main chain carboxyl of amino and second amino-acid residue of the main chain of first amino-acid residue.Term " N-terminal " and " C-terminal " refer to comprise the polypeptide zone of the part in the N-terminal of polypeptide and C-terminal zone respectively with regard to peptide sequence.The sequence of part that comprises the N-terminal zone of polypeptide comprises mainly from half amino acid of the N-terminal of polypeptide chain, but is not limited thereto kind of a sequence.For example, the N-terminal sequence can comprise the internal portion of peptide sequence, comprises from half base of the N-terminal of polypeptide and C-terminal.This is applied to the C-terminal zone equally.N-terminal and C-terminal zone can but need not to comprise respectively the final N-terminal that limits polypeptide and the amino acid of C-terminal.

As used herein, term " recombinant protein " or " recombinant polypeptide " refer to by recombinant DNA molecules expressed protein molecule.By contrast, term " natural protein " is used in reference in this article from naturally occurring (that is non-reorganization) the isolating protein of originating.Protocols in Molecular Biology can be used to produce the protein that relatively has the recombinant forms of same nature with the protein of natural form.

As used herein, term " cell ", " clone ", " host cell " are used interchangeably, and all this kind names comprise the offspring or the potential offspring of these names." transformant " means the cell that nucleic acid molecule of the present invention has been introduced (or in its ancestors) in it.Randomly, nucleic acid molecule of the present invention can be introduced in the suitable clone, so that preparation can be produced the clone by the stable transfection of the protein of genes encoding or polypeptide.The carrier, cell and the method that are used to make up this kind clone are well-known in the art.Word " transformant " or " transformant " comprise the former generation transformant that derives from original transformant, with to shift number irrelevant.Because have a mind to or sudden change unintentionally, all offsprings may not be accurately identical aspect dna content.Yet when screening in original transformant, the sudden change offspring with same functionality is included in the definition of transformant.

Term " homology " refers to the complementary degree between two or more sequences.Can there be portion homologous or complete homology (that is identity).Homology (is for example used sequence analysis software usually, Genetics Computer Group.University of WisconsinBiotechnology Center.1710 University Avenue.Madison, the sequence analysis software bag of WI 53705) measure.This kind software is by specifying the homology degree to mate similar sequences to various replacements, disappearance, insertion and other modifications.Conservative replacement generally comprises the replacement in following group: glycine, L-Ala; Xie Ansuan, Isoleucine, leucine; Aspartic acid, L-glutamic acid, l-asparagine, glutamine; Serine, Threonine; Methionin, arginine; And phenylalanine, tyrosine.

When term " isolating " uses with regard to polypeptide, in when " isolating protein " or " isolated polypeptide " uses, refer to be identified and with its source in the polypeptide that separates of associating at least a pollutent usually with it.Therefore, isolated polypeptide is with the form that is different from it and finds at occurring in nature or be provided with and exist.By contrast, the status discovery that exists at occurring in nature with them of non-isolated polypeptide (for example, protein and enzyme).

Term " purifying " or " purifying " mean that for example protein or nucleic acid are removed the result of any process of some pollutent from the purpose component.The per-cent of purified components increases in sample thus.

As used herein, " pure " mean the purpose kind be exist preponderate kind (promptly, on mole foundation, it is abundanter than any other the single kind in the composition), and preferably basically the fraction of purifying be that wherein the purpose kind comprises the composition at least about 50% (on mole foundation) of all macromole kinds of existence.Usually, it is about 80% that " pure basically " composition will comprise surpassing of all macromole kinds of existing in the composition, more preferably surpasses about 85%, about 90%, about 95% and about 99%.Most preferably, the purpose kind is purified to basic homogeneity (pollutant kind can't detect in composition by the conventional sense method), and wherein composition is made up of single macromole kind basically.

As used herein, term " is operably connected " and refers to that nucleotide sequence connects by this way: promptly, make generation can instruct the synthetic nucleic acid molecule of given gene transcription and/or desired protein molecule.This term refers to that also the sequence of coded amino acid connects by this way: promptly make to produce functional (for example, enzymatic activity, can with binding partner binds, can suppress etc.) protein or polypeptide.

As used herein, term " polyhistidyl bundle " or (His mark) refer to comprise the molecule of 2-10 histidine residues, for example the polyhistidyl bundle of 5-10 residue.The polyhistidyl bundle allow covalently bound molecule the fixed metal for example on nickel, zinc, cobalt or copper, the chelate column or by with the interactional affinity purification of the another kind of molecule antibody of His labeled reactant (for example with).

" protein removes critical sequences " includes but not limited to, the PEST sequence, for example from cyclin, for example the PEST sequence of mitotic cell cyclin, uridylic permease or ODC, from fugitive protein ODC, early response protein for example c-myc or c-fos, MyoD, HMG CoA reductase enzyme of cytokine, lymphokine, proto-oncogene for example for example, or the sequence in the C-terminal zone of S-adenylyl methionine decarboxylase, CL sequence, cyclin are destroyed box or N-degron.

As used herein, " marker gene " or " reporter gene " is that the cell of expressing this gene is given different phenotypes and therefore allowed to have the cell of this gene and the gene that does not have the cell differentiation of this gene.Optional or the selection markers but this kind gene can be encoded, this depends on to mark whether to give and can pass through chemical process, promptly by (for example using selective agent, weedicide, microbiotic etc.) proterties of ' selection ', or whether it just can promptly pass through " reporter " proterties that ' screening ' identified by observing or test.The element of present disclosure is by using the detailed illustration of expression of specific marker genes.Certainly, many examples of suitable marker gene or reporter gene are known in the art, and can use in practice of the present invention.Therefore, be to be understood that following discussion is exemplary rather than detailed.According to technology disclosed herein and general recombinant technology known in the art, the invention enables to change any gene.Exemplary report protein is by the nucleic acid molecule encoding that comprises modified reporter gene, include but not limited to the neo gene, β-gal gene, the gus gene, the cat gene, the gpt gene, the hyg gene, the hisD gene, the ble gene, the mprt gene, the bar gene, nitrilase gene, the galactopyranoside gene, the xylosidase gene, thymidine kinase gene, the arabinofuranosidase/xylosidase gene, mutant acetolactate synthase gene (ALS) or etheric acid (acetoacid) synthase gene (AAS), methotrexate resistance dhfr gene, dalapon dehalogenase gene, give sudden change anthranilic acid synthase gene (WO97/26366) to the resistance of 5-methyl tryptophan, R-locus gene, the β-Nei Xiananmei gene, the xylE gene, alpha-amylase gene, tyrosinase cdna, luciferase (luc) gene (for example, sea pansy (Renilla reniformis) luciferase genes, the Lampyridea luciferase genes, or Pleonomus luciferase (Pyrophorus plagiophthalamus) gene), aequorin gene, the red fluorescent protein gene, or the modification of green fluorescence protein gene.

All amino-acid residues that this paper identifies are natural L-configuration.Consistent with standard polypeptide name, be shown in the following correspondence table about the abbreviation of amino-acid residue.

Correspondence table

1-letter 3-letter amino acid

Y Tyr L-tyrosine

G Gly L-glycine

F Phe L-phenylalanine

M Met L-methionine(Met)

A Ala L-L-Ala

S Ser L-Serine

I Ile L-Isoleucine

L Leu L-leucine

T Thr L-Threonine

V Val L-Xie Ansuan

P Pro L-proline(Pro)

K Lys L-Methionin

H His L-Histidine

Q Gln L-glutaminate

E Glu L-L-glutamic acid

W Trp L-tryptophane

R Arg L-arginine

D Asp L-aspartic acid

N Asn altheine

C Cys L-halfcystine

I. Luciferase residue or the regional method of evaluation to modifying tolerance

Numerous methods can be used for identifying site such in the luciferase genes and/or zone, it for example can be modified and to destroy, and still produce hope when transcribing and translate, for example the gene product that can detect easily.For example, amplified reaction can be used for removing and/or inserting in luciferase genes the Nucleotide of one or more amino-acid residues.Alternately, transposon can be used to prepare the library of inserting sudden change.Transposon is the moving DNA sequence of finding in prokaryotic organism and Eukaryotic genome.Transposon tagging has been considered to be used for the random assignment primer binding site for a long time, produces gene and " knocks out ", and physical markings or genetic marker are introduced strong research tool in the big target DNA s.The insertion that is used for preparing the reporter gene of the modified luciferase of the present invention is such, promptly is to insert in the frame of the inside in the coding region of luciferase.

A kind of transposon system of frequent use is an isolating Tn5 system from Gram-negative bacteria.The Tn5 transposase is to have cloned and be purified to high specific acitivity, and need not the host cell factor and carry out little, the single subunit enzyme of swivel base.In addition, it is height random that the Tn5 transposon in target DNA inserts, and is undertaken by simple procedure.The Tn5 transposase is included in its short 19 chimeric ends of base pair (Mosaic End, ME) any dna sequence dna between the Tn5 transposase recognition sequence with swivel base.

GPS-M mutagenesis system uses the TnsABC* transposase inserting at random in the DNA target based on the transposon of Tn7.Target DNA can be the chromosomal DNA of plasmid, clay, BAC or purifying.If insert the site in the constant gene segment C of translation, this will cause invalid (afunction) sudden change usually so.Have the minimum site preference that inserts, therefore the destruction of any open reading-frame (ORF) all is possible.Because the target immunity, the distance of about 190kb is last in vivo only exists 1 insertion/dna molecular.Therefore, vitro reactions produces the target DNA molecule colony that is included in the transposable element on the different positions separately.

The transposon donor can by add or replace microbiotic for example the kalamycin resistance mark modify.Donor plasmid can be grown in the standard laboratory coli strain, and the carrier main chain carries for example Amp of the antibiotic marker different with transposon ^rAnd replication orgin.In order to destroy unreacted donor molecule and to avoid undesirable reaction product, donor can by with rare nickase for example PI-SceI (VDE) digestion destroy.Be transformed into application in the natural competence biology (its picked-up wall scroll DNA chain) for mutagenized dna wherein, breach is filled and connects.

In case identified in the luciferase sequence to modifying the site of tolerance, insertion, disappearance and conversion or its any combination that just can prepare sequence.With regard to the sequence of conversion, people such as Plainkum (2003) report, because avtive spot sterically hindered, have new N with C-terminal and the ribonuclease A of circular permutation form that comprises the peptide linker of the proteolytic enzyme recognition site that is connected original N and C-terminal have the ribonuclease activity of minimizing.People such as Plainkum find can increase this activity of proteins with proteolytic enzyme cutting circular permutation ribonuclease A, and supposition is to realize by the blocking-up of removing for avtive spot.Under the situation of luciferase, N and C-terminal separate about 40 dusts, and this is the distance that equates with 5-6 amino acid.Preparation circular permutation Lampyridea luciferase, one of them has the recognition site at the new N-terminal on the Asp (234) and new C-terminal on Pro (233) and proteolytic enzyme enteropeptidase, described enteropeptidase the cutting of the C-terminal side of Asp (4) Lys (referring to the U.S. openly apply for 20050153310 and PCT/US2004/032705).The activity of the mutain that merges increases about 90-about 150 times (Fig. 3) by handling with enteropeptidase.The other biological transmitter comprise insert circular permutation Lampyridea luciferase or Pleonomus luciferase (CP1:R=Asn401 and CP2:R=Arg223) (referring to the U.S. openly apply for 20050153310 and PCT/US2004/032705) in Caspase-3DEVD cleavage site (Fig. 3), the PSA cleavage site is Ala-Asn-Lys-Ile-Ser-Tyr-Gln-Ser-Ser-Ser-Thr-Glu (SEQ IDNO:17) for example, the rhinovirus protease site is Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro (SEQ ID NO:19) for example, with SARS virus protease site TSAVLQSGFR (SEQID NO:20) for example.CP2 has the insertion on the 234th bit position in the corresponding luciferase in the Pleonomus luciferase.As mentioned below, preparation circular permutation sea pansy luciferase.

Biosensor of the present invention include but not limited to following these: wherein the allogeneic amino acid sequence comprises the protein bound structural domain, for example in conjunction with the structural domain of IL-17RA, IL-17A for example, or the IL-17A binding domains of IL-17RA, the Jun binding domains of Erg, or the EG binding domains of Jun; Potassium channel voltage sensor structural domain, for example be used to detect the structural domain that protein conformation changes, the GTP enzyme binding domains of Cdc42 or rac target, or other GTP enzyme binding domainss, the structural domain relevant with kinases or phosphatase activity, modulability myosin light chain for example, PKC δ, the PH and the DEP structural domain that comprise pleckstrin, other phosphorylation recognition structure territory and substrates; The conjugated protein structural domain of glucose, the conjugated protein structural domain of L-glutamic acid/aspartic acid, PKA or cAMP dependency bound substrates, InsP3 acceptor, GKI, PDE, estrogen receptor ligands binding domains, apoK1-er, or calmodulin binding domains.

In one embodiment, biosensor is used to detect the GTP enzyme, for example the DMCA of IBB, the CRaf1 of Cdc42 or Rac and EBFP, EGFP PAK fragment, Raichu-Rac, Raichu-Cdc42, beta 2 integrin alpha v β 3, input albumen-a or NBD-Ras (being used for the Ras activation), have the combining of binding domains of the Ras/Rap Ral RBD of Ras isoprenylation sequence.In one embodiment, biosensor (for example detects PI (4,5) P2, use PH-PCL δ 1, PH-GRP1), (for example, PH-OSBP), PI (3 for PI (4,5) P2 or PI (4) P, 4,5) P3 (for example, using PH-ARNO or PH-BTK or PH-born of the same parents mucoprotein 1), PI (3,4,5) P3 or PI (3,4) P2 (for example, using PH Akt), PI (3) P are (for example, use FYVE-EEA1) or Ca2+ (cytosol) (for example, the C2 structural domain of use calmodulin or PKC.

In one embodiment, structural domain be have a phosphorylation tyrosine (for example, at Src, among Ab1 and the EGFR) structural domain, it detects the phosphorylation of ErbB2, Src, tyrosine phosphorylation among Ab1 and the EGFR, the activation of MKA2 (for example, use MK2), cAMP inductive phosphorylation, the activation of PKA (for example using the KID of CREG), the phosphorylation of CrkII (for example using SH2 structural domain pTyr peptide), bZIP transcription factor and REL protein (for example bFos and bJun ATF2 and Jun, or p65NF κ B) combination, or microtubule is in conjunction with (for example using kinesin).

Therefore, the present invention includes the luciferase biosensor, comprise the circular permutation luciferase, described luciferase sequence can comprise the disappearance of original (wild-type) N or C-terminal or the residue on both, for example in the disappearance of the 1-3 on the N-terminal or more residues or the 1-6 on C-terminal or more residues, and with the direct or indirect interactional sequence of molecules of interest.

II. Exemplary polynucleotide and protein

The present invention includes the modified luciferase that comprises any following aminoacid sequence, but described aminoacid sequence provide reorganization or synthetically synthetic have detection of active, the polypeptide of luminescence activity for example, with and protein fragments.The luciferase sequence of modified luciferase is identical or substantially the same with the aminoacid sequence of the luciferase of corresponding unmodified.Having substantially the same polypeptide of sequence or peptide, to mean aminoacid sequence identical to a great extent but may not be identical, and keep the functionally active of the sequence relevant with it.Generally speaking, if two aminoacid sequences have at least 70% identity, or have at least 80%, 90%, 95% or more identity, then they are substantially the same or homologous basically.

Homology or identity (are for example used sequence analysis software usually, Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, the sequence analysis software bag of WI 53705) measure.This kind software is by specifying the homology degree to mate similar sequences to various disappearances, replacement and other modifications.Term " homology " and " identity " in two or more nucleic acid or peptide sequence background refer to, when measuring when the sequence comparison algorithm that uses any number or by artificial comparison and range estimation, when on comparison window or designated area with regard to maximum correspondence relatively and during comparison, two or more sequences or the subsequence of same amino acid residue identical or that have prescribed percentage or Nucleotide.

For sequence relatively, usually a sequence is served as the canonical sequence that compares with cycle tests.When using sequence comparison algorithm, will test with canonical sequence and import in the computer, specify subsequence coordinate and specified sequence algorithm routine parameter when needing.The default procedures parameter can be used, maybe alternative parameter can be specified.Sequence comparison algorithm is subsequently based on the sequence identity per-cent of program parameter calculating with respect to the cycle tests of canonical sequence.

The sequence alignment method that is used for comparison is well-known in the art.The optimal sequence comparison that is used for comparison can be undertaken by following: people's (1988) such as people's (1970) such as local homology's algorithm of people such as Smith (1981), Needleman homology alignment algorithm, Person similarity searching method, the computerize of these algorithms realize (Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, the GAP among the WI, BESTFIT, FASTA and TFASTA) or manually compare and estimate.

The computer realization of these mathematical algorithms can be used for comparative sequences to measure sequence identity.This kind realization includes but not limited to: the CLUSTAL in the PC/Gene program (can be from Intelligenetics, Mountain View, California acquisition); ALIGN program (version 2 .0) and Wisconsin genetics software package, version 8 (can be from Genetics ComputerGroup (GCG), 575 Science Drive, Madison, Wisconsin, USA obtains) in GAP, BESTFIT, BLAST, FASTA and TFASTA.Use the algorithm of these programs can use default parameter to carry out.The CLUSTAL program is by people such as Higgins (1988); People such as Higgins (1989); People such as Corpet (1988); People such as Huang (1992); Fully describe with people (1994) such as Pearson.The ALIGN program is based on the algorithm of Myers and Miller (1988).People's such as Altschul (1990) blast program is based on the algorithm of Karlin and Altschul (1990).

Being used to carry out the software that BLAST analyzes can be by the open acquisition of state-run biotechnology information center (http://www.ncbi.nlm.nih.gov/).This algorithm relates to the short word that at first is tested and appraised length W in the search sequence and identifies the higher assessment sub-sequence to (HSPs), when with database sequence in word when comparison of equal length, its coupling or satisfy some on the occasion of threshold value score T.T is called neighborhood word score threshold value (people such as Altschul, 1990).These initial neighborhood word are hit and are served as seed and be used for initial search to find the longer HSPs that comprises them.Word hits subsequently and extends along each sequence on both direction, as long as accumulation comparison score can increase.For nucleotide sequence, accumulation score operation parameter M (the award score of a pair of coupling residue; Always〉0) and N (the punishment score of mispairing residue; Always＜0) calculate.For aminoacid sequence, rating matrix is used for calculating the accumulation score.Word on each direction hits stops when extending in following situation: accumulation comparison score is compared reduction X with the maximum value that it reaches, because one or more negative scoring residues comparison, the accumulation score becomes below zero or zero, or reaches the end of arbitrary sequence.

Except sequence of calculation identity per-cent, the BLAST algorithm also carry out two similaritys between the sequence statistical study (referring to for example, Karlin ﹠amp; Altschul (1993).A kind of similarity measurement value that is provided by the BLAST algorithm is minimum and probability (P (N)), and it provides coupling between two Nucleotide or the aminoacid sequence with the indication of occurrent probability.For example, if the test nucleotide sequence with reference to the minimum in the comparison of nucleotide sequence and probability less than about 0.1, more preferably less than about 0.01, most preferably less than about 0.001, then test nucleotide sequence and be regarded as similar to canonical sequence.

In order to obtain to be used for the jagged comparison of comparison purpose, can described in people such as Altschul (1997), use Gapped BLAST (in BLAST 2.0).Alternately, PSI-BLAST (in BLAST 2.0) can be used to carry out the iterative searching of the remote relation between the detection molecules.Referring to people such as Altschul, the same.When using BLAST, GappedBLAST, PSI-BLAST, can use the default parameter of program (for example BLASTN is used for nucleotide sequence, and BLASTX is used for protein) separately.BLASTN program (for nucleotide sequence) uses the comparison of word length (W) 11, expected value (E) 10, cutoff 100, M=5, N=-4 and 2 chains as default value.For aminoacid sequence, the BLASTP program use word length (W) 3, expected value (E) 10 and BLOSUM62 rating matrix as default value (referring to Henikoff ﹠amp; Henikoff, 1989).Referring to Http:// www.ncbi.nlm.nih.gov

Especially, except conservative variations, polypeptide can be relevant basically.Conservative variations refers to that amino-acid residue is by the similar residue replacement of another biology.The example of conservative variations comprises that for example Isoleucine, Xie Ansuan, leucine or methionine(Met) replace another to a hydrophobic residue, or a polar residues replaces another, and for example arginine replaces Methionin, L-glutamic acid replaces aspartic acid or glutamine replaces l-asparagine etc.Conservative other illustrative examples that replace comprise following variation: L-Ala is changed into Serine; Arginine is changed into Methionin; L-asparagine is changed into glutamine or Histidine; Aspartic acid is changed into L-glutamic acid; Halfcystine is changed into Serine; Glutamine is changed into l-asparagine; L-glutamic acid is changed into aspartic acid; Glycine is changed into proline(Pro); Histidine is changed into l-asparagine or glutamine; Isoleucine is changed into leucine or Xie Ansuan; Leucine is changed into Xie Ansuan or Isoleucine; Methionin is changed into arginine, glutamine or L-glutamic acid; Methionine(Met) is changed into leucine or Isoleucine; Phenylalanine is changed into tyrosine, leucine or methionine(Met); Serine is changed into Threonine; Threonine is changed into Serine; Tryptophane is changed into tyrosine; Tyrosine is changed into tryptophane or phenylalanine; Xie Ansuan is changed into Isoleucine and is changed into leucine.

In one embodiment, polynucleotide of the present invention are optimized for expressing in specific host.As used herein, optimize and to comprise codon optimized and in eukaryotic cell, the introducing of Kozak sequence and/or one or more introns.Therefore, nucleic acid molecule can have and surpassing 30%, 35%, 40% or surpass 45%, for example 50%, 55%, 60% or more codon on be different from the codon composition of wild-type nucleic acid sequence of the luciferase of the unmodified of encode.The preferred codon that is used for using in the present invention is than those of the more frequent use of at least one other codon for the same amino acid of particular organisms, and access to your password son and be not to be used to clone or screen the low son that accesses to your password that nucleic acid molecule is expressed in this biology of low in more preferably, neither the sort of biology.In addition, the preferred codon of some amino acid (that is those amino acid that, have 3 or more codons) can comprise two or more codons than the more frequent use of other (not preferred) codons.In the nucleic acid molecule in a kind of biology the existence than the codon of more frequent use in the another kind of biology cause such nucleic acid molecule: promptly when in the cell of introducing the biology use those codons more continually, described nucleic acid molecule expression levels in those cells is higher than wild-type or the expression of parental nucleic acid sequence in those cells.

In one embodiment of the invention, different codons is those of more frequent use in Mammals, and in another embodiment, and different codons is those of more frequent use in plant.The Mammals of particular type, for example the people can have and the Mammals of another kind of type on the same group preferred codon not.Similarly, the plant of particular type can have and the plant of another kind of type on the same group preferred codon not.In one embodiment of the invention, the great majority in the different codon are the preferred codons in required host cell.The preferred codon that is used for Mammals (for example, the people) and plant is (for example, people such as Wada, 1990) known in the art.For example, preferred people's codon includes but not limited to, CGC (Arg), CTG (Leu), TCT (Ser), AGC (Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCC (Ala), GGC (Gly), GTG (Val), ATC (Ile), ATT (Ile), AAG (Lys), AAC (Asn), CAG (Gln), CAC (His), GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys) and TTC (Phe) (people such as Wada, 1990).Therefore, preferred " humanization " synthetic nucleic acid molecule of the present invention has the codon composition that is different from the wild-type nucleic acid sequence by having the preferred people's codon that increases number, and described preferred people's codon is CGC, CTG, TCT, AGC, ACC, CCA, CCT, GCC, GGC, GTG, ATC, ATT, AAG, AAC, CAG, CAC, GAG, GAC, TAC, TGC, TTC or its any combination for example.For example, with respect to the wild-type nucleic acid sequence, nucleic acid molecule of the present invention can have GCC or GCT codon or its any combination of the ACC of the AGC of the CCA of the ATC of the GGC of the GTG of the leucic CTG of coding that increases number or TTG codon, coding Xie Ansuan or GTC codon, coding glycine or GGT codon, coding Isoleucine or ATT codon, coding proline(Pro) or CCT codon, the arginic CGC of coding or CGT codon, encoding serine or TCT codon, coding Threonine or ACT codon, coding L-Ala.Similarly, have the nucleic acid molecule of the codon of more frequent use in plant that increases number and have the codon composition that is different from the wild-type nucleic acid sequence by having the vegetable codon that increases number, described vegetable codon includes but not limited to CGC (Arg), CTT (Leu), TCT (Ser), TCC (Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCT (Ser), GGA (Gly), GTG (Val), ATC (Ile), ATT (Ile), AAG (Lys), AAC (Asn), CAA (Gln), CAC (His), GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys), TTC (Phe), or its any combination (people such as Murray, 1989).Preferred codon can be different people such as (, 1990) Wada for dissimilar plants.

Luciferase protein matter that the present invention is modified or fusion rotein can be prepared by recombination method or by solid state chemistry peptide synthetic method.This kind method has been (Merrifield known in the art from 20 generation level sixties in early days, 1963) (also referring to people such as Stewart, Solid PhasePeptide Synthesis, the 2nd edition, Pierce Chemical Co., Rockford, Ill., the 11-12 page or leaf)), and in the recent period be used for the laboratory peptide design and the synthetic agent box (Cambridge Research Biochemicals) that are obtained commercially.The laboratory reagent box that this kind is obtained commercially generally uses people's (1984) such as Geysen instruction, and is provided for synthetic peptide on the tip of many " bar (rods) " or " pin (pins) ", and all these bars or pin are connected with single plate.When using this kind system, the plate of bar or pin is squeezed and inserts in second block of plate of respective aperture or holder, and described hole or holder comprise that the tip that is used to make suitable amino acid and pin or bar is adhered to or the solution of grappling.By repeating this kind procedure of processing, for example to be inverted and the tip of bar and pin is inserted in the suitable solution, amino acid is built into required peptide.In addition, many available FMOC peptide synthesis systems are available.For example, polypeptide or segmental assembling can be used Applied Biosystems, and Inc.Model 431 A automated peptide synthesizers carry out on solid support.This kind equipment is by directly synthesizing or can using a series of fragments of other known technology link coupled to prepare peptide of the present invention easily by synthetic.

III. To the useful fusion partner of the modified luciferase of the present invention

The polynucleotide of the present invention of modified luciferase of encoding can use with other nucleotide sequences, described other nucleotide sequences are native sequences cDNA or carried out the sort of of manipulation in vitro for example for example, described operational example is as being used to prepare N-terminal, C-terminal or N and C-terminal fusion rotein, for example with by different reporter genes, comprise the fusions of selective marker encoded protein matter.Many examples of suitable fusion partner are known in the art and can use in practice of the present invention.

Fusion partner includes but not limited to affine structural domain or other functional protein sequences, for example has those of enzymatic activity.For example, the functional protein sequence kinase catalytic structural domain (Hanks and Hunter, 1995) of can encoding, generation add the fusion rotein of phosphoric acid part can for the specific amino acids enzymatic, the Src homology 2 of maybe can encoding (SH2) structural domain (people such as Sadowski, 1986; Mayer and Baltimore, 1993), produce and phosphorylated tyrosine specificity bonded fusion rotein.

Affine structural domain generally be can with binding partners, for example be fixed on the interactional peptide sequence of binding partners on the solid support.The for example dna sequence dna of Histidine of a plurality of continuous monamino acids of encoding, when merging with expressed protein, can by with resin column for example the high-affinity of nickel agarose combine the single step purification that is used for recombinant protein.Encoded peptide, the sequence of chitin binding domains (it combines with chitin), glutathione-S-transferase (it combines with gsh), vitamin H (it combines with avidin and Streptavidin) etc. for example can also be used to promote the purifying of target protein matter.Affine structural domain can separate by method well-known in the art and target protein matter, and described method comprises the intein (use of protein self-splicing element (people such as Chong, 1997).Exemplary affine structural domain comprises HisV5 (HHHHH) (SEQ ID NO:4), HisX6 (HHHHHH) (SEQ ID NO:5), C-myc (EQKLISEEDL) (SEQ ID NO:6), Flag (DYKDDDDK) (SEQ ID NO:7), SteptTag (WSHPQFEK) (SEQ ID NO:8), hemagglutinin, HA mark (YPYDVPDYA) (SEQ ID NO:9) for example, GST, Trx, cellulose binding domain, RYIRS (SEQ ID NO:10), Phe-His-His-Thr (SEQ ID NO:11), the chitin binding domains, the S-peptide, the T7 peptide, the SH2 structural domain, the terminal RNA mark of C-, WEAAAREACCRECCARA (SEQ ID NO:12), the melts combine structural domain, for example zinc binding domains or calcium binding domains, for example from calcium binding protein those, described calcium binding protein is calmodulin for example, TnC, calcinerin B, myosin light chain, recoverin, S-regulates albumen (modulin), visinin, VILIP, neurocalcin, the hippocampus calsequestrin, common albumen (frequenin), caltractin, the big subunit of calpain, S100 protein, parvalbumin, calcium binding protein D _9K, calcium binding protein D _28K, and calretinin, intein, vitamin H, Streptavidin, MyoD, Id, leucine zipper sequence, and maltose binding protein.In one embodiment, fusion partner is the sequence that is used for purified fusion protein, for example His or GST mark, and in one embodiment, the N of purifying mark and circular permutation luciferase or C-terminal merge.

Another kind of fusion partner comprises by reporter gene encoded protein matter, described reporter gene includes but not limited to the neo gene, β-gal gene, the gus gene, the cat gene, the gpt gene, the hyg gene, the hisD gene, the ble gene, the mprt gene, the bar gene, nitrilase gene, the galactopyranoside gene, the xylosidase gene, thymidine kinase gene, the arabinofuranosidase/xylosidase gene, mutant acetolactate synthase gene (ALS) or etheric acid synthase gene (AAS), methotrexate resistance dhfr gene, dalapon dehalogenase gene, give sudden change anthranilic acid synthase gene (WO 97/26366) to the resistance of 5-methyl tryptophan, R-locus gene, the β-Nei Xiananmei gene, the xylE gene, alpha-amylase gene, tyrosinase cdna, coral polyp luciferase (luc) gene (for example, sea pansy luciferase genes), aequorin gene, the red fluorescent protein gene, or green fluorescence protein gene.The gene of the coding " selectable marker " that also comprises in the optional or screenable marker gene at term, its secretion can be used as to be identified or selects the method for transformant to detect.But example comprises the mark of the secretion antigen that coding can interact be identified by antibody, but or even the Secretases that can detect by its catalytic activity.But secretory protein is included in many classifications, for example comprise can by ELISA detect little, can spread protein and insert or be captured in protein in the cytolemma.

IV. The encode modified luciferase or the carrier and the host cell of its fusions

In case behind the luciferase that preparation coding is modified or the required nucleic acid molecule of its fusions, the modified luciferase of preparation coding or comprise the Expression of Fusion Protein box of modified luciferase just.For example, comprise that the nucleic acid molecule of nucleotide sequence of the modified luciferase of coding is optional to be operably connected with transcriptional regulatory sequences, to form expression cassette, described transcriptional regulatory sequences is one or more enhansers, promotor, transcription termination sequence or its combination for example.Nucleic acid molecule or expression cassette can be introduced carrier for example in plasmid or the virus vector; the optional selectable marker gene that comprises of described carrier; and carrier is introduced in the purpose cell; described purpose cell is prokaryotic cell prokaryocyte intestinal bacteria for example for example; streptomyces species (Streptomyces spp.); genus bacillus species (Bacillus spp.); staphylococcus species (Staphylococcus spp.) etc.; and eukaryotic cell comprises plant (dicotyledons or monocotyledons); fungi; yeast is Pichia (Pichia) for example; saccharomyces (Saccharomyces) or Schizosaccharomyces (Schizosaccharomyces), or mammalian cell.Preferred mammalian cell comprises ox, goat, sheep, dog, cat, non-human primate for example ape and monkey and people's cell.Preferred mammal cell line includes but not limited to CHO, COS, 293, Hela, CV-1, SH-SY5Y, HEK293 and NIH3T3 cell.

The expression of the modified luciferase of coding can be by any promotor control that can express in prokaryotic cell prokaryocyte or eukaryotic cell.Preferred promoter in prokaryote includes but not limited to SP6, T7, T5, tac, bla, trp, gal, lac or maltose promoter.Preferred promoter in eukaryote includes but not limited to constitutive promoter, viral promotors for example, for example CMV, SV40 and RSV promotor, and adjustment type promotor, for example for example tet promotor, hsp70 promotor and the synthetic promoter regulated by CRE of the induction type or the type promotor of checking.Nucleic acid molecule of the present invention, expression cassette and/or carrier can be introduced in the cell by any method, and described method includes but not limited to the conversion, electroporation, microinjection, fat transfection of calcium mediation etc.

V. Exemplary purposes

Modified luciferase or its fusions are useful for following any purpose, include but not limited to detect the amount of specific molecular or have (biosensor), separate specific molecular, detect in the specific molecular for example because the conformational change that combination, phosphorylation or ionization cause, promote high or low flux screening, detect protein-protein, protein-DNA or other based on protein interactions, or selection or evolution biosensor.For example, modified luciferase or its fusions for example are used for external or (for example detect specific kinases based on the mensuration of cell, by the kinases site is inserted in the report protein), RNAi (for example, insert in the proteinic encoding sequence of report by suspecting, monitor the reporter activity after RNAi adds subsequently by the sequence of RNAi identification), or the amount of proteolytic enzyme, existence or activity, for example detect the existence of specific virus proteolytic enzyme, it is the indicator of the existence of virus or antibody successively; Screening inhibitor, for example proteinase inhibitor; Identify recognition site or detect substrate specificity, for example use to have the modified luciferase of selected recognition sequence or have a plurality of not homotactic modified luciferase libraries and single molecules of interest or a plurality of molecule (for example, library); Select or evolution biosensor or molecules of interest, for example proteolytic enzyme; External or based on the method for cell in via complementary or in conjunction with detecting protein-protein interaction.In one embodiment, the modified luciferase that comprises insertion is contacted with the random library or the sudden change library of molecule, and identify and insert interactional molecule.In another embodiment, the modified luciferase library with a plurality of insertions is contacted with molecule, and the modified luciferase of evaluation and interaction of molecules.In one embodiment, modified luciferase or its fusions for example are used for external or detect the amount of cAMP or cGMP or (for example exist based on the mensuration of cell, by cAMP or cGMP binding site are inserted in the circular permutation luciferase), screening inhibitor or activator, for example cAMP or cGMP inhibitor or activator, inhibitor or activator with cAMP binding site bonded cAMP, or the inhibitor or the activator of g protein coupled receptor (GPCR), identify recognition site or detect substrate specificity, for example use and have the modified luciferase of selected recognition sequence or (for example have a plurality of not homotactic modified luciferase libraries and single molecules of interest or a plurality of molecule, the library), with selection or evolution cAMP or cGMP binding site, or be used for full animal imaging.

The present invention also provides and has used modified luciferase or its fusion rotein, expression, location and/or the transportation of monitoring molecule in cell, and the method for monitoring the variation of intracellular microenvironment.In one embodiment, modified luciferase comprises the recognition site of molecule, and when this molecule and recognition site interaction, causes active increase, and therefore can be used to detect or measure the existence or the amount of molecule.For example, in one embodiment, modified luciferase comprises the inside of containing two structural domains and inserts, and described two structural domains are interact with each other under certain conditions.In one embodiment, a structural domain in the insertion comprise can phosphorylation amino acid, and another structural domain is the phosphoamino acid binding domains.In the presence of suitable kinases or Phosphoric acid esterase, two domain interactions in the insertion and change the conformation of modified luciferase, but cause the change of the detection of active of modified luciferase.In another embodiment, modified luciferase comprises the recognition site of molecule, and when this molecule and recognition site interaction, causes active increase, and therefore can be used to detect or measure the existence or the amount of another kind of molecule.

Two-hybrid system is to be used for detecting in vivo protein: protein interaction and evaluation relate to protein: the strong especially method of the residue/structural domain of protein interaction.The basis of two-hybrid system is the adjustment structure territory of finding in some transcription factor: with specific dna sequence bonded DNA binding domains and with the interactional transcriptional activation domains of basic transcription mechanism (Sadowski, 1988).Can promote assembling and the increase of rna plymerase ii mixture on the TATA box to transcribe with the associating transcriptional activation domains of DNA binding domains.At CheckMate ^TMMammals two-hybrid system (Promega Corp., Madison, WI) in, when a kind of protein (" X ") with the fusion of DNA binding domains interacts with second kind of protein (" Y ") (described second kind of protein and transcriptional activation domains merge), combine closely by DNA binding domains and transcriptional activation domains that the plasmid that separates produces.In this system, the interaction between protein X and the Y causes reporter gene or selectable marker gene transcription.Especially, the pBIND carrier comprises the yeast GAL4DNA binding domains of upstream, polyclone zone, and the pACT carrier comprises the hsv VP16 activation domain of upstream, polyclone zone.In addition, pBIND vector expression sea pansy luciferase.With 2 kinds of gene clones of 2 kinds of potential interactional target protein matter of coding in pBIND and pACT carrier, with the fusion rotein of the activation domain that produces the DNA binding domains that has GAL4 respectively and VP16.The pG5luc carrier comprises 5 GAL4 binding sites of minimum TATA box upstream, and it is the upstream of Lampyridea luciferase genes (luc+) successively.With pGAL4 and pVP16 fusion constructs together with the transfection of pG5luc carrier in mammalian cell.After the transfection 2-3 days, cell was cleaved, and the amount of sea pansy luciferase and Lampyridea luciferase can be used Dual-Luciferase

Reporter is measured system (Promega catalogue #E1910) and is carried out quantitatively.Interaction between 2 kinds of test proteins such as GAL4 and the VP16 fusion constructs causes the Lampyridea luciferase expression to surpass the increase of negative control.The luciferase that the present invention is modified, for example the sort of (the N-terminal fragment) that lacks on site that modification is tolerated or zone merges with the DNA binding domains, and rest part of luciferase (C-terminal fragment) and transcriptional activation domains fusion.

The present invention also provides screening can regulate for example method of the reagent of the amount of ring nucleus thuja acid (" test " reagent) of molecules of interest." adjusting " refers to the change of character; Active this kind of biological or chemical strengthens or suppresses, and wherein changing can be accidental in the generation of particular event, for example activation of signal transduction pathway, and/or only in particular cell types, show." conditioning agent " refers to reagent (naturally occurring or the non-natural existence), the biological example macromole (for example, nucleic acid, protein, non-peptide or organic molecule), small molecules, by the biomaterial extract of bacterium, plant, fungi or animal (particularly Mammals) cell or tissue preparation for example, or any other reagent.Conditioning agent is by being included in the lateral reactivity of assessing in the screening assay described herein as the inhibitor or the activator (directly or indirectly) (for example, agonist, partial antagonist, partial agonist or antagonist) of bioprocess.The activity of conditioning agent can be known, unknown or part known.This kind conditioning agent can use the inventive method to screen.Term " test agent " refers to by the reagent of one or more screening method tests of the present invention for the supposition conditioning agent.Usually, various predetermined concentrations are used for screening, for example 0.01 μ M, 0.1 μ M, 1.0 μ M and 10.0 μ M.Contrast can be included in the signal measurement under the situation that does not have test agent, with the reagent of known adjusting target relatively, or with before test agent contact, in and/or afterwards with sample (for example cell, tissue or biology) comparison.

In one embodiment, this method comprises the reagent of screening adjusting protease activity.For example, in one embodiment, provide and identified the compositions and methods that to regulate apoptosis.Caspase family protein enzyme is relevant with apoptosis.Therefore, this method comprises making and suspects the sample comprise Caspase family protein enzyme and suspect that regulating the active reagent of Caspase contacts with the modified luciferase with the cleavage site that can be cut by Caspase.The activity of modified luciferase is detecting in sample with before test agent contacts and back.Contact the reagent that the active increase indication in back suppresses apoptosis with reagent, and active minimizing indication activates the reagent of apoptosis.

Therefore, the invention provides and be used for indentifying substance and detect its active screening system, described reagent is regulated the cutting of the recognition sequence that exists in the modified luciferase protein matter of the present invention.This allows rapid screening protein active conditioning agent.The use of screening system described herein provides identifies adjusting (for example, inhibition or activation) proteolytic enzyme for example sensitivity and the fast method of the reagent of Caspase family protein.

Therefore the modified luciferase protein matter of the present invention be used as substrate, with insertion in the modified luciferase protein matter of research adjusting and interactional reagent or the condition between the molecules of interest.Especially, the present invention has considered wherein to insert the modified luciferase protein matter that comprises a kind of aminoacid sequence, and described aminoacid sequence is the cleavage site of purpose enzyme.Therefore, when molecules of interest is proteolytic enzyme, insert the peptide that comprises the cutting recognition sequence that comprises proteolytic enzyme.The cutting recognition sequence of proteolytic enzyme is the specific amino acids sequence of being discerned by proteolytic enzyme during the proteolysis cutting.Therefore, the invention provides the method for the amount of the proteolytic enzyme in the working sample, of the change of this method by making the modified luciferase polypeptide of sample and the present invention contact and measure luciferase activity.The modified luciferase protein matter of the present invention can be used to monitor the intracellular protease activity of expressing modified luciferase etc.

In one embodiment, therefore the modified luciferase of the present invention is used as substrate, regulate ring-type nucleotide binding site in the modified luciferase and the molecules of interest for example reagent that exists or measure or the condition of the interactional reagent between the ring nucleus thuja acid or condition, adjusting ring-type Nucleotide with research, perhaps regulate and the concentration dependent molecule of cell the cyclic nucleotides for example reagent or the condition of acceptor.Especially, the present invention has considered wherein to insert the modified luciferase protein matter that comprises cAMP or cGMP binding site.Therefore, when molecules of interest is cAMP or cGMP, the invention provides cAMP or the existence of cGMP or the method for amount in the working sample, this method is the change by making the modified luciferase polypeptide of sample and the present invention contact and measure luciferase activity.The modified luciferase protein matter of the present invention can be used to monitor amount or the existence of cAMP or cGMP, perhaps changes the amount of molecule of the amount of intracellular cAMP with modified luciferase or cGMP or existence or existence or the like.

Mensuration of the present invention can be used for screening of medicaments, with the amount of identifying Change Example such as ring nucleus thuja acid or the bonded compound that changes ring nucleus thuja acid and ring-type nucleotide binding site.In one embodiment, be determined at and external the sample that comprises cAMP carried out.Modified luciferase and the test agent of the sample that makes the cAMP that comprises known quantity and the present invention mixed.The amount of the luciferase activity in the working sample subsequently.The active amount of every mole of cAMP in the presence of test agent can compare with the active amount of every mole of cAMP under the situation that does not have test agent subsequently.The amount of difference indication test agent change cAMP or cAMP combine with the cAMP binding site.

In one embodiment, cell is regulated with reagent or is contacted with reagent, and described reagent suspection is directly or indirectly regulated for example cAMP amount or combination.Cell in the cultivation carries out cracking and measures the cAMP amount.For example, the modified luciferase of the lysing cell sample that makes the cAMP that comprises known or unknown quantity and the present invention mixes.The amount of cAMP in the sample is measured subsequently as mentioned above, and this mensuration is the degree by luciferase activity modified in the lysing cell sample of measuring contrast or untreated samples and processing.Active or inhibition can be calculated based on every microgram in the sample or milligram protein.Usually, difference is proofreaied and correct to produce the absolute magnitude of cAMP at canonical measure.

Being used for measuring the material and the composition that use in the present invention is ideally suited in the preparation test kit.This kind test kit can comprise the carrier instrument, and it contains one or more container instruments, and routine phial, pipe etc., each container tool kit contain one of separate elements of using in the method.One of container comprises the modified luciferase of the present invention or the polynucleotide form of carrier (for example with).Second container can comprise the substrate of modified luciferase.

The present invention will be described further by following non-limiting example.

Example I

The site that in Pleonomus and Lampyridea luciferase, modification is tolerated

In Pleonomus and the Lampyridea luciferase to the position of modifying tolerance and some modified luciferase be disclosed in the U.S. openly apply for 20050153310 and PCT/US2004/032705 in, the disclosure of described patent is integrated with this paper (also referring to Fig. 1 and table 1) as a reference.

Table 1

The active % of insertion in the Lampyridea luciferase behind the amino acid

7 10

121 5-10

233 50-75

267 2

294 3

303 5-10

361 3-5

540 15

541 75

Example II

Circular permutation Lampyridea luciferase fusions with the cAMP binding site

CAMP is one of most important second messenger who is used for cell signalling.CAMP measures acceptor (GPCR) the drug development particularly important for the G albumen coupling.In order to identify the biosensor of cAMP, make cAMP binding site and circular permutation Lampyridea luciferase (CPM-FFLuc) merge (Fig. 5 A-B) (pBFB8, pBFB9, pBFB10, pBFB11, pBFB22, pBFB40, pBFB41, pBFB42).A kind of CPM-FF Luc cAMP binding site fusions uses the cAMP binding site (by the direct activated exchanger of cAMP matter) (Bos, 2003) from people Epac1.Before studies show that from people Epac1 single-chain fragment (residue 157-316) in conjunction with cAMP (Nikolaev, J.Biol.Chem., 279, 37215 (2004)).Second kind of CPM-FF Luc/cAMP binding site fusions uses the B structural domain (CPM-FF Luc/RII β B) from people PKA modulability IIB type subunit.

Materials and methods

The dna fragmentation of the residue 157-316 of composite coding people Epac1, it comprises that some reticent Nucleotide changes with the potential expression (Fig. 5 C) that is increased in the intestinal bacteria.Two primers are used to be created in 5 ' and 3 ' end on have the PCR fragment of the EPAC1 in XhoI and NcoI site: 5 ' primer: atgcctcgagGAAGAAGAACTTGCTGAAGCTG (SEQ ID NO:22), 3 ' primer: atgccatggAACTCCATGTTCTTCTAAACGC (SEQ ID NO:23) respectively

Resulting PCR fragment digests and is cloned in the XhoI and NcoI site of circular permutation beetle luciferase construct.Resulting plasmid expression has modified Lampyridea (pSPLuc+, the Promega Corporation) luciferase that inserts the EPAC1 between original N and the C-terminal.The correct size of fusion rotein is verified (Fig. 6) by acellular expression of TnT and SDS-PAGE.This construct is accredited as FF105.

The DNA of coding RII β B is inserted in the new expression vector of coding CPM-FF Luc/RII β B fusions [Luc 2.0 (234-544)-joint X-people RII β (residue 266-414)-joint Y-Luc (4-233)].By using the unique combination of restriction enzyme, produce various constructs with RII β B, described RII β B and the CPM-FF Luc with various X/Y peptide linker length merge.

CPM-FF Luc expression plasmid synthetic that is used for follow-up insertion RII β B

The synthetic 1816bp fragment (DNA2.0 of coding CPM-FF Luc; SEQ ID NO:16 is referring to Figure 20) digest with HindIII/XbaI, and be connected with the 3265bp HindIII/XbaI fragment of pGL4.74 (PromegaCorp.).Synthetic luciferase (the Luc2.0 that resulting plasmid-encoded synthetic peptide by 42 amino acid whose Gly/Ser of being rich in and the amino acid 544 of Lampyridea luciferase are connected with 4; Promega Corp.) circular permutation mutant [Luc2.0 (234-544)-42aa is rich in peptide-Luc2.0 (4-233) of Gly/Ser] (pBFB8).Fig. 5 A has described this parent CPM-FF Luc expression plasmid (pBFB8) and has been used to prepare various terminal length and unique restriction site of insertion cAMP structural domain.This fusion rotein can use T7 or HSV-TK promotor to express in external or body respectively.In addition, SgfI and PmeI restriction enzyme sites be included in 5 ' and 3 ' end on, to promote this open reading-frame (ORF) subsequent transfer to other plasmid (Flexi carrier system; Promega Corp.).

Coding has (X=4, Y=4; PBFB9), (X=10, Y=10; PBFB10) and (X=20, Y=20; PBFB11) the CPM-FF Luc/RII β B of the X/Y joint length of individual amino-acid residue merges egg White plasmid is synthetic

The middle unique restriction enzyme that exists of multiple clone site (MCS) that above-mentioned plasmid DNA construction body and function connects the dna fragmentation of coding Luc2.0 (233-544) and Luc2.0 (4-233) digests, to allow the synthetic (X=4 that has, Y=4), (X=10, Y=10) and (X=20, Y=20) the CPM-FF Luc/RII β B expression construct of the X/Y joint length of individual amino-acid residue.Fig. 5 B described side joint RII β B structural domain with the joint length of preparation pBFB9 (X=4, Y=4), pBFB10 (X=10, Y=10) and pBFB11 (X=20, Y=20).

Have in order to synthesize (X=4, the Y=4) construct of joint length, primer 5 '-AAAAAAGTC GAC CGG AAT GTA TGA AAG CTT TAT TGA GTC ACT GCC-3 ' (SEQ ID NO:25; BFB51) and 5 '-AAA AAA GAG CTC CCA ACA ATATCC ATG TTC GTT CCA AAC-3 ' (SEQ ID NO:26; BFB20) be used for the RII β B DNA of amplification from ATCC 10625233 (Genbank ID BC075800).Resulting product digests with the SalI/SacI restriction enzyme and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/SacI.This novel constructs is accredited as pBFB9.

Have in order to synthesize (X=10, the Y=10) construct of joint length, primer 5 '-AAA AAATCC GGA ATG TAT GAA AGC TTT ATT GAG TCA CTG CC-3 ' (SEQ IDNO:211; BFB21) and 5 '-AAA AAA AGG CCT ACA ATA TCC ATG TTCGTT CCA AAC-3 ' (SEQ ID NO:27; BFB22) be used for the RII β B DNA of amplification from ATCC10625233 (Genbank ID BC075800).Resulting product digests with the BspEI/StuI restriction enzyme and is connected in the parent CPM-FFLuc expression plasmid (pBFB8) that digests with BspEI/ZraI.This novel constructs is accredited as pBFB10.

Have in order to synthesize (X=20, the Y=20) construct of joint length, primer 5 '-AAA AAACCC GGG ATG TAT GAA AGC TTT ATT GAG TCA CTG CC-3 ' (SEQ IDNO:28; BFB23) and 5 '-AAA AAA TCC GGA CCC AAC AAT ATC CATGTT CGT TCC AAA C-3 ' (SEQ ID NO:29; BFB24) be used for the RII β B DNA of amplification from ATCC10625233 (Genbank ID BC075800).Resulting product digests with the BspEI/SmaI restriction enzyme and is connected in the parent CPM-FFLuc expression plasmid (pBFB8) that digests with AgeI/NruI.This novel constructs is accredited as pBFB11.

Have (X=4, Y=4), (X=10, Y=10) and (X=20, Y=20) X/Y of individual amino-acid residue The CPM-FF Luc/RII β B Expression of Fusion Protein of joint length

The synthetic use of the fusion rotein of expection size

T7 link coupled malt extract system (Promega Corp.) confirms to have (X=4, Y=4 together with the external translation Mk system of FluoroTect GreenLys (Promega Corp.); PBFB9), (X=10, Y=10; PBFB10) and (X=20, Y=20; PBFB11) the CPM-FF Luc/RII β B fusion rotein of the X/Y joint length of individual amino-acid residue.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 400ng plasmid DNA

10 μ LTnT malt extracts

0.8 μ L TNT reaction buffer

0.4 μ L T7 polysaccharase

0.4 μ L aminoacid mixture

0.4μL rRNasin

0.4 μ L Fluoro Tect GreenLys mark

DH ₂O to 20 μ L cumulative volume

After 1.5 hours, 5 μ L TNT reaction is differentiated via SDS-PAGE according to the scheme (NuPAGENovex 4-12% bis-tris gel, Invitrogen Corp.) of manufacturers in 30 ℃ of incubations.The protein of translation manifests via fluorescence imaging instrument (fluorimager) (Typhoon VariableMode Imager, Amersham Biosciences) subsequently.Spectrodensitometry is analyzed CPM-FFLuc/RII β B fusion rotein that (ImageQuant, GE Healthcare) point out to have variable X/Y joint length and is similar to peptide (pBFB8) with 42 amino acid whose Gly/Ser of being rich in and the CPM-FF Luc expressing fusion protein of Epac1 (FF105).

Have (X=4, Y=4; PBFB9), (X=10, Y=10; PBFB10) and (X=20, Y=20; PBFB11) the CPM-FF Luc/RII β B of the X/Y joint length of individual amino-acid residue merges egg White function characterizes

For having (X=4, Y=4; PBFB9), (X=10, Y=10; PBFB10) and (X=20, Y=20; PBFB11) individualThe CPM-FFLuc/RII β B fusion rotein of the X/Y joint length of amino-acid residue uses T7 link coupled reticulocyte lysate system (Promega Corp.) measures in the existence of 100 μ M cAMP and the luciferase activity not.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 400ng plasmid DNA

10 μ L rabbit reticulocyte extracts

0.8 μ LTNT reaction buffer

0.4 μ L T7 polysaccharase

0.4 μ L aminoacid mixture

0.4μL rRNasin

DH ₂O to 20 μ L cumulative volume

After 1.5 hours, fusion rotein separately carries out incubation in the existence of 100 μ M cAMP or not in 30 ℃ of incubations, and this incubation is by making 9 μ L

Reaction and 1 μ L1mM cAMP stoste or dH ₂The O combination.At room temperature incubation added 100 μ L luciferase assay reagent (LAR with 1 μ L sample after 10 minutes; Promega Corp.) solution+/-100 μ M cAMP (90 μ L LAR+10 μ L 1mM cAMP stoste or dH ₂Among the O.Luminous use Veritas microtiter plate photometer (Turner Biosystems; Program Bright-Glo) measures.

Use has (X=4, Y=4; PBFB9), (X=10, Y=10; PBFB10) and (X=20, Y=20; PBFB11) the CPM-FF Luc/RII β B of the X/Y joint length of individual amino-acid residue merges egg White dose response experiments

Have (X=4, Y=4; PBFB9), (X=10, Y=10; PBFB10) and (X=20, Y=20; PBFB11) individualThe cAMP dose response of the CPM-FF Luc/RII β B fusion rotein of the X/Y joint length of amino-acid residue uses

T7 link coupled reticulocyte lysate system (Promega Corp.) is measured after expression.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 2000ng plasmid DNA

50 μ L rabbit reticulocyte extracts

4 μ L TNT reaction buffers

2 μ L T7 polysaccharases

2 μ L aminoacid mixtures

2μL rRNasin

DH ₂O to 100 μ L cumulative volume

After 2 hours, fusion rotein separately is with the cAMP incubation of various concentration in 30 ℃ of incubations, and this incubation is by making 9 μ L Reaction and the combination of 1 μ L cAMP stoste (final concentrations of 0,0.01,0.025,0.1,0.25,1,2.5,10,25 or 100 μ M cAMP).At room temperature behind incubation 〉=25 minute, 1 μ L sample is added 100 μ L luciferase assay reagent (LAR with cAMP concentration (90 μ LLAR+10 μ L cAMP stoste) separately; PromegaCorp.) in the solution.Luminous use Veritas microtiter plate photometer (Turner Biosystems; Program Bright-Glo) measures.

Have (X=10, Y=10; PBFB10) CPM-FF of the X/Y joint length of individual amino-acid residue The selectivity of Luc/RII β B fusion rotein

With respect to other ring nucleus thuja acids, have (X=10, Y=10; PBFB10) individualThe CPM-FF Luc/RII β B fusion rotein of the X/Y joint length of amino-acid residue uses for cAMP activatory selectivity

T7 link coupled reticulocyte lysate system (Promega Corp.) is measured after expression.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 6000ng plasmid DNA

150 μ L rabbit reticulocyte extracts

12 μ L TNT reaction buffers

6 μ L T7 polysaccharases

6 μ L aminoacid mixtures

6μL rRNasin

DH ₂O to 300 μ L cumulative volume

After 2.3 hours, fusion rotein is with cAMP, cGMP or the N6-benzoyl cAMP incubation of various concentration in 30 ℃ of incubations, and this incubation is by making 9 μ L Reaction and the combination of 1 μ L ring nucleus thuja acid stoste (final concentrations of 0,0.01,0.025,0.1,0.25,1,2.5,10,25 or 100 μ M cAMP).At room temperature behind incubation 〉=29 minute, 1 μ L sample is added 100 μ L luciferase assay reagent (LAR with ring-type nucleotide concentration (90 μ L LAR+10 μ L ribonucleotide stoste) separately; Promega Corp.) in the solution.Luminous use Veritas microtiter plate photometer (Turner Biosystems; Program Bright-Glo) measures.

The result

Protein kinase A modulability II β type subunit (PRKAR2B) has two cAMP binding sites, i.e. A and B.CAMP binding site from B structural domain (RII β B) is used to prepare the circular permutation luciferase (CPM-FF Luc) (CPM-FFLuc/RII β B) with RII β B.Have (X=4, Y=4; PBFB9), (X=10, Y=10; PBFB10)With (X=20, Y=20; PBFB11)The CPM-FFLuc/RII β B fusion rotein of the X/Y joint length of individual amino-acid residue is presented under the existence of 100 μ M cAMP luciferase activity 23,58 and 39 times induce respectively separately.As expection, do not observe cAMP for the CPM-FF Luc fusion rotein (pBFB8) of peptide and regulate with 42 amino acid whose Gly/Ser of being rich in.Except RII β B, be used to produce cAMP transmitter (FF105) from the cAMP binding site of Epac1.Yet, luciferase activity induce multiple less than transmitter (Fig. 7) based on RII β B.

Induce the effective concentration of multiple for 50% maximum, every kind of CPM-FF Luc/RII β B fusion rotein demonstrates the unique dose response (Fig. 8 A) with variate-value.With respect to other ring nucleus thuja acids, have (X=10, Y=10; PBFB10) the CPM-FF Luc/RII β B fusion rotein of the X/Y joint length of individual amino-acid residue demonstrate for cAMP bonded enhanced selectivity (Fig. 8 B).

EXAMPLE III

Circular permutation sea pansy luciferase with cAMP binding site

Materials and methods

4 kinds of humanization sea pansy luciferase dna fragmentations are increased by pF5RK or phRL empty carrier (Promega Corp.), and are cloned in the CPM-FF Luc fusion protein construct=[peptide-Luc2.0 (4-233) of Luc2.0 (234-544)-42 an amino acid whose Gly/Ser of being rich in] (pBFB8; Fig. 5 A), to be created in splitted circular permutation sea pansy luciferase open reading-frame (ORF) (CPM-hRL) (Fig. 5 D) between position Ser91/Tyr92 or the Ile223/Pro224.The sequencing primer that is used to produce 4 kinds of humanization sea pansy luciferase dna fragmentations is: 5 '-ATGGGCGATCGCCatgtatcgcctcctggatcactacaag-3 ' (hRL92 SgfI; FF273; SEQ ID NO:110); 5 '-ATGGGCGATCGCCatgcctctcgttaagggaggcaagc-3 ' (hRL224 SgfI; FF277; SEQ ID NO:111); 5 '-gcatCTCGAGccctgctcgttcttcagcacgcgc-3 ' (hRL311/XhoI; FF294; SEQID NO:112); 5 '-atgcGAGCTCaggagcttccaaggtgtacgacccg-3 ' (hRL2SacI; FF295; SEQ ID NO:113); 5 '-TTGTGTTTAAACtgagccattcccgctcttgccg-3 ' (hRL91/PmeI; FF276; SEQ ID NO:114); With 5 '-TTGTGTTTAAACgatctcgcgaggccaggagagg-3 ' (hRL223 PmeI; FF278; SEQ ID NO:115).Primer is to FF273/FF294 and FF277/FF294 be used to the to increase C-terminal fragment (being respectively hRL 92-311 and hRL224-311) of humanization sea pansy luciferin enzyme dna.Resulting product digests with the SgfI/XhoI restriction enzyme, and be connected to parent CPM-FF Luc fusion protein construct=[peptide-Luc2.0 (4-233) of Luc2.0 (234-544)-42 an amino acid whose Gly/Ser of being rich in], in the pBFB8 with SgfI/XhoI digestion.Primer is to FF276/FF295 and FF278/FF295 be used to the to increase N-terminal fragment (being respectively hRL 2-91 and hRL 2-223) of humanization sea pansy luciferin enzyme dna.Resulting product digests with the SacI/PmeI restriction enzyme, and is connected in the intermediate product CPM-FF Luc/hRL plasmid of the coding [peptide-Luc2.0 (4-233) of-42 amino acid whose Gly/Ser of being rich in of hRL (92-311 or 224-311)] that digests with SacI/PmeI.This causes the generation of CPM-hRL expression vector, and wherein circular permutation hRL luciferase fragment is by peptide (peptide that is rich in Gly/Ser that is equal to Fig. 5 A, the 201325.15.A1 (CPM91) of 42 amino acid whose Gly/Ser of being rich in; 201325.15.B6 (CPM223)) merge.To encode the sequence clone of people RII β B amino acid 266-414 (Genbank ID BC075800) in the subclass of unique restriction enzyme sites, the amino acid (Fig. 5 D) of described site coding as existing in the peptide of preamble for the described Gly/Ser of being rich in of CPM-FF Luc/RII β B cAMP transmitter.Resulting construct coding have respectively merge with the N of RII β B and C-terminal, have an X=4, Y=20 (201325.44.H6 (CPM91); 201325.33.C9 (CPM223)), X=10, Y=4 (201325.50.D12 (CPM91); 201325.54.E2 (CPM223)) or X=10, Y=20 (201325.58.E11 (CPM91); 201325.54.E12 (CPM223)) the individual CPM-hRL/RII β B syzygy (Fig. 5 D) that is rich in the joint of Gly/Ser.In addition, total length hRL open reading-frame (ORF) is cloned in the SgfI/PmeI site of CPM-FF Luc expression plasmid (201325.50.A7, Fig. 5 A) of peptide-Luc2.0 (4-233) of coding Luc2.0 (234-544)-42 an amino acid whose Gly/Ser of being rich in.

The plasmid DNA of 1 μ g purifying/50 μ l Fructus Hordei Germinatus

(Promega catalogue #L4140) reaction is used for the marking protein product.Fructus Hordei Germinatus Be reflected at FluoroTect ^TMGreen _LysCarried out 1 hour in 30 ℃ under the existence of tRNA (Promega catalogue #L5001).The CPM-hRL construct is expressed together with following contrast: have X=10, the CPM-FF Luc/RII β B of Y=4 (pBFB41), total length sea pansy luciferase (201325.50.A7) and " no DNA " (negative control).Every kind of lysate of 15 μ l and 1.5 μ l 1mM cAMP (Promega catalogue #V642A, 100 μ M final concentrations) or water are mixed, and incubation 10 minutes at room temperature.(5X sea pansy luciferase assay lysis buffer (Promega catalogue #E291A)+water (Promega catalogue #P119C) adds in the reaction of sea pansy luciferase and " no DNA " sample with 75 μ l1X sea pansy luciferase assay lysis buffers, mix, and every kind of mixture of 20 μ l is added in the white flat underside of 96 holes in triplicate.2 μ l are had X=10, and the CPM-FF Luc/RII β B sample (pBFB41) of Y=4 joint adds in the white flat underside of 96 holes in triplicate.With 100 μ l sea pansy luciferase assay damping fluids+1X sea pansy luciferase assay substrate (Promega Corp.; Catalogue #E2820) adds in each sea pansy luciferase and " no DNA " hole.100 μ l luciferase assay damping fluids are added luciferase assay substrate (PromegaCorp.; Catalogue #E1500) adds to comprise and have X=10, in each hole of the CPM-FFLuc/RII β B (pBFB41) of Y=4 joint.Luminous use Veritas photometer is measured.Before the cAMP incubation, every kind of lysate of 10 μ l size fractionation on the SDS-PAGE gel is separated.The fluorescence protein product manifests on the Typhoon imager.

The result

Fructus Hordei Germinatus

Reaction obtains every kind of construct protein of about equivalent.There is not visible protein from " no DNA " sample.Total length sea pansy luciferase construct (201325.50.A7) causes Duoing than CPM-hRL91-42aa construct (201325.15.A1) about 100 times luminous and Duo about 100,000 times luminous than CPM-hRL223-42aa construct (201325.15.B6).RII β B construct CPM-hRL91-4aa-RII β B-20aa (201325.44.H6) and CPM-hRL91-10aa-RII β B-20aa (201325.58.E11) produce during with 100 μ M cAMP incubations than water more luminous (be respectively 115-146 doubly and 100 times).RII β B construct CPM-hRL223-4aa-RII β B-20aa (201325.33.C9), CPM-hRL223-10aa-RII β B-4aa (201325.54.E2) and CPM-hRL223-10aa-RII β B-20aa (201325.54.E12) produce during with 100 μ M cAMP incubations than water and many doubly luminous of 1.7-2.1.With water relatively, total length sea pansy luciferase (201325.50.A7), CPM-hRL91-42aa (201325.15.A1) and CPM-hRL223-42aa construct (201325.15.B6) change during with the cAMP incubation and are no more than 1.3 times.CPM-FF Luc/RII β B transmitter (pBFB41) with construct of X=10, a Y=4 joint produces doubly luminous of many 85-90 in the presence of cAMP." no DNA " reaction has low luminous (littler by 1,000 than total length sea pansy luciferase, 000 times) and does not have change (referring to Figure 10) during with the cAMP incubation.

EXAMPLE IV

With CPM-FF Luc/RII β B cAMP biosensor vitro detection cAMP

Materials and methods

For confirm cAMP measure in cell lysate and the cell lysate stain remover in the presence of effect, have (X=10, Y=10; The CPM-FFLuc/RII β B fusion rotein of X/Y joint length pBFB10) uses

T7 link coupled reticulocyte lysate system (Promega Corp.) is expressed.In brief, following component is assembled according to the scheme of manufacturer recommendation and in 30 ℃ of incubations 1.5 hours:

The 1000ng plasmid DNA

25 μ L rabbit reticulocyte extracts

2 μ L TNT reaction buffers

1 μ L T7 polysaccharase

1 μ L aminoacid mixture

1μL rRNasin

DH ₂O to 50 μ L cumulative volume

In order to simulate the experiment condition that cAMP measures after the lysis of stain remover mediation, following component is at room temperature mixed with the cAMP of final concentration 0,0.01,0.025,0.1,0.25,1,2.5,10 and 25 μ M.

0.5 μ L

The cAMP transmitter of expressing

19.5 μ L malt extract (Promega Corp.; Catalogue #L4140, part #L411A)

5 μ L cAMP stostes

25 μ L Bright-Glo measure reagent (Promega Corp., catalogue #E2610)

Assembly reaction mixes immediately and luciferase activity uses Turner 20/20 photometer to carry out continuously measured (Turner Biosystems) 1 measurement/second.

In some experiments, for enhancing signal stability and luminous, reaction mixture comprises 4mM luciferin (Promega Bioscience), 2mM coenzyme A (Sigma), 10mM ATP (Pharmacia), 10mM DTT (Promega), 16mM sal epsom, 150mM HEPES, pH8.0 (Fisher), 1% Tergitol N101 (Sigma), 1% Mazu DF101 and 1mMCDTA (Sigma).In vitro translated CPM-FF Luc/RII β B cAMP biosensor uses

Link coupled rabbit reticulocyte system (Promega) synthesizes, and wherein uses 1 μ g plasmid DNA for 50 μ l total reaction volume, and adds (per 100 μ l measure reagent and add 1 μ l translation product) in the reaction mixture before being about to measure cAMP.Subsequently 100 μ l mensuration reagent+transmitter is added the cAMP that 100 μ l cell cultures or 100 μ l dilute in perfect medium (DMEM/F12+10% FBS).

Cell cultures

For analyzed in vitro, the HEK-293 cell is bed board in 96 orifice plates, and in having the 100 μ l DMEM/F12 (Invitrogen) of 10%FBS (Hyclone) 37 ℃ with 5% CO ₂Condition under grow to 50-90% and converge.Cell stimulates with 0.02-250 μ M Fu Sikelin (Sigma), and wherein Fu Sikelin passes through 2 times of dilutions in perfect medium.

Use the typical curve of cAMP

Use the concentration range of 0.005-50 μ M cAMP that 1mM cAMP (Promega) is diluted in the complete DMEM/F12 substratum with 10% FBS, wherein cAMP carries out serial dilution by 2 times of dilutions.Make the cAMP luminescence assays reagent mix of 100 μ l cAMP and 100 μ l homogeneous.

The result

CPM-FF Luc/RII β B cAMP transmitter works in various lysis buffers and with various luciferase reagent.In addition, CPM-FF Luc/RII β B cAMP transmitter is used at vitro detection cAMP (Fig. 9,11A and 11B) with homogeneous mensuration form.For example, use malt extract, the dosage dependence value of luciferase activity took place in about 3 minutes, had the dynamicrange (Fig. 9) of the cAMP detection of 0.025-25 μ M cAMP.In use optimizing the other example of reagent formulation, for having X=10, the CPM-FFLuc/RII β B cAMP transmitter of the X/Y joint length of Y=4 (pBFB41), the vitro detection of cAMP shows that signal and background ratio are 20, EC ₅₀Be 1.28 μ M (Figure 11 A).Similarly, use identical optimization reagent formulation, for having X=10, the CPM-FF Luc/RII β B cAMP transmitter of the X/Y joint length of Y=10 (pBFB10), the vitro detection of cAMP shows that signal and background ratio are 11, EC ₅₀Be 0.64 μ M (Figure 11 A).This cAMP measures has following advantage: reduce the noclilucence of compound interferential and read; Homogeneous single step form; And need the bonded specificity and induce the ability of conformational change.

EXAMPLE V

Use CPM-FF Luc/RII β B cAMP biosensor detects cAMP concentration in cell Change

Cell cultures

Cell in having the 60mlDMEM/F12 of HEPES damping fluid (Invitrogen) and 10% FBS 37 ℃ with 5% CO ₂Under cultivate.

Plasmid

Coding is had (X=10, the ORF based on the cAMP biosensor of CPM-FFLuc/RII β B of X/Y joint length Y=0) is transferred to Flexi carrier pF4K (Flexi carrier system; Promega Corp.).Resulting plasmid construction body (pBFB141) utilizes upstream CMV promotor to be used for expressing relevant cAMP biosensor at mammalian cell.

Transfection

Cell uses 0.3 μ l

The hole of reagent and 0.15 μ g DNA/96 orifice plate is used

Reagent (MIRUS) carries out transfection.Allow the cell grow overnight and measured at second day.

The adjusting of biosensor

After the transfection about 1 day, from incubator, take out cell and balance to room temperature.The 5 μ l aliquots containigs of 100mM luciferin EF add altogether in 90 μ l cell culture+transfection reagents, to produce the final concentration of about 5mg/mL luciferin.Cell is incubation at least 90 minutes at room temperature subsequently.After at room temperature 90 minutes, luminous baseline measures uses 96 hole Veritas photometer (TurnerBiosystems; 0.5 the integrating time in second/hole) measure.Cell uses 10 μ M Racemic isoproterenols (CalBiochem), 50mM Fu Sikelin (Sigma) to induce subsequently, or do not induce (0.1% DMSO, Sigma), and luminous about 30 minutes of continuously measured.After 30 minutes, 10mM Proprasylyte (Sigma) adding is had in the different third adrenal cell, and 0.1%DMSO is added in the every other sample.Luminous continuously measured in ensuing 30 minutes subsequently.The final additive of 50 μ M Fu Sikelin is added in different third suprarenal gland/Proprasylyte sample, and 0.1% DMSO is added in the every other sample.Luminous in ensuing continuously measured half an hour subsequently.Sample is measured in 12 multiple groups.The 10x stoste of Racemic isoproterenol, Proprasylyte, Fu Sikelin and DMSO is prepared in 1x PBS (Invitrogen).

The result

Variation for the IC of measuring cAMP, HEK 293 cells CPM-FFLuc/RII β B (X=10, Y=0, pBFB141) construct carries out transient transfection, handle with compound subsequently, described compound is known to increase cAMP concentration (Racemic isoproterenol in the cell by the GPCR activation, be the moving agent of a kind of beta-adrenergic receptor kinase 1), suppress to reduce cAMP concentration (Proprasylyte, promptly a kind of B-adrenergic receptor antagonist) in the cell or the activation by adenylate cyclase increases cAMP concentration (Fu Sikelin) in the cell by GPCR.Independent Racemic isoproterenol and Fu Sikelin handle the light output that makes from transfectional cell increases about 2 times, the increase (Figure 11 C) of cAMP concentration in the reflection cell.In addition, after the of short duration reaction of the variation of cAMP concentration,, handle transfectional cell (Figure 11 C) for Fu Sikelin subsequently with Racemic isoproterenol, Proprasylyte.Also test the CPM-FF Luc/RII β B fusion rotein of the peptide (pBFB8) of wild-type luciferase and 42 amino acid whose Gly/Ser of being rich in of expression, and do not shown the specific reaction of the interpolation of the known conditioning agent of cAMP concentration in the pair cell.

Example VI

Light output and induce multiple as about based on the cAMP transmitter of CPM-FF Luc/RII β B The function of X/Y peptide linker length changes

A. the coding have variable X/Y peptide linker length the cAMP based on CPM-FF Luc/RII β B The plasmid of transmitter is synthetic

Has [2x (x=0-5) in order to produce, 2y (y=0-5)] the cAMP sensor groups based on CPM-FF Luc/RII β B of variable X/Y peptide linker length, coding (X=0, Y=0, pBFB89), (X=2, Y=2, pBFB96), (X=6, Y=6, pBFB108) and (X=8, Y=8, the plasmid of transmitter pBFB115) use the overlapping extension PCR of montage (SOE PCR) to synthesize.When needing, the standard molecule clone technology is used for the exchange coding and has (X=0, Y=0), (X=2, Y=2), (X=4, Y=4), (X=6, Y=6), (X=8, Y=8) and (X=10, Y=10) peptide linker based on the dna fragmentation between the plasmid of the cAMP transmitter of CPM-FF Luc/RII β B, to be created in all the other clones in this group.In addition, SOE PCR is used to synthesize the clone's (table 2) in [10+2n (n=0-5), 0] and [10 ,-2n (n=1-7)] group.

Table 2

pBFB89 X＝0，Y＝0 RIIβB(SEQ ID NO：124)

pBFB90 X＝0，Y＝2 RIIβB-SG(SEQ ID NO：125)

pBFB91 X＝0，Y＝4 RIIβB-GSSG(SEQ ID NO：126)

pBFB92 X＝0，Y＝6 RIIβB-SGGSSG(SEQ ID NO：127)

pBFB93 X＝0，Y＝8 RIIβB-GGSGGSSG(SEQ ID NO：128)

pBFB94 X＝0，Y＝10 RIIβB-GSGGSGGSSG(SEQ ID NO：129)

pBFB95 X＝2，Y＝0 GS-RIIβB(SEQ ID NO：130)

pBFB96 X＝2，Y＝2 GS-RIIβB-SG(SEQ ID NO：131)

pBFB97 X＝2，Y＝4 GS-RIIβB-GSSG(SEQ ID NO：132)

pBFB98 X＝2，Y＝6 GS-RIIβB-SGGSSG(SEQ ID NO：133)

pBFB99 X＝2，Y＝8 GS-RIIβB-GGSGGSSG(SEQ ID NO：134)

pBFB100 X＝2，Y＝10 GS-RIIβB-G SGGSGGSSG(SEQ ID NO：135)

pBFB101 X＝4，Y＝0 GSTG-RIIβB(SEQ ID NO：136)

pBFB102 X＝4，Y＝2 GSTG-RIIβB-SG(SEQ ID NO：137)

pBFB9 X＝4，Y＝4 GSTG-RIIβB-GSSG(SEQ ID NO：138)

pBFB103 X＝4，Y＝6 GSTG-RIIβB-SGGSSG(SEQ ID NO：139)

pBFB104 X＝4，Y＝8 GSTG-RIIβB-GGSGGSSG(SEQ ID NO：140)

pBFB39 X＝4，Y＝10 GSTG-RIIβB-GSGGSGGSSG(SEQ ID NO：141)

pBFB105 X＝6，Y＝0 GSTGGS-RIIβB(SEQ ID NO：142)

pBFB106 X＝6，Y＝2 GSTGGS-RIIβB-SG(SEQ ID NO：143)

pBFB107 X＝6，Y＝4 GSTGGS-RIIβB-GSSG(SEQ ID NO：144)

pBFB108 X＝6，Y＝6 GSTGGS-RIIβB-SGGSSG(SEQ ID NO：145)

pBFB109 X＝6，Y＝8 GSTGGS-RIIβB-GGSGGSSG(SEQ ID NO：146)

pBFB110 X＝6，Y＝10 GSTGGS-RIIβB-GSGGSGGSSG(SEQ ID NO：147)

pBFB111 X＝8，Y＝0 GSTGGSGG-RIIβB(SEQ ID NO：148)

pBFB112 X＝8，Y＝2 GSTGGSGG-RIIβB-SG(SEQ ID NO：149)

pBFB113 X＝8，Y＝4 GSTGGSGG-RIIβB-GSSG(SEQ ID NO：150)

pBFB114 X＝8，Y＝6 GSTGGSGG-RIIβB-SGGSSG(SEQ ID NO：151)

pBFB115 X＝8，Y＝8 GSTGGSGG-RIIβB-GGSGGSSG(SEQ ID NO：152)

pBFB116 X＝8，Y＝10 GSTGGSGG-RIIβB-GSGGSGGSSG(SEQ ID NO：153)

pBFB117 X＝10，Y＝0 GSSGGSGGSG-RIIβB(SEQ ID NO：154)

pBFB118 X＝10，Y＝2 GSSGGSGGSG-RIIβB-SG(SEQ ID NO：155)

pBFB41 X＝10，Y＝4 GSSGGSGGSG-RIIβB-GSSG(SEQ ID NO：156)

pBFB119 X＝10，Y＝6 GSSGGSGGSG-RIIβB-SGGSSG(SEQ ID NO：157)

pBFB120 X＝10，Y＝8 GSSGGSGGSG-RIIβB-GGSGGSSG(SEQ ID NO：158)

pBFB10 X＝10，Y＝10 GSSGGSGGSG-RIIβB-GSGGSGGSSG(SEQ ID NO：159)

pBFB128 X＝10，Y＝-2 GSSGGSGGSG-RIIβB(266-412)(SEQ ID NO：160)

pBFB129 X＝10，Y＝-4 GSSGGSGGSG-RIIβB(266-410)(SEQ ID NO：161)

pBFB130 X＝10，Y＝-6 GSSGGSGGSG-(266-408)(SEQ ID NO：162)

pBFB131 X＝10，Y＝-8 GSSGGSGGSG-RIIβB(266-406)(SEQ ID NO：163)

pBFB132 X＝10，Y＝-10 GSSGGSGGSG-RIIβB(266-404)(SEQ ID NO：164)

pBFB133 X＝10，Y＝-12 GSSGGSGGSG-RIIβB(266-402)(SEQ ID NO：165)

pBFB134 X＝10，Y＝-14 GSSGGSGGSG-RIIβB(266-400)(SEQ ID NO：166)

pBFB135 X＝12，Y＝0 GSSGGSGGSGGG-RIIβB(SEQ ID NO：167)

pBFB136 X＝14，Y＝0 GSSGGSGGSGGGSG-RIIβB(SEQ ID NO：168)

pBFB137 X＝16，Y＝0 GSSGGSGGSGGGSGGS-RIIβB(SEQ ID NO：169)

pBFB138 X＝18，Y＝0 GSSGGSGGSGGGSGGSGG-RIIβB(SEQ ID NO：170)

pBFB139 X＝20，Y＝0 GSSGGSGGSGGGSGGSGGSG-RIIβB(SEQ ID NO：171)

(the amino acid 266-414 of the corresponding Genbank I of RII β B D AAH75800)

I. Coding lacks peptide linker (X=0, Y=0; PBFB89) based on CPM-FF Luc/RII β B The plasmid of cAMP transmitter synthetic

For synthetic lack peptide linker (X=0, construct Y=0), with 3 primers that separate to the RII β B DNA that increases, to produce 3 kinds of PCR products that separate.Primer to 5 '-CCT CGAACA CCG AGC GAC C-3 ' (SEQ ID NO:31) and 5 '-GCA GTG ACT CAATAA AGC TTT CAT ACA TCT TCT TGG CCT TAA TGA GAA TCTCG-3 ' (SEQ ID NO:18) is used to produce product #1; Primer to 5 '-CGA GAT TCT CATTAA GGC CAA GAA GAT GTA TGA AAG CTT TAT TGA GTC ACTGC-3 ' (SEQ ID NO:32) and 5 '-GGC CCT TCT TAA TGT TTT TGG CTACAA TAT CCA TGT TCG TTC CAA ACA G-3 ' (SEQ ID NO:33) is used to produce product 2; And primer to 5 '-CTG TTT GGA ACG AAC ATG GAT ATTGTA GCC AAA AAC ATT AAG AAG GGC C-3 ' (SEQ ID NO:34) and 5 '-GTA TCT TAT CAT GTC TGC TCG AAG CG-3 (SEQ ID NO:35) is used to produce product 3.The SOE PCR of 3 kinds of products produces total length PCR product, and it digests with the SgfI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FFLuc expression plasmid (pBFB8) that digests with SgfI/XbaI.

Ii. Coding has (X=2, Y=2; PBFB96) peptide linker length based on CPM-FFLuc/RII β B The plasmid of cAMP transmitter synthetic

Have peptide linker (3 primers that separate are to being used to the RII β B that increases, to produce 3 kinds of PCR products that separate for X=2, construct Y=2) for synthetic.Primer to 5 '-CCT CGA ACACCG AGC GAC C-3 ' (SEQ ID NO:36; BFB31) and 5 '-CAA TAA AGC TTTCAT ACA TCG AGC CCT TCT TGG CCT TAA TGA GAA TCT CG-3 ' (SEQ ID NO:37; BFB120) be used to produce product 1; Primer to 5 '-CGA GAT TCTCAT TAA GGC CAA GAA GGG CTC GAT GTA TGA AAG CTT TATTG-3 ' (SEQ ID NO:38; BFB119) and 5 '-CTT CTT AAT GTT TTT GGCACC GGA TAC AAT ATC CAT GTT CGT TCC AAA CAG-3 ' (SEQ IDNO:39; BFB122) be used to produce product 2; And primer to 5 '-CTG TTT GGA ACGAAC ATG GAT ATT GTA TCC GGT GCC AAA AAC ATT AAG AAG-3 ' (SEQ ID NO:40; BFB122) and 5 '-GTA TCT TAT CAT GTC TGC TCGAAG CG-3 ' (SEQ ID NO:41; BFB34) be used to produce product 3.The SOE PCR of 3 kinds of products produces total length PCR product, and it digests with the SgfI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with SgfI/XbaI.

Iii. Coding has (X=6, Y=6; DBFB108) peptide linker length based on CPM-FF The plasmid of the cAMP transmitter of Luc/RII β B is synthetic

Have peptide linker in order to synthesize (X=6, construct Y=6), primer 5 '-AAA AAA AAAGTC GAC CGG AGG TTC AAT GTA TGA AAG CTT TAT TGA GTCACT GC-3 ' (SEQ ID NO:42; BFB123) and 5 '-AAA AAA GAG CTC CCTCCA GAT ACA ATA TCC ATG TTC GTT CCAAAC AG-3 ' (SEQ ID NO:43; BFB124) be used for pcr amplification RII β B DNA.Resulting product digests with the SalI/SacI restriction enzyme, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/SacI.

Iv. Coding has (X=8, Y=8; PBFB115) peptide linker length based on CPM-FF The plasmid of the cAMP transmitter of Luc/RII β B is synthetic

Have peptide linker in order to synthesize (X=8, construct Y=8), primer 5 '-AAA AAA GTCGAC CGG AGG TTC AGG CGG TAT GTA TGA AAG CTT TAT TGAGTC ACT GC-3 ' (SEQ ID NO:44; BFB125) and 5 '-AAA AAA GAG CTCCCT CCA GAT CCA CCT ACA ATA TCC ATG TTC GTT CCA AACAG-3 ' (SEQ ID NO:116; BFB126) be used for pcr amplification RII β B DNA.Resulting product digests with the SalI/SacI restriction enzyme, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/SacI.

V. Coding has all the other of peptide linker length in the set [2x (x=0-5), 2y (y=0-5)] Plasmid based on the cAMP transmitter of CPM-FF Luc/RII β B is synthetic

Coding had (X=0, Y=0), (X=2, Y=2), (X=4, Y=4), (X=6, Y=6), (X=8, Y=8) and (X=10, the plasmid based on the cAMP transmitter of CPM-FF Luc/RII β B of peptide linker length Y=10) carry out XhoI/XbaI or the digestion of XmnI/XbaI restriction enzyme.In each case, restriction enzyme digestion produces two fragments: C-terminal part, joint Y and the Luc 2.04-233 of coding RII β B are segmental than small segment; With the big fragment of all the other elements that comprise original plasmid, comprise the sequence of the N-terminal part of coding Luc2.0234-544, joint X and RII β B.In order to produce all the 36 kinds of clones in [2x (x=0-5), 2y (y=0-5)] set, make less fragment and being connected from various restriction enzyme digestion than big fragment.

Vi. Coding have peptide linker length in the set [10+2n (n=1-5), 0] based on CPM-FF The plasmid of the cAMP transmitter of Luc/RII β B is synthetic

Have peptide linker length (X=12, Y=0 in order to synthesize; PBFB135) the plasmid based on the cAMP transmitter of CPM-FFLuc/RII β B, two primers that separate are to being used to the RII β B that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAA AAA TCC GGA GGA GGTATG TAT GAA AGC TTT ATT GAG TCA CTG C-3 ' (SEQ ID NO:46BFB142) and 5 '-GGC CCT TCT TAA TGT TTT TGG CTA CAA TAT CCATGT TCG TTC CAA ACA G-3 ' (SEQ ID NO:47; BFB118) be used to produce product #1; Primer to 5 '-CTG TTT GGA ACG AAC ATG GAT ATT GTA GCCAAA AAC ATT AAG AAG GGC C-3 ' (SEQ ID NO:48; BFB117) and 5 '-GTA TCT TAT CAT GTC TGC TCG AAG CG-3 ' (SEQ ID NO:49; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the BspEI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with BspEI/XbaI.

Have peptide linker length (X=14, Y=0 in order to synthesize; PBFB136) the plasmid based on the cAMP transmitter of CPM-FFLuc/RII β B, two primers that separate are to being used to the RII β B that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAA AAA TCC GGA GGA GGTTCT GGC ATG TAT GAA AGC TTT ATT GAG TCA CTG C-3 ' (SEQ IDNO:45; BFB143) and 5 '-GGC CCT TCT TAA TGT TTT TGG CTA CAATAT CCA TGT TCG TTC CAA ACA G-3 ' (SEQ ID NO:21; BFB118) be used to produce product 1; Primer to 5 '-CTG TTT GGA ACG AAC ATG GAT ATTGTA GCC AAA AAC ATT AAG AAG GGC C-3 ' (SEQ ID NO:24; BFB117) and 5 '-GTA TCT TAT CAT GTC TGC TCG AAG CG-3 ' (SEQ IDNO:30; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the BspEI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with BspEI/XbaI.

Have peptide linker length (X=16, Y=0 in order to synthesize; PBFB137) the plasmid based on the cAMP transmitter of CPM-FFLuc/RII β B, two primers that separate are to being used to the RII β B that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-ATAAAT TCC GGA GGA GGTTCT GGC GGA TCA ATG TAT GAA AGC TTT ATT GAG TCA CTG C-3 ' (SEQ ID NO:50; BFB144) and 5 '-GGC CCT TCT TAA TGT TTT TGGCTA CAA TAT CCA TGT TCG TTC CAA ACA G-3 ' (SEQ ID NO:51; BFB118) be used to produce product 1; Primer to 5 '-CTG TTT GGA ACG AAC ATG GATATT GTA GCC AAA AAC ATT AAG AAG GGC C-3 ' (SEQ ID NO:52; BFB117) and 5 '-GTA TCT TAT CAT GTC TGC TCG AAG CG-3 ' (SEQ IDNO:53; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the BspEI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with BspEI/XbaI.

Have peptide linker length (X=18, Y=0 in order to synthesize; PBFB138) the plasmid based on the cAMP transmitter of CPM-FFLuc/RII β B, two primers that separate are to being used to the RII β B that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAA AAT TCC GGA GGA GGTTCT GGC GGA TCA GGC GGT ATG TAT GAA AGC TTT ATT GAGTCA CTG C-3 ' (SEQ ID NO:54; BFB145) and 5 '-GGC CCT TCT TAA TGTTTT TGG CTA CAA TAT CCA TGT TCG TTC CAA ACA G-3 ' (SEQ IDNO:55; BFB118) be used to produce product 1; Primer to 5 '-CTG TTT GGA ACG AACATG GAT ATT GTA GCC AAA AAC ATT AAG AAG GGC C-3 ' (SEQ IDNO:56; BFB117) and 5 '-GTA TCT TAT CAT GTC TGC TCG AAG CG-3 ' (SEQ ID NO:57; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the BspEI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with BspEI/XbaI.

Vii. Coding have peptide linker length in the set [10 ,-2n (n=1-7)] based on CPM-FF The plasmid of the cAMP transmitter of Luc/RII β B is synthetic

For synthetic N-terminal peptide linker length with (X=10), lack the C-terminal peptide linker, have RII β B residue 266-412 (10 ,-2; PBFB128) the plasmid based on the cAMP transmitter of CPM-FF Luc/RII β B, two primers that separate are to being used to the RII β B DNA that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAA AAA GTC GAC CGG AGGTTC AGG CGG TTC-3 ' (SEQ ID NO:58; BFB127) and 5 '-GGC CCT TCTTAA TGT TTT TGG CAT CCA TGT TCG TTC CAAACA GG-3 ' (SEQ IDNO:59; BFB128) be used to produce product 1; Primer to 5 '-CCT GTT TGG AAC GAACAT GGA TGC CAA AAA CAT TAA GAA GGG CC-3 ' (SEQ ID NO:60; BFB129) and 5 '-GTA TCT TAT CAT GTC TGC TCG AAG CG-3 ' (SEQ IDNO:61; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the SalI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/XbaI.

For synthetic N-terminal peptide linker length with (X=10), lack the C-terminal peptide linker, have RII β B residue 266-410 (10 ,-4; PBFB129) the plasmid based on the cAMP transmitter of CPM-FF Luc/RII β B, two primers that separate are to being used to the RII β B DNA that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAAAAAGTCGACCGGAGGTTCAGGCGGTTC-3 ' (SEQ ID NO:62; BFB127) and 5 '-GGCCCTTCTTAATGTTTTTGGCGTTCGTTCCAAACAGGGCAACTAAC-3 ' (SEQ ID NO:63; BFB130) be used to produce product #1; Primer to 5 '-GTTAGTTGCCCTGTTTGGAACGAACGCCAAAAACATTAAGAAGGGCC-3 ' (SEQ ID NO:64; BFB131) and 5 '-GTATCTTATCATGTCTGCTCGAAGCG-3 ' (SEQ ID NO:65; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the SalI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/XbaI.

For synthetic N-terminal peptide linker length with (X=10), lack the C-terminal peptide linker, have RII β B residue 266-408 (10 ,-6; PBFB130) the plasmid based on the cAMP transmitter of CPM-FF Luc/RII β B, two primers that separate are to being used to the RII β B DNA that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAAAAAGTCGACCGGAGGTTCAGGCGGTTC-3 ' (SEQ ID NO:66; BFB127) and 5 '-GGCCCTTCTTAATGTTTTTGGCTCCAAACAGGGCAACTAACTGTTCTTC-3 ' (SEQ ID NO:67; BFB132) be used to produce product 1; Primer to 5 '-GAAGAACAGTTAGTTGCCCTGTTTGGAGCCAAAAACATTAAGAAGGGCC-3 ' (SEQ ID NO:68; BFB133) and 5 '-GTATCTTATCATGTCTGCTCGAAGCG-3 ' (SEQ ID NO:69; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the SalI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/XbaI.

For synthetic N-terminal peptide linker length with (X=10), lack the C-terminal peptide linker, have RII β B residue 266-406 (10 ,-8; PBFB131) the plasmid based on the cAMP transmitter of CPM-FF Luc/RII β B, two primers that separate are to being used to the RII β B DNA that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAAAAAGTCGACCGGAGGTTCAGGCGGTTC-3 ' (SEQ ID NO:70; BFB127) and 5 '-GGCCCTTCTTAATGTTTTTGGCCAGGGCAACTAACTGTTCTTCATAGG-3 ' (SEQ ID NO:71; BFB134) be used to produce product 1; Primer to 5 '-CCTATGAAGAACAGTTAGTTGCCCTGGCCAAAAACATTAAGAAGGGCC-3 ' (SEQ ID NO:72; BFB135) and 5 '-GTATCTTATCATGTCTGCTCGAAGCG-3 ' (SEQ ID NO:73; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the SalI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/XbaI.

For synthetic N-terminal peptide linker length with (X=10), lack the C-terminal peptide linker, have RII β B residue 266-404 (10 ,-10; PBFB132) the plasmid based on the cAMP transmitter of CPM-FF Luc/RII β B, two primers that separate are to being used to the RII β B DNA that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAAAAAGTCGACCGGAGGTTCAGGCGGTTC-3 ' (SEQ ID NO:74; BFB127) and 5 '-GGCCCTTCTTAATGTTTTTGGCAACTAACTGTTCTTCATAGGTAGCGATG-3 ' (SEQ ID NO:75; BFB136) be used to produce product 1; Primer to 5 '-CATCGCTACCTATGAAGAACAGTTAGTTGCCAAAAACATTAAGAAGGGCC-3 ' (SEQ ID NO:76; BFB137) and 5 '-GTATCTTATCATGTCTGCTCGAAGCG-3 ' (SEQ ID NO:77; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the SalI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/XbaI.

For synthetic N-terminal peptide linker length with (X=10), lack the C-terminal peptide linker, have RII β B residue 266-402 (10 ,-12; PBFB133) the plasmid based on the cAMP transmitter of CPM-FF Luc/RII β B, two primers that separate are to being used to the RII β B DNA that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAAAAAGTCGACCGGAGGTTCAGGCGGTTC-3 ' (SEQ ID NO:78; BFB127) and 5 '-GGCCCTTCTTAATGTTTTTGGCCTGTTCTTCATAGGTAGCGATGTTCC-3 ' (SEQ ID NO:79; BFB138) be used to produce product 1; Primer to 5 '-GGAACATCGCTACCTATGAAGAACAGGCCAAAAACATTAAGAAGGGCC-3 ' (SEQ ID NO:80; BFB139) and 5 '-GTATCTTATCATGTCTGCTCGAAGCG-3 ' (SEQ ID NO:81; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the SalI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/XbaI.

For synthetic N-terminal peptide linker length with (X=10), lack the C-terminal peptide linker, have RII β B residue 266-400 (10 ,-14; PBFB134) the plasmid based on the cAMP transmitter of CPM-FF Luc/RII β B, two primers that separate are to being used to the RII β B DNA that increases, to produce 2 kinds of PCR products that separate.Primer to 5 '-AAAAAAGTCGACCGGAGGTTCAGGCGGTTC-3 ' (SEQ ID NO:82; BFB127) and 5 '-GGCCCTTCTTAATGTTTTTGGCTTCATAGGTAGCGATGTTCCTTTTC-3 ' (SEQ ID NO:83; BFB140) be used to produce product 1; Primer to 5 '-GAAAAGGAACATCGCTACCTATGAAGCCAAAAACATTAAGAAGGGCC-3 ' (SEQ ID NO:84; BFB141) and 5 '-GTATCTTATCATGTCTGCTCGAAGCG-3 ' (SEQ ID NO:85; BFB34) be used to produce product 2.The SOE PCR of 2 kinds of products produces total length PCR product, and it digests with the SalI/XbaI restriction enzyme subsequently, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/XbaI.

B. the cAMP sensing that has variable X/Y peptide linker length based on CPM-FF Luc/RII β B The function of device characterizes

I. Have X/Y peptide linker length in the set [2x (x=0-5), 2y (y=0-5)] based on The function of the cAMP transmitter of CPM-FF Luc/RII β B characterizes

Using

After T7 link coupled reticulocyte lysate system (Promega Corp.) is expressed, measurement have in the set [2x (x=0-5), 2y (y=0-5)] X/Y joint length CPM-FF Luc/RII β B cAMP transmitter cAMP exist and not in the presence of luciferase activity.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 400ng plasmid DNA

10 μ L rabbit reticulocyte extracts

0.8 μ L TNT reaction buffer

0.4 μ L T7 polysaccharase

0.4 μ L aminoacid mixture

0.4μL rRNasin

DH ₂O to 20 μ L cumulative volume

After 1.5 hours, fusion rotein separately carries out incubation in the existence of 100 μ M cAMP or not in 30 ℃ of incubations, and this incubation is by making 9 μ L

Reaction and 1 μ L 1mM cAMP stoste or dH ₂The O combination.At room temperature behind incubation 〉=15 minute, 1 μ L sample is added 100 μ L luciferase assay reagent (LAR; Promega Corp.) solution+/-100 μ M cAMP (90 μ L LAR+10 μ L 1mM cAMP stoste or dH ₂O) in.Luminous use Veritas microtiter plate photometer (Turner Biosystems; Program Bright-Glo) measures.Generally speaking, with having set [2x (x=0-5), 2y (y=0-5)] in the CPM-FF Luc/RII β B fusions of X/Y joint length observe a kind of trend, wherein in the existence of 100 μ M cAMP or not, along with peptide linker length increases, the uciferase activity that measures increases (Figure 12).In addition, observe second kind of trend, wherein in the presence of 100 μ M cAMP, the multiple of inducing of luciferase activity increases (Figure 13) with the peptide linker length that increases.

Ii. Have set [10 ,-2n (n=1-7)], [10,2n (n=1-5)] and [10+2n (n= 0-5), 0] the X/Y peptide linker in the individual amino-acid residue based on CPM-FF Luc/RII β B's The function of cAMP transmitter characterizes

Using

After T7 link coupled reticulocyte lysate system (Promega Corp.) is expressed, measurement has set [10,-2n (n=1-7)], [10,2n (n=1-5)] and [10+2n (n=0-5), 0] in the X/Y joint length CPM-FF Luc/RII β B cAMP transmitter cAMP exist and not in the presence of luciferase activity.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 400ng plasmid DNA

10 μ L rabbit reticulocyte extracts

0.8 μ L TNT reaction buffer

0.4 μ L T7 polysaccharase

0.4 μ L aminoacid mixture

0.4μL rRNasin

DH ₂O to 20 μ L cumulative volume

After 1 hour, fusion rotein separately carries out incubation in the existence of 100 μ M cAMP or not in 30 ℃ of incubations, and described incubation is by making 9 μ L

Reaction and 1 μ L 1mM cAMP stoste or dH ₂The O combination.At room temperature behind incubation 〉=9 minute, 1 μ L sample is added 100 μ L luciferase assay reagent (LAR; Promega Corp.) solution+/-100 μ M cAMP (90 μ L LAR+10 μ L 1mM cAMP stoste or dH ₂O) in.Luminous use Veritas microtiter plate photometer (Turner Biosystems; Program Bright-Glo) measures.Generally speaking, for the CPM-FF Luc/RII β B cAMP transmitter that lacks the C-terminal peptide linker, in the existence of 100 μ M cAMP or the luciferase activity not follow the RII β B C-terminal brachymemma of increase and reduce (Figure 14).In addition, for the CPM-FF Luc/RII β B cAMP transmitter of set [10 ,-2n (n=1-7)] and [10,2n (n=1-5)], it is to have (X=10, Y=0 that the maximum in the presence of 100 μ M cAMP is induced multiple; The transmitter of peptide linker pBFB117).In addition, for having (X=10, Y=0; PBFB117) transmitter of the peptide linker of individual amino-acid residue, the CPM-FF Luc/RII β B cAMP transmitter of set [10+2n (n=0-5), 0] show the maximum multiple (Figure 15) of inducing.

Example VII A

Has circular permutation Pleonomus luciferase and from the B structure of PKA modulability II β type subunit The cAMP biosensor in territory

A . be used for the closing of CPM Pleonomus Luc expression plasmid (pBFB53) of follow-up insertion RII β B Become

For synthetic embodiment X, the Pleonomus variant of synthetic plasmid in the part A, primer 5 '-TATAATGCTAGCGATCGCCATGGGCGTGACTGTGCTGGTGTATC-3 ' (SEQ ID NO:86; BFB94) and 5 '-TTTTTTCTCGAGCCGCCGCCAGCTTTTTCGAGG-3 ' (SEQ ID NO:87; BFB95) be used for amplification coding from plasmid pCBG68-basis (Genbank Acc#AY258593; Promega Corp) the segmental Pleonomus Equivalent of Lampyridea luciferase of residue 234-544 (Pleonomus luciferase amino acid 231-542).Resulting product digests with the NheI/XhoI restriction enzyme, and is connected in parent CPM-FF Luc (pBFB8) expression plasmid that digests with NheI/XhoI, to produce plasmid intermediate product 1.Subsequently, primer 5 '-AAAAAAGAGCTCCGGTGAAAAGAACGTGATCTACGGCC-3 ' (SEQ ID NO:88; BFB96) and 5 '-AAAAAATCTAGAGTTTAAACAGGGATCAATTGAGTACCCACAC-3 ' (SEQ ID NO:89; BFB97) be used for amplification coding from plasmid pCBG68-basis (Genbank Acc# AY258593; Promega Corp) the segmental Pleonomus Equivalent of Lampyridea luciferase of residue 4-233 (Pleonomus luciferase amino acid 5-230).Resulting product digests with the SacI/XbaI restriction enzyme, and is connected in the above-described plasmid intermediate product 1 that digests with SacI/XbaI.

B. coding has (X=4, Y=4; PBFB54) and (X=10, Y=4; PBFB55) individual ammonia Synthesizing of the plasmid of the CPM Pleonomus Luc/RII β B fusion rotein of the peptide linker of the sour residue of base

Have in order to synthesize (X=4, the Y=4) construct of joint length, primer 5 '-AAA AAAGTC GAC CGG AAT GTA TGA AAG CTT TAT TGA GTC ACT GCC-3 ' (SEQ ID NO:90; BFB51) and 5 '-AAA AAA GAG CTC CCA ACA ATATCC ATG TTC GTT CCA AAC-3 ' (SEQ ID NO:91; BFB20) be used for the RII β B DNA of amplification from ATCC 10625233 (Genbank ID BC075800).Resulting product digests with the SalI/SacI restriction enzyme and is connected in the parent CPM Pleonomus Luc (pBFB53).

Have in order to synthesize (X=10, the Y=4) construct of joint length, primer 5 '-AAA AAAGAG CTC CCA ACA ATA TCC ATG TTC GTT CCA AAC-3 ' (SEQ IDNO:92; BFB20) and 5 '-AAA AAA TCC GGA ATG TAT GAA AGC TTTATT GAG TCA CTG CC-3 ' (SEQ ID NO:93; BFB21) be used for the RII β B DNA of amplification from ATCC10625233 (Genbank ID BC075800).Resulting product digests with the BspEI/SacI restriction enzyme, and is connected in parent CPM Pleonomus Luc (pBFB53) expression plasmid that digests with BspEI/SacI.

B. have (X=4, Y=4; PBFB54) and (X=10, Y=4; PBFB55) individual amino acid The function of the CPM Pleonomus Luc/RII β B fusion rotein of the peptide linker of residue characterizes

Have (X=4, Y=4; PBFB54) and (X=10, Y=4; PBFB55) the cAMP dose response of the CPM Pleonomus Luc/RII β B fusion rotein of the X/Y joint length of individual amino-acid residue uses

T7 link coupled reticulocyte lysate system (Promega Corp.) is measured after expression.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 2400ng plasmid DNA

60 μ L rabbit reticulocyte extracts

4.8 μ L TNT reaction buffer

2.4 μ L T7 polysaccharase

2.4 μ L aminoacid mixture

2.4μL rRNasin

DH ₂O to 120 μ L cumulative volume

After 1.5 hours, fusion rotein separately carries out incubation with the cAMP of various concentration in 30 ℃ of incubations, and this incubation is by making 9 μ L

Reaction and the combination of 1 μ L cAMP stoste (final concentrations of 0,0.01,0.025,0.1,0.25,1,2.5,10 and 25 μ M cAMP).At room temperature balance comprises the 100 μ L luciferase assay reagent (LAR of the cAMP of concentration (90 μ LLAR+10 μ L cAMP stoste) separately with the adding of 1 μ L sample after about 20 minutes; PromegaCorp.) in the solution.Luminous use Veritas microtiter plate photometer (Turner Biosystems; Program Bright-Glo) measures.Have (X=4, Y=4; PBFB54) and (X=10, Y=4; PBFB55) to demonstrate the multiple of inducing of luciferase activity under 25 μ M cAMP respectively be 4.0 and 5.5 to the CPM Pleonomus Luc/RII β B fusion rotein of the X/Y joint length of individual amino-acid residue.Yet, under all concentration of test, induce multiple less than inducing multiple (Figure 16) based on the transmitter of Lampyridea luciferase based on the cAMP transmitter of Pleonomus luciferase.

Example VII A I

Utilize circular permutation Lampyridea luciferase and from the B structure of PKA modulability I α type subunit The cAMP biosensor in territory

The DNA that will encode from the B structural domain of people PKA modulability I α type subunit (RI α B) is connected in the expression vector of coding CPM-FF Luc/RI α B fusions [Luc2.0 (234-544)-joint X-people RI α (residue 245-381)-joint Y-Luc2.0 (4-233)].

A. have (X=4, Y=4; PBFB56) and (X=20, Y=20; PBFB58) individual amino Synthesizing of the CPM-FF Luc/RI α B fusion rotein of the peptide linker of acid residue

Have in order to synthesize (X=4, the Y=4) construct of joint length, primer 5 '-ATATAACTCGAGCGGAATGTATGAGGAATTCCTTAGTAAAGTCTCTATTTTAG-3 ' (SEQ ID NO:94; BFB98) and 5 '-AAAAAAGAGCTCCCGACAGACAGTGACACAAAACTGTTGTAC-3 ' (SEQ ID NO:95; BFB99) the RI α B DNA (Genbank Acc#BC036285) that is used to increase.Resulting product digests with the XhoI/SacI restriction enzyme, and is connected in parent CPM-FF Luc (pBFB8) expression vector that digests with XhoI/SacI.

Have in order to synthesize (X=20, the Y=20) construct of joint length, primer 5 '-ATTAAACCCGGGATGTATGAGGAATTCCTTAGTAAAGTCTCTATTTTAG-3 ' (SEQ ID NO:96; BFB102) and 5 '-AAAAAATCCGGACCCGACAGACAGTGACACAAAACTGTTGTAC-3 ' (SEQ ID NO:97; BFB103) the RI α B DNA (Genbank Acc#BC036285) that is used to increase.Resulting product digests with the SmaI/BspEI restriction enzyme, and is connected in parent CPM-FF Luc (pBFB8) expression vector that digests with NruI/AgeI.

B. have (X=4, Y=4; PBFB56) and (X=20, Y=20; PBFB58) individual amino The function of the CPM-FF Luc/RI α B fusion rotein of the peptide linker of acid residue characterizes

Have (X=4, Y=4; DBFB56) and (X=20, Y=20; PBFB58) individualThe cAMP dose response of the CPM-FF Luc/RI α B fusion rotein of the X/Y joint length of amino-acid residue uses

T7 link coupled reticulocyte lysate system (Promega Corp.) is measured after expression.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 2400ng plasmid DNA

60 μ L rabbit reticulocyte extracts

4.8 μ L TNT reaction buffer

2.4 μ L T7 polysaccharase

2.4 μ L aminoacid mixture

2.4μL rRNasin

DH ₂O to 120 μ L cumulative volume

After 1.5 hours, fusion rotein separately carries out incubation with the cAMP of various concentration in 30 ℃ of incubations, and this incubation is by making 9 μ L

Reaction and the combination of 1 μ L cAMP stoste (final concentrations of 0,0.01,0.025,0.1,0.25,1,2.5,10,25 and 100 μ M cAMP).At room temperature behind balance 〉=10 minute, 1 μ L sample added comprise the 100 μ L luciferase assay reagent (LAR of the cAMP of concentration (90 μ L LAR+10 μ L cAMP stoste) separately; Promega Corp.) in the solution.Luminous use Veritas microtiter plate photometer (TurnerBiosystems; Program Bright-Glo) measures.Have (X=4, Y=4; PBFB56) (X=20, Y=20; DBFB58) individualThe CPM-FFLuc/RI α B fusion rotein of the X/Y joint length of amino-acid residue demonstrates luciferase activity under 100 μ M cAMP the multiple of inducing is 1.8.Yet, when concentration 〉=0.025 μ M, induce multiple less than inducing multiple (Figure 17) based on the transmitter of RII β B based on the cAMP transmitter of RI α B.

Example I X

Utilize the heat-resisting luciferase of circular permutation and from the B structural domain of RKA modulability II β type subunit The cAMP biosensor

A. the heat-resisting Luc expression plasmid of CPM (pBFB45) synthetic that is used for follow-up insertion RII β B

For synthetic heat-resisting luciferase (UltraGlo luciferase, Promega Corp.), primer 5 '-AATTAAGCTAGCGATCGCCATGACGTCAGCAATTTTAACGGTAATACC-3 ' (SEQ ID NO:98; BFB88) and 5 '-TTTTTTCTCGAGCCATTGGTGTGTTTTTCTAACATTTGTCTTAAC-3 ' (SEQ ID NO:99; BFB89) be used for the segmental UltraGlo luciferase of the Lampyridea luciferase Equivalent of amplification coding residue 234-544 (UltraGlo luciferase residue 233-543).Resulting product digests with the NheI/XhoI restriction enzyme, and is connected in parent CPM-FF Luc (pBFB8) expression plasmid that digests with NheI/XhoI, to produce plasmid intermediate product 1.Subsequently, primer 5 '-AATTTTGAGCTCCGGTGATAAGAATATTTTATATGGGCCCGAAC-3 ' (SEQ ID NO:100; BFB90) and 5 '-AAAAAATCTAGAGTTTAAACGGGATTAATTGCGTTACCAAAAGTAG-3 (SEQ ID NO:101; BFB91) be used for the segmental Pleonomus Equivalent of Lampyridea luciferase of amplification coding residue 4-233 (UltraGlo luciferase residue 3-232).Resulting product digests with the SacI/XbaI restriction enzyme, and is connected in the above-described plasmid intermediate product 1 that digests with SacI/XbaI.

B. coding has (X=4, Y=4; PBFB51) and (X=20, Y=20; PBFB52) individual The plasmid of the heat-resisting Luc/RII β of the CPM of the peptide linker of amino-acid residue B fusion rotein is synthetic

For composite coding have (X=4, the Y=4) plasmid of the heat-resisting Luc/RII β of the CPM of joint length B fusion rotein, primer 5 '-AAA AAA GTC GAC CGG AAT GTA TGAAAG CTT TAT TGA GTC ACT GCC-3 ' (SEQ ID NO:102; BFB51) and 5 '-AAA AAA GAG CTC CCA ACA ATA TCC ATG TTC GTT CCAAAC-3 ' (SEQ ID NO:103; BFB20) be used for the RII β B DNA of amplification from ATCC 10625233 (Genbank ID BC075800).Resulting product digests with the SalI/SacI restriction enzyme, and is connected in the heat-resisting Luc expression plasmid of above-described parent CPM (pBFB45) that digests with XhoI/SacI.

For composite coding have (X=20, the Y=20) plasmid of the heat-resisting Luc/RII β of the CPM of joint length B fusion rotein, primer 5 '-AAA AAA CCC GGG ATG TAT GAA AGCTTT ATT GAG TCA CTG CC-3 ' (SEQ ID NO:104; BFB23) and 5 '-AAAAAA TCC GGA CCC AAC AAT ATC CAT GTT CGT TCC AAA C-3 ' (SEQ ID NO:105; BFB24) be used for the RII β B DNA of amplification from ATCC10625233 (GenbankID BC075800).Resulting product digests with the BspEI/SmaI restriction enzyme, and is connected in the heat-resisting Luc expression plasmid of above-described parent CPM that digests with AgeI/NruI.

C. have (X=4, Y=4; PBFB51) and (X=20, Y=20; PBFB52) individual amino The function of the heat-resisting Luc/RII β of the CPM B fusion rotein of the peptide linker of acid residue characterizes

Have (X=4, Y=4; DBFB51) and (X=20, Y=20; DBFB52) individualThe cAMP dose response of the heat-resisting Luc/RII β of the CPM B fusion rotein of the X/Y joint length of amino-acid residue uses

T7 link coupled reticulocyte lysate system (Promega Corp.) is measured after expression.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 2400ng plasmid DNA

60 μ L rabbit reticulocyte extracts

4.8 μ L TNT reaction buffer

2.4 μ L T7 polysaccharase

2.4 μ L aminoacid mixture

2.4μL rRNasin

DH ₂O to 120 μ L cumulative volume

After 1.5 hours, fusion rotein separately carries out incubation with the cAMP of various concentration in 30 ℃ of incubations, and this incubation is by making 9 μ L

Reaction and the combination of 1 μ L cAMP stoste (final concentrations of 0,0.01,0.025,0.1,0.25,1,2.5,10,25 and 100 μ M cAMP).At room temperature behind balance 〉=19 minute, 1 μ L sample added comprise the 100 μ L luciferase assay reagent (LAR of the cAMP of concentration (90 μ L LAR+10 μ L 1mM cAMP stoste) separately; Promega Corp.) in the solution.Luminous use Veritas microtiter plate photometer (TurnerBiosystems; Program Bright-Glo) measures.Have X=4, Y=4The heat-resisting Luc/RII β of the CPM B fusion rotein of the X/Y joint length of individual amino-acid residue (pBFB51)The multiple of inducing that demonstrates luciferase activity under 100 μ M cAMP is 1.5 (Figure 18).Yet, have X=20, Y=20The heat-resisting Luc/RII β of the CPM B fusion rotein of the X/Y joint length of individual amino-acid residue (pBFB52)To cAMP reactionless (Figure 18).Under 2 kinds of situations, when concentration 〉=0.025 μ M, induce multiple less than inducing multiple (Figure 18) based on the transmitter of Lampyridea luciferase based on the cAMP transmitter of the heat-resisting Luc/RII β of CPM B.

Embodiment X

Use in CPM sea pansy luciferase/RII β B biosensor cell and detect cAMP concentration Variation (Fu Sikelin titration)

Cell cultures

100 μ l HEK-293 cells bed board in 96 orifice plates, and in having the DMEM/F12 of HEPES damping fluid (Invitrogen) and 10% FBS 37 ℃ with 5% CO ₂Under grow to 70-90% and converge.

Transfection

Cell uses 0.3 μ l

(hole with (X=4, the CPM-hRL/RII β B cAMP biosensor (201325.78.E5) of X/Y peptide linker length Y=20))/96 orifice plates is used for reagent and 0.15 μ g DNA Reagent (MIRUS) carries out transfection.Allow the cell grow overnight and measured at second day.

The adjusting of biosensor

After the transfection about 1 day, from incubator, take out cell and balance to room temperature.10 μ l aliquots containigs of 600 μ MEnduRen viable cell substrates (Promega) add altogether in the 100 μ l cell cultures, to produce the final concentration of about 60 μ M coelentrazine.Cell is incubation at least 15 minutes at room temperature subsequently.After at room temperature 15 minutes, luminous baseline measures uses 96 holes Veritas photometer (Turner) to measure with 0.5 second/hole.Cell uses 0.025 μ M-250 μ M Fu Sikelin (Sigma) to induce subsequently, or do not induce (0.1% DMSO, Sigma), and luminous about 30 minutes (Figure 19) of continuously measured.Sample is measured in the group of 5 repetition/Fu Sikelin concentration.EC ₅₀S uses GraphPad Prism for Windows, and edition 4 calculates.

The result

After stimulating with Fu Sikelin, the light output increase of the DNA cells transfected of use by oneself, described dna encoding has that (X=4, the CPM-hRL/RII β B cAMP biosensor (201325.78.E5) of X/Y peptide linker Y=20) is (Figure 19).The maximum horizontal inductive light output ratio untreated cell of Fu Sikelin is high 3.6 times.In addition, the EC of Fu Sikelin reaction ₅₀Be 0.059 μ M (Figure 19).

Embodiment XI

Utilize circular permutation Lampyridea luciferase and from cGMP activatory protein kinase (GKI-B) or the cGMP of the B structural domain of human phosphodiester enzyme 2A (PDE2A) is biological passes Sensor

CGMP is the important cell second messenger with various physiological functions, particularly in cardiovascular and neural system.A series of cGMP transmitters are prepared by circular permutation Lampyridea luciferase and cGMP binding domains are merged.

A. coding have (X=4, Y=4) and (X=10, Y=10) peptide of individual amino-acid residue connects The plasmid of the CPM-FF Luc/GKI-B fusion rotein of head is synthetic

Have in order to synthesize (X=4, the Y=4) construct of joint length, primer 5 '-AAAAAACTCGAGCGGATTAAAAAGCGTTCCAACATTCCAG-3 ' (SEQ ID NO:106; BFB151) and 5 '-AAAAAAGAGCTCCCAGACAGCTTCAGGTTGGCGAAG-3 ' (SEQ IDNO:107; BFB163) be used to increase people GKI-B DNA (Origene, catalogue #TC116252; Genbank Acc#NM_006258), and for example corresponding residue 231-350 (pBFB164, pBFB165) or 231-373 (pBFB171, DNA pBFB172).Resulting product digests with the XhoI/SacI restriction enzyme, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with XhoI/SacI.

Have in order to synthesize (X=10, the Y=10) construct of joint length, primer 5 '-AAAAAATCCGGATTAAAAAGCGTTCCAACATTCCAG-3 ' (SEQ IDNO:108; BFB153) and 5 '-AAAAAAAGGCCTGACAGCTTCAGGTTGGCGAAG-3 ' (SEQ IDNO:109; BFB164) be used to increase people GKI-B DNA (Origene, catalogue #TC116252; Genbank Acc#NM_006258).Resulting product digests with the BspEI/StuI restriction enzyme, and is connected in the parent CPM-FF Luc expression plasmid (pBFB8) that digests with BspEI/ZraI.

B. have (X=4, Y=4) and (X=10, Y=10) the X/Y joint length of individual amino-acid residue The function of CPM-FF Luc/GKI-B fusion rotein characterize

Use

T7 link coupled reticulocyte lysate system (Promega Corp.), after expression, measure and have (X=4, Y=4) and (X=10, Y=10) the CPM-FF Luc/GKI-B fusion rotein of the X/Y joint length of amino-acid residue 100 μ M cGMP exist and not in the presence of luciferase activity.In brief, following component is assembled according to the scheme of manufacturer recommendation:

The 400ng plasmid DNA

10 μ L rabbit reticulocyte extracts

0.8 μ L TNT reaction buffer

0.4 μ L T7 polysaccharase

0.4 μ L aminoacid mixture

0.4μL rRNasin

DH ₂O to 20 μ L cumulative volume

After 1 hour, fusion rotein separately carries out incubation in the existence of 100 μ M cGMP or not in 30 ℃ of incubations, and this incubation is by making 9 μ L

Reaction and 1 μ L 1mM cGMP stoste or dH ₂The O combination.At room temperature behind incubation 〉=10 minute, 1 μ L sample is added 100 μ L luciferase assay reagent (LAR; Promega Corp.) solution+/-100 μ M cGMP (90 μ L LAR+10 μ L 1mM cGMP stoste or dH ₂O) in.Luminous use Veritas microtiter plate photometer (Turner Biosystems; Program Bright-Glo) measures.In the presence of 100 μ M cGMP, have that (X=4, Y=4) the CPM-FF Luc/GKI-B fusion rotein (pBFB171) of joint length demonstrates 2 times of increases of luciferase activity.In addition, in the presence of 100 μ M cGMP, have that (X=10, Y=10) the CPM-FFLuc/GKI-B fusion rotein (pBFB172) of joint length demonstrates 1.5 times of increases of luciferase activity.

Table 2

pBFB	Splice combinations	Use the RLU of 100 μ McGMP	Do not use the RLU of cGMP
				pBFB 171	(X＝4，Y＝4)	247,801	497,938
pBFB 172	(X＝10，Y＝10)	1,148,496	1,707,449

C. CPM-FF Luc/ human phosphodiester enzyme 2A (PDE2A encodes; Genbank NM 002599; Amino-acid residue 416-549) synthesizing of plasmid

Dna sequence dna that coding has the circular permutation Lampyridea luciferase construct of the through engineering approaches N on residue 234 and 233 and C-terminal respectively and the sequence of coding people PDE2A are merged, and described people PDE2A has with respect to the different protein folding of RII β B structural domain [Met-(Luc2.0234-544)-(joint X)-(people PDE2A416-549)-(joint Y)-(Luc2.04-233)-Val].The extended familys that belong to the small molecules bonding unit that is called the GAF structural domain from the cGMP binding domains of people PDE2A.Preparation has (pBFB167; X=4, Y=4) (pBFB168; X=10 is Y=10) with (pBFB169; X=20, Y=20) construct (Figure 21) of the X/Y joint length of individual amino-acid residue.In the presence of 100 μ M cGMP, for having (pBFB168; X=10 is Y=10) with (pBFB169; X=20, the Y=20) construct of individual amino acid joint uses T7 link coupled reticulocyte lysate system behind vivoexpression, identify luciferase activity based on the biosensor of PDE2A and have 2 and 11 times respectively and induce (Figure 22).In addition, in the experiment of using T7 link coupled reticulocyte lysate system separating after the expression, find that these biosensors are dose-dependentlys via the activation of cGMP, and be (pBFB169 optionally cAMP; Figure 23).

Therefore, these cGMP transmitters are used for the variation in vitro detection cGMP concentration, and these biosensors come in handy for the variation that detects the cGMP concentration in the viable cell, are used for using or being used for full animal imaging research in cell culture experiments.

Embodiment XII

Luciferase calcium biosensor

The calcium biosensor is prepared by the sequence that makes coding circular permutation Lampyridea luciferase combines the protein domain of calcium with coding sequence fusion, and described Lampyridea luciferase has the N and the C-terminal of the through engineering approaches on residue 234 and 233 respectively.One type calcium biosensor utilization quick chicken skeletal troponin C (TnC) (amino acid/11 5-163; N109D, D111N, N145D, D147N; Genbank NM_205450) mutant of [Met-(Luc2.0234-544)-(joint X)-(TnC)-(joint Y)-(Luc2.04-233)-Val], second type calcium biosensor utilizes people's calmodulin (CaM) (amino acid 5-148; Genbank BC005137) [Met-Luc+ (234-544)-(joint X)-people's calmodulin (5-148)-(joint Y)-Luc+ (4-233)].

CPM-FF Luc/TnC and CPM-FF Luc/CaM construct with variable X/Y peptide linker length use T7 link coupled reticulocyte lysate system at external expression the (pBFB225, pBFB226, pBFB227, pBFB7; Figure 24).Reaction replenishes 10mM CaCl subsequently ₂Or 10mM EDTA adds 2.5mM EGTA.For having (X=8, Y=8; PBFB7) the visible maximum reaction of CPM-FF Luc/CaM biosensor, wherein X=LEGSGGGG (SEQ ID NO:306) and Y=GGGGSGPW (SEQ ID NO:307), luciferase activity reduces above 60 times in the presence of calcium.(pBFB227) visible similar reaction is although magnitude is lower for pBFB225, pBFB226 for the CPM-FF Luc/CaM construct with different X/Y peptide linker length.Do not observe reaction (pBFB8 and pBFB22 for contrast construct or for wild-type Lampyridea luciferase with 42 amino acid whose joints at random; Figure 25).

These biosensors come in handy for the variation that detects calcium concn in external and viable cell.

Embodiment XIII

Use the cAMP biosensor of a plurality of decorating sites in the Lampyridea luciferase

Other decorating site, for example circular permutation can be used to develop Lampyridea luciferase biosensor, for example the cAMP biosensor.Hereinbefore, the cAMP biosensor uses and has primary structure Met-(Luc2.0 residue 234-544)-GSSGGSGGSGGG-RII β B-(Luc2.0 residue 4-233)-Val (SEQ ID NO:184; RII β B is the B cAMP binding domains from people PKA modulability II β type structural domain amino acid 266-414) the circular permutation mutant of Lampyridea luciferase be prepared.Similar construct uses the Lampyridea luciferase mutant of circular permutation on other residue to be prepared.Generally speaking, 23 kinds of fusion rotein of the following type of encoding independently construct: Met-(Luc2.0 residue X-544)-GSSGGSGGSGGG-RII β B-(Luc2.0 residue 4-Y)-Val (the corresponding SEQ ID of GSSGGSGGSGGG NO:121 have been tested; Figure 26 has listed the X/Y value about various constructs).For in these constructs each, get rid of construct with the circular permutation on residue 255, for circular permutation, use PDB file 1LCI ( Http: //www.rcsb.org/pdb/home/home.do), be chosen in that for example βZhe Die or α spiral are site in the solvent exposed surface ring on border by secondary structure.The solvent exposed surface ring is as decorating site, for example circular permutation than be embedded in protein core in the heart the site or the site that participates in α or beta structure may more comply with.This is by supported for the active shortage of the construct visible with the circular permutation on 255, and wherein Tyr255 is the component that is embedded in protein core α spiral in the heart.This construct set is illustrated in visible great majority in the 1LCI crystalline structure but is not that all surfaces is folding.

After using TNT T7 link coupled reticulocyte lysate system expression, many different circular permutation sites have been identified, wherein in the presence of 100 μ M cAMP, surpass the luminescence activity of photometric background detection level and induce multiple to surpass 2 times of (CPM sites: 37,47,75,83,107,144,160,188,225,233,242,268,308,358,377,403 and 490).In addition, identified that the multiple of inducing of luminescence activity surpasses the construct of CPM233, wherein in this experiment maximum multiple activation number above 200 times.Select the DNA of construct to be transferred to the mammalian expression vector (pF9A that comprises the CMV promotor coding; PromegaCorp.) in.Construct is: pBFB317 (CPM site 268), pBFB318 (CPM site 358), pBFB319 (CPM site 47), pBFB321 (CPM site 225), pBFB322 (CPM site 233), pBFB325 (CPM site 308), pBFB326 (CPM site 377), pBFB327 (CPM site 403), pBFB328 (CPM site 75) and pBFB329 (CPM site 83) (about X, Y value referring to Figure 26).Behind the DNA transient transfection with coding various Met-(Luc2.0 residue X-544)-GSSGGSGGSGGG-RII β B-(Luc2.0 residue 4-Y)-Val (the corresponding SEQ ID of GSSGGSGGSGGG NO:121) construct, the HEK293 cell is handled with activation of endogenous sexual gland thuja acid cyclase with 50 μ M Fu Sikelin.Behind the incubation 16 minutes, measurement is luminous from viable cell colony.As expection, various constructs serve as the cAMP biosensor in viable cell.What is interesting is, show that in cell the highest construct of inducing multiple is not to have the highest same construct (comparison diagram 27-28) of inducing multiple external.

Embodiment XIV

Non-conversion sea pansy luciferase cAMP biosensor

As described herein, circular permutation sea pansy luciferase construct can be used as biosensor.Prepared and have the non-conversion sea pansy luciferase construct of insertion to the RII β B in the site of modifying tolerance, described site is for example between the residue 91/92,223/224 or 229/230.Construct produces as mentioned above.They are: hRL (1-91)-4 amino acid whose peptide linker-RII β B-4 amino acid whose peptide linker-hRL (92-311) (201360.17.A3), hRL (1-91)-4 amino acid whose peptide linker-RII β B-20 amino acid whose peptide linker-hRL992-311) (201360.17.A12), hRL (1-91)-10 amino acid whose peptide linker-RII β B-4 amino acid whose joint-hRL (92-311) (201360.17.D7), hRL (1-91)-42 amino acid whose peptide linker-hRL (92-311) (201325.165.A2), hRL (1-223)-4 amino acid whose peptide linker-RII β B-4 amino acid whose joint-hRL (224-311) (201360.24.A1), hRL (1-223)-4 amino acid whose peptide linker-RII β B-20 amino acid whose joint-hRL (224-311) (201360.24.A10), hRL (1-223)-10 amino acid whose peptide linker-B-4 amino acid joint-hRL of RII β (224-311) (201360.24.C5), hRL (1-223)-10 amino acid whose peptide linker-RII β B-20 amino acid whose joint-hRL (224-311) (201360.24.E11), hRL (1-223)-42 amino acid whose peptide linker-hRL (224-311) (201325.177.B7), hRL (1-229)-4 amino acid whose peptide linker-RII β B-4 amino acid whose joint-hRL (230-311) (201360.19.E9), hRL (1-229)-4 amino acid whose peptide linker-RII β B-20 amino acid whose joint-hRL (230-311) (201360.54.A1), hRL (1-229)-42 amino acid whose peptide linker-hRL (230-311) be (Figure 29) (201325.165.C5).

Protein uses TNT T7 link coupled Fructus Hordei Germinatus lysate system to be expressed by construct, makes 17 μ L TNT reaction and is supplemented with 3.4 μ L1mM cAMP stoste or dH ₂The 17 μ L 300mM HEPES/200mM thiocarbamides (pH about 7.5) of O mix; Permission was reacted at room temperature incubation about 10 minutes.Every kind of sample of 10 μ L is added in 96 orifice bores in triplicate, and luminous use 100 μ L sea pansy luciferase assay reagent are measured on the Glomax photometer.

HRL (1-91)-joint-RII β B-joint-hRL (92-311) protein is induced about 12-23 doubly, hRL (1-223)-joint RII β B-joint-hRL (224-311) protein is not induced, and hRL (1-229)-joint-RII β B-(230-311) protein is induced about 2-9 doubly.None is induced in the construct of 42 amino acid whose joints, and (Figure 30) do not induced in total length sea pansy luciferase construct (201325.50.A7) and " no DNA " contrast yet.

Embodiment XV

Light is exported and is induced the multiple conduct about the cAMP sensing based on CPM-hRL91Luc/RII β B The function of the X/Y peptide linker length of device changes

Having produced the construct that coding has variable X/Y peptide linker length based on the cAMP transmitter of CPM-hRL91Luc/RII β B.Protein uses TNT T7 link coupled Fructus Hordei Germinatus lysate system to be expressed by construct, makes 17 μ L TNT reaction and is supplemented with 3.4 μ L1mM cAMP stoste or dH ₂The 17 μ L 300mM HEPES/200mM thiocarbamides (pH about 7.5) of O mix; Permission was reacted at room temperature incubation about 10 minutes.Every kind of sample of 10 μ L is added in 96 orifice bores in triplicate, and luminous use 100 μ L sea pansy luciferase assay reagent are measured on the Glomax photometer.As shown in Figure 32, light is exported and is induced multiple to become with joint length.Inducing multiple is about 87-331.(Figure 32) do not induced in the construct of 42 amino acid whose joints, total length sea pansy luciferase construct and " no DNA " contrast.

Embodiment XVI

Utilize circular permutation sea pansy luciferase and from the B structural domain of PKA modulability I α type subunit Or the cAMP biosensor of GAF structural domain

Coding is connected to coding CPM-hRL91 Luc/RI α B fusions [hRL (92-311)-joint X-humanRI α (residue 245-381)-joint Y-hRL (1-91)] from the DNA of the B structural domain (RI α B) of people PKA modulability I α type subunit; (X=4, Y=20; PBFB210), (X=4, Y=4; PBFB211), (X=10, Y=10; PBFB212) and (X=20, Y=20; PBFB213) in the expression vector (Figure 33).Protein uses TNT T7 link coupled Fructus Hordei Germinatus lysate system to be expressed by construct, makes 17 μ L TNT reaction and is supplemented with 3.4 μ L 1mMcAMP stoste or dH ₂The 17 μ L 300mM HEPES/200mM thiocarbamides (pH about 7.5) of O mix; Permission was reacted at room temperature incubation about 10 minutes.Every kind of sample of 10 μ L is added in 96 orifice bores in triplicate, and luminous use 100 μ L sea pansy luciferase assay reagent are measured on the Glomax photometer.As shown in Figure 34, light is exported and is induced multiple to become with joint length.Inducing multiple is about 2.8-6.8.(Figure 34) do not induced in the construct of 42 amino acid whose joints (201325.15.A1), total length sea pansy luciferase construct (201325.50.A7) and " no DNA " contrast.

Use circular permutation sea pansy luciferase (hRL) and GAF structural domain to make up the cAMP biosensor of other type.The plasmid DNA construction body is encoded following fusion rotein: Met-(hRL92-311)-GSSGGSGGSGGGSGGSGGSG-(from the GAF A structural domain of Bruce trypanosome (Trypanosoma brucei) PDE; Genbank AF192755 amino acid 241-375)-GSGGSGGSGGTSGGSGGSSG-A-(hRL3-91)-Val (SEQID NO:185) [clone pBFB232].After using T7 link coupled reticulocyte lysate system expression, luminescence activity is measured in the existence of external source cAMP or not.In the presence of cAMP, the activity of measurement is 7595 RLU; Under the situation that does not have cAMP, the activity of measurement is 298 RLU (about 25 times of variations).These results point out that other structural domain can use in the generation at biosensor in CPM hRL construct.Such reagent can allow to monitor the variation of the cAMP concentration in the viable cell, and it can also provide the different advantages that surpass existing cAMP biosensor based on FRET in the sort of mensuration form.In addition, because the GAF structural domain is the folding of high conservative in the character of being responsible in conjunction with the molecule of broad range, so the CPM hRL biosensor of type can use this folding being prepared in addition.

Embodiment XVII

Use the cAMP biosensor of a plurality of decorating sites in the sea pansy luciferase

CAMP biosensor with circular permutation mutant of sea pansy luciferase is combining the increase that the back shows luminescence activity with cAMP, the circular permutation mutant of described sea pansy luciferase has primary structure Met-(hRL 92-311)-GSTG-RII β B-GSGGSGGSGGTSGGSGGSSG (hRL 2-91)-Val (SEQ ID NO:186; RII β B is the B cAMP binding domains from people PKA modulability II β type structural domain amino acid 266-414).Similar construct, promptly " division " protein or circular permutation protein can use the sea pansy luciferase mutant of modifying on other site to produce.Generally speaking, 14 kinds of fusion rotein of the following type of encoding independently circular permutation construct: Met-(hRL X-311)-GSTG-RII β B-GSGGSGGSGGTSGGSGGSSG (hRL 2-Y)-Val (the corresponding SEQ ID of GSTG NO:122 have been tested; The corresponding SEQ ID of GSGGSGGSGGTSGGSGGSSG NO:123).Following table provides the peptide X/Y value of 14 kinds of constructs.

Table 3

The CPM site	The X value	The Y value	Clone ID
					31	32	30	pBFB276
42	43	41	pBFB277
				69	70	68	pBFB278
111	112	110	pBFB279
				151	152	150	pBFB280
169	170	168	pBFB281
				193	194	192	pBFB282
208	209	207	pBFB283 *
				251	252	250	pBFB284
259	260	258	pBFB285
				274	275	273	pBFB286
91	92	91	PBFB287 and 201325.44.H6

223	224	223	201325.33.C9
				229	230	229	201325.86.B1

^*Annotate: for construct pBFB283, last amino acid on C-terminal is PFSEFKPD (SEQ ID NO:120) rather than PFK, and does not insert Val before terminator codon.

For whole (except 4 kinds) in these constructs, use the homology model of sea pansy luciferase, use 1BN6 (Rhod species) (Rhodococcus sp.) and 2DHD (xanthobacter autotrophicus) (Xanthobacter autotrophicus) haloalkane dehalogenation enzyme crystal structure to be used for circular permutation as the site that template is chosen in the solvent exposed surface ring.The solvent exposed surface ring as decorating site for example circular permutation than be embedded in protein core in the heart the site or participate in α or the site of beta structure may more be complied with.This hypothesis is by supported for the active shortage of the Lampyridea luciferase construct visible with the circular permutation on 255, and wherein Tyr255 is the component that is embedded in protein core α spiral in the heart.The set of this construct be illustrated in the homology model structure visible some but be not that all surfaces is folding.Select 4 CPM sites based on previous report (people such as Kaihara, 2003, people such as Remy, 2005 and people such as Paulmurugan, 2003): 91,111,223 and 229.Construct uses TNT T7 link coupled reticulocyte lysate system or TnT T7 link coupled malt extract system to express, and at external the test (Figure 35 and 36).

The result points out that many different circular permutation sites can be used to produce for example cAMP biosensor of biosensor.Identified the alternative site of circular permutation, wherein do not induce/the inductive activity level surpasses the initial construct (CPM 91) with the circular permutation on 91.In addition, the multiple of inducing of having identified luminescence activity surpasses the construct of CPM 91.In addition, because the utmost point low-solubility of CPM 91 when at expression in escherichia coli, will be tested with this construct and compare the deliquescent increase of other construct.The solvability that increases can promote for example exploitation of cAMP detection reagent of external biological transmitter.

The result points out that also many sites can't be used for circular permutation.All sites between the residue 169-274 has low inducing and not derivative activity, and the multiple of inducing of luminescence activity is about 2 times or lower.

Construct is at carrier main chain (pF5A; Promega Corp.) design in, it allows vivoexpression (T7 promotor) and Mammals to express (CMV promotor).With coding various Met-(hRL residue X-311)-GSTG-RII β B-GSGGSGGSGGTSGGSGGSSG-(hRL residue 2-Y)-Val (the corresponding SEQ ID of GSTG NO:122; The corresponding SEQ ID of GSGGSGGSGGTSGGSGGSSG NO:123) behind the DNA transient transfection of construct (pBFB276, pBFB277, pBFB278, pBFB279, pBFB280, pBFB287), the HEK293 cell is handled with activation of endogenous sexual gland thuja acid cyclase with 100 μ M Fu Sikelin.Behind the incubation 14 minutes, measurement is luminous from viable cell colony.As expection, various constructs serve as the cAMP biosensor in viable cell.What is interesting is that construct CPM 31 is presented at the highest external multiple of inducing, yet this not like this in cell.Yet generally speaking, light is exported and is induced multiple to show in vitro and in vivo similarly trend (Figure 37).

Embodiment XVIII

Prepare many different genetic constructs, use bent firefly luciferase (Gluc) (the Genbank AAG54095 that lacks 17 amino acid whose N-terminal peptides that serve as secretion signal with test; Amino acid/11 8-185) possibility of preparation biosensor.When being measured by viable cell, with respect to other luciferases, the bent firefly luciferase that contains or do not contain the N-terminal signal peptide has been reported the bigger light intensity of generation (people such as Tannous, 2005; People such as Remy, 2006).In addition, the fragment of Gluc has used in the complementary action of protein system that (Gluc divides on amino-acid residue 110; People such as Remy, 2006); Therefore, possible Gluc also will comply with the circular permutation on this site or other sites.

In order to prepare Gluc cAMP biosensor, the prediction of secondary protein structure is used to select each site of Gluc circular permutation: Met-(Gluc A-185)-(joint X)-(people RII β B Genbank BC075800 amino-acid residue 266-414)-(joint Y)-(Gluc 18-B).

Table 4

The CPM site	The A residue	The B residue	Joint length X	Joint length Y	pBFB#
							100	101	99	4	4	pBFB290
100	101	99	10	10	pBFB291
						100	101	99	20	20	pBFB292
110	111	109	4	4	pBFB293
						110	111	109	10	10	pBFB294
110	111	109	20	20	pBFB295
						48	49	47	4	4	pBFB296
48	49	47	10	10	pBFB297
						48	49	47	20	20	pBFB298
68	69	67	4	4	pBFB299
						68	69	67	10	10	pBFB300
68	69	67	20	20	pBFB301
						84	85	83	4	4	pBFB302
84	85	83	10	10	pBFB303
						84	85	83	20	20	pBFB304
91	92	90	4	4	pBFB305
						91	92	90	10	10	pBFB306
91	92	90	20	20	pBFB307
						114	115	113	4	4	pBFB308
114	115	113	10	10	pBFB309
						114	115	113	20	20	pBFB310
126	127	125	4	4	pBFB311
						126	127	125	10	10	pBFB312
126	127	125	20	20	pBFB313
						162	163	161	4	4	pBFB314
162	163	161	10	10	pBFB315
						162	163	161	20	20	pBFB316

Wherein the various terminal combination has sequence:

Table 5

Joint length	Sequence
		(X＝4，Y＝4)	GSTG-RIIβB-GSSG(SEQ ID NO：187)
(X＝10，Y＝10)	GSSGGSGGSG-RIIβB-GSGGSGGSSG(SEQ ID NO：188)
		(X＝20，Y＝20)	GSSGGSGGSGGGSGGSGGSG-RIIβB-GSGGSGGSGGTSGGSGGSSG(SEQ ID NO：189)

The site useful for Gluc cAMP can replace, to produce the biosensor that uses this circular permutation site to be used for other molecules.In addition, complying with the site of circular permutation in a kind of oar foot animal luciferase may be for example useful in the luciferase from Metridia longa at other oars foot animal luciferases.

Embodiment XIX

Be used for to relate to the direct detection of intracellular signal transduction incident based on the GPCR method for measuring of cell.The most successful comprise use fluorescence dye or aequorin are used for monitoring in real time the method for intracellular Ca2+.Yet similar techniques lacks the dynamic (dynamical) detection of cAMP in the cell.Circular permutation Lampyridea luciferase with allosteric RII β B cAMP binding domains of protein kinase A is can send and the proportional luminous transmitter of cAMP concentration.Use viable cell, the zero step GPCR of this transmitter to measure the variation that the clone detection of dynamic cAMP concentration of stable or transient transfection is used in permission.In addition, can develop single step homology mensuration form is used at vitro detection cAMP (Figure 38).

The ORF that comes leisure to be called the pBFB 135 under the biosensor classification of " CPM-FF Luc/RII β B " is used to produce instantaneous and stable cell lines described below.These clones are called " CP234-Luc/RIIB ", " cAMP LucSensor ", " LucSensor " and " FF cAMP transmitter ".

The HEK293 cell of stably express CP234-Luc/RIIB (ORF derives from pBFB135) is resuspended in the perfect medium, and mixes with 5mg/mL luciferin-EF.Cell is with 1x 10 ⁵Cells/well is bed board in 96 orifice plates, and balance was to room temperature 1.5 hours.After the Fu Sikelin stimulation, the luminous GloMax that in the time of 15 minutes, uses ^TMPhotometer is measured.The result shows that this mensuration produces the EC of 0.36 μ M for Fu Sikelin ₅₀Value (Figure 38).

Measure for Z ', with 2 x 10 ⁴Individual cells/well is distributed in 384 orifice plates and uses similar scheme to carry out balance.Half plate is induced with 20 μ M Fu Sikelin, and second half maintenance is not induced.The luminous TECAN GENios Pro that after inducing, uses ^TMPhotometer was caught 15 minutes.Induce multiple be 6.1 and Z ' be 0.83.Because have the better quality that the mensuration that surpasses 0.5 Z ' is considered as high throughput screening (HTS), so comply with HTS based on the mensuration of cAMP biosensor.

The HEK293 cell of stably express d1 dopamine receptor carries out transient transfection with the plasmid DNA of coding CP234-Luc/RIIB or R361K mutant (sudden change in the cAMP binding domains) (ORFs derives from pBFB135 and pBFB147 respectively).Cell carries out bed board and carries out balance with luciferin-EF as mentioned above, and will add (10 μ M) in each hole from the compound of LOPAC library (plate 6).Behind the incubation 50 minutes, plate is at TECAN GENiosPro ^TMCarry out reading on the photometer.Also use the luciferase reporter gene to measure hitting that (CRE response element) identify with red display (Figure 39).The great majority of being identified by the cAMP biosensor assay hit with relevant by hitting of CRE-Luc reporter mensuration evaluation, have confirmed the biological cognation that cAMP biosensor GPCR measures.

The same bed board of cell and carry out balance with luciferin-EF, then after compound adds, luminous use GloMax ^TMPhotometer was measured in the time of 40 minutes.The EC that uses the cAMP biosensor assay to produce ₅₀And IC ₅₀The pharmacokinetic parameter of value with use those of additive method report related good in the literature, reconfirm the biological cognation (Figure 40) of cAMP biosensor GPCR mensuration.

Reaction is under differing temps (Figure 42) and test in the cell of various agonists and antagonist (Figure 43) incubation also.The HEK293 cell of expressing cAMP LucSensor (ORF derives from pBFB135) and d1 dopamine receptor, contacts with agonist or antagonist room temperature or 37 ℃ of following incubations 1.5 hours then with luciferin.Be reflected on the photometer and measure.When cell at physiological condition for example 37C and CO ₂Under when carrying out incubation, have quicker and dynamic reaction to compound.Those that cAMP kinetics is expected in the result under 37 ℃ is being similar to for cell in nature.At room temperature, exist for the slower reaction of lower dynamics range, this may be useful for extensive screening.

Figure 44 be presented at cAMP LucSensor stable transfection and with the cell of the Dopamine HCL of different amounts contact in about inducing the time-histories of multiple.The result shows that this system allows to monitor in real time the cAMP kinetics in the viable cell.In addition, the result among Figure 45 shows that this system allows the assessment compound to render a service, and this is consistent relatively on different time points.Figure 46 provides the effectiveness ordering (EC about various agonists ₅₀) and the result, and show that some compound is a partial agonist.Render a service (IC about antagonist ₅₀) data presentation in Figure 47.

CAMP LucSensor can also be used for measuring the adjusting of the GPCR (endogenous GPCR) that has expressed at host cell.Example uses expresses beta 2-adrenergic receptor and with the HEK 293 cells demonstration of cAMP LucSensor stable transfection.After the similar scheme of describing for Dopamine Receptors, Figure 48-49 shows the effectiveness ordering of various agonists and antagonist respectively.

Carry out the comparison that 3 kinds of noclilucence GPCR measure.About the results are shown among Figure 50 of those mensuration and agonist.About the results are shown among Figure 51 of 3 kinds of bioluminescence assays using antagonist.Ordering about test compounds is identical in all 3 kinds of mensuration.

The luminous increase of cAMP LucSensor can be the result of the efficient that increases the conformational change from open to the outside world to " closure " in the presence of cAMP.

The HEK293 cell of stably express d1 dopamine receptor is also with the CPM-hRL Luc/RII β B X=4 that is coded under the control of CMV promotor (201325.78.E5) or TK promotor (201325.44.H6), the plasmid DNA of Y=20 is carried out transient transfection, stimulates with Fu Sikelin, SKF38393 or Dopamine HCL then.The same CPM-hRL Luc that tests wild-type sea pansy luciferase and do not contain RII β B structural domain, and do not show the specific reaction (data not shown) that cAMP is regulated.Cell is used in the T75 flask

Reagent (MIRUS) carries out transfection, wherein uses 60 μ L

Reagent and 30 μ gDNA/ flasks allow overnight growth and measured at second day.After the transfection about 1 day, from incubator, take out cell and be subjected to trypsin treatment, counting, and with 10, the DMEM/F12 with 10% FBS and 60 μ M EnduRen viable cell substrates of 000 cells/well bed board in 96 orifice plates (the HEPES damping fluid, Invitrogen) in.EnduRen viable cell substrate (Promega) reconstruct and add in the perfect medium of preheating final concentration in 100 μ L DMSO to 60 μ M.Cell is subsequently 37 ℃ of following incubations at least 1 hour and be cooled to room temperature subsequently.After at room temperature 15 minutes, luminous baseline measures uses 96 hole GloMax ^TMPhotometer is measured with 0.5 second/hole.Cell is used in Fu Sikelin (Sigma), the SKF38393 (Sigma) for preparing in the perfect medium subsequently, the 10x stoste of Dopamine HCL (Sigma) is induced, or does not induce (0.1% DMSO (Sigma)), and luminous about 30 minutes of continuously measured.Sample is measured in the group of 4 repetition/Fu Sikelin, Dopamine HCL or SKF38393 concentration.EC ₅₀Data representation was induced back 15 minutes, and used GraphPad Prism for Windows, and edition 4 calculates.

Be similar to CPM-FF Luc/RII β B biosensor, use CPM-hRL Luc/RII β BX=4, the EC that Y=20 biosensor (201325.44.H6 and 201325.78.E5) produces ₅₀Value with use those of additive method report related good in the literature, reconfirm the biological cognation (Figure 52 A-D) of cAMP biosensor GPCR mensuration.

Embodiment XX

Use the change that detects cAMP concentration in the CPM-hRL Luc/RII β B cAMP biosensor cell Change

Cell cultures

Cell in having the 2mlDMEM/F12 of HEPES damping fluid (Invitrogen) and 10% FBS 37 ℃ with 5% CO ₂In 6 orifice plates, cultivate down.

Plasmid

3 kinds of constructs describing among the embodiment XVII are used to detect interior variation of cell of cAMP concentration.The construct that uses is: pBFB277, pBFB279 and pBFB287.Stably express CPM91-hRL/RII β B (ORF derives from the HEK293 cell of 201325.44.H6) also uses in these experiments.

Transfection

The HEK293 cell uses 6 μ l

The hole of reagent and 2 μ g DNA (pBFB277, pBFB279 and pBFB287)/6 orifice plates is used

Reagent (MIRUS) carries out transfection.Allow the cell grow overnight and measured at second day.

The adjusting of biosensor

After the transfection about 1 day, cell was subjected to trypsin treatment, be resuspended among the fresh DMEM/F12 that contains HEPES damping fluid (Invitrogen) and 1% FBS, and with about 10,000 cells/well bed board in 96 orifice plates.Alternately, the HEK293 clone of stably express CP91-hRL/RII β B is with about 10,000 cells/well bed board in 96 orifice plates.The 10 μ L aliquots containigs of 600 μ M EnduRen are added altogether in the 100 μ L cell cultures, to produce the final concentration of about 5.5 μ M EnduRen.Cell is subsequently at 37C and 5% CO ₂Under carry out incubation.After 5 hours, plate takes out and allows to be cooled to room temperature at least 20 minutes from incubator.After 20 minutes, luminous baseline measures uses 96 hole Veritas photometer (Turner Biosystems; 0.5 the integrating time in second/hole) measure.Cell uses 10 μ M Racemic isoproterenols (CalBiochem), 50mM Fu Sikelin (Sigma) to induce subsequently, or do not induce (0.1% DMSO, Sigma), and luminous about 30 minutes of continuously measured.After 30 minutes, 10mM Proprasylyte (Sigma) adding has been used in the different third suprarenal gland inductive cell, and 0.1% DMSO has been added in the every other sample.Luminous continuously measured in ensuing 30 minutes subsequently.The final additive of 50 μ M Fu Sikelin is added in different third suprarenal gland/Proprasylyte sample, and 0.1% DMSO is added in the every other sample.Luminous in ensuing continuously measured half an hour subsequently.Sample is measured in 4-6 multiple group.The 10x stoste of Racemic isoproterenol, Proprasylyte, Fu Sikelin and DMSO is prepared in the DMEM/F12 that contains HEPES damping fluid (Invitrogen) and 1% FBS.

The result

Variation for the IC of measuring cAMP, HEK 293 cells are with 3 kinds of CPM-hRL Luc/RII β B (X=4, Y=20) construct (circular permutation on the different positions in the sea pansy luciferase) carries out transient transfection, handle with compound subsequently, described compound is known to increase cAMP concentration (Racemic isoproterenol in the cell by the GPCR activation, be the moving agent of a kind of beta-adrenergic receptor kinase 1), suppress to reduce cAMP concentration (Proprasylyte, promptly a kind of B-adrenergic receptor antagonist) in the cell by GPCR, or by cAMP concentration (Fu Sikelin) in the activation increase cell of adenylate cyclase.Racemic isoproterenol and Fu Sikelin handle the light output that makes separately from transfectional cell increases about 2 times, the increase (Figure 53) in the reflection cell in the cAMP concentration.In addition, the of short duration reaction of the variation of cAMP concentration by use Racemic isoproterenol, is used Proprasylyte subsequently, handle cell observation with Fu Sikelin subsequently and arrive (Figure 53).Use the detection of the cAMP adjusting of sea pansy luciferase biosensor also in the HEK293 cell of stably express CPM91-hRL/RII β B, to be confirmed.About 5 times of increases (Figure 53) of the light output that these data presentation response Racemic isoproterenols and Fu Sikelin handle.Be similar to the cell of transient transfection, the of short duration reaction of the variation of cAMP concentration by use Racemic isoproterenol, is used Proprasylyte subsequently, handle cell observation with Fu Sikelin subsequently and arrive (Figure 53).

Embodiment XXI

Non-conversion Lampyridea luciferase cAMP biosensor

Prepared and have the various non-conversion Lampyridea luciferase construct of direct insertion to the RII β B in the site of modifying tolerance, described site is for example between the residue 233/234,355/359,82/83 and 307/308.With the coding following fusion rotein dna clone in carrier pF9A:

Table 6

pBFB403	Met-(Luc2.04-233)-GSTG-RIIβB-GSSG-(Luc2.0234-544)(SEQ ID NO：172)
		pBFB404	Met-(Luc2.04-233)-GSSGGSGGSG-R2βB-GSGGSGGSSG-(Luc2.0234-544)(SEQ ID NO：173)
pBFB405	Met-(Luc2.04-233)-GSSGGSGGSGGGSGGSGGSG-R2βB-GSGGSGGSGGTSGGSGGSSG-(Luc2.0234-544)(SEQ ID NO：174)
		pBFB406	Met-(Luc2.04-355)-GSTG-RIIβB-GSSG-(Luc2.0359-544)(SEQ ID NO：175)

pBFB407	Met-(Luc2.04-355)-GSSGGSGGSG-R2βB-GSGGSGGSSG-(Luc20359-544)(SEQ ID NO：176)
		pBFB408	Met-(Luc2.04-355)-GSSGGSGGSGGGSGGSGGSG-R2βB-GSGGSGGSGGTSGGSGGSSG-(Luc2.0359-544)(SEQ ID NO：177)
pBFB409	Met-(Luc2.04-82)-GSTG-RIIβB-GSSG-(Luc2.083-544)(SEQ ID NO：178)
		pBFB410	Met-(Luc2.04-82)-GSSGGSGGSG-R2βB-GSGGSGGSSG-(Luc2.083-544)(SEQ ID NO：179)
pBFB411	Met-(Luc2.04-82)-GSSGGSGGSGGGSGGSGGSG-R2βB-GSGGSGGSGGTSGGSGGSSG-(Luc2.083-544)(SEQ ID NO：180)
		pBFB412	Met-(Luc2.04-307)-GSTG-RIIβB-GSSG-(Luc2.0308-544)(SEQ ID NO：181)
pBFB413	Met-(Luc2.04-307)-GSSGGSGGSG-R2βB-GSGGSGGSSG-(Luc2.0308-544)(SEQ ID NO：182)
		pBFB414	Met-(Luc2.04-307)-GSSGGSGGSGGGSGGSGGSG-R2βB-GSGGSGGSGGTSGGSGGSSG-(Luc2.0308-544)(SEQ ID NO：183)

Luc2.0=is by the North America Lampyridea luciferase (referring to Genbank IDAY738222) of luc2.0 genes encoding; The residue 266-414 (GenbankBC075800) of RII β B=people PKA modulability II β type subunit

Protein uses TnT T7 link coupled reticulocyte lysate system to be expressed by these constructs.After the expression, make 9 μ L TNT reaction and 1 μ L 1mM cAMP stoste or H ₂O mixes, and allows to react at room temperature incubation about 15 minutes.Behind the incubation, 2 μ L solution are distributed in each hole of 96 orifice plates in triplicate.The luminous Glomax photometer that uses after injecting 100 μ L luciferase assay reagent (0.5 second integrating time) is measured.

The result points out that the cAMP biosensor can produce (referring to Figure 54) by RII β B directly being inserted in any one in 4 selected insertion sites.The result also points out the site of circular permutation tolerance is seemed also to tolerate for direct insertion, thereby produces feasible biosensor.

Embodiment XXII

Non-conversion and conversion thorn shrimp luciferase cAMP biosensor

The oxidation of thin angle thorn shrimp (Oplophorus gracilirostris) luciferase (OpLuc) catalysis coelentrazine is to send blue light.The mature form of enzyme is 18.7kD (169aa).Original ORF comprises that representative is used for 27 extra residues of excretory putative signal peptide.The removal of the 27aa signal peptide of supposing causes the about 50 times increase of luciferase activity.Because its small size, OpLuc complies with especially as biosensor or in PCA and uses.

OpLuc is active and stable, and prerequisite is that it is present in the extract or Bacillus coli cells lysate of no TnT cell.Yet it is deactivation immediately behind purifying.Gel-filtration show luciferase (, not being contained in the 35kD protein of finding in the natural biological) at expression in escherichia coli 13.7 and 29kD protein standard between wash-out.The MW of enzyme is 18.7kD.Therefore, seem if express, and do not contain 35kD protein that luciferase is kept as monomer so.It is active that this enzyme keeps under pH7.5-9, and active the beginning under pH9.5 reduces.

Prepared and have various non-conversion thorn shrimp luciferase (OpLuc) construct of direct insertion to the RII β B in the site of modifying tolerance, described site is for example between the residue 50/51 and 84/85.With the coding following fusion rotein dna clone in carrier pF5K:

Table 7

pBFB397	Met-(OpLuc1-50)-GSTG-R2βB-GSSG-(OpLuc51-169)(SEQ ID NO：190)
		pBFB398	Met-(OpLuc1-50)-GSSGGSGGSG-R2βB-GSSGGSGGSG-(OpLuc51-169)(SEQ ID NO：191)
pBFB399	Met-(OpLuc1-50)-GSSGGSGGSGGGSGGSGGSG-R2βB-GSGGSGGSGGTSGGSGGSSG-(OpLuc51-169)(SEQ ID NO：192)
		pBFB400	Met-(OpLuc1-84)-GSTG-R2βB-GSSG-(OpLuc85-169)(SEQ ID NO：193)
pBFB401	Met-(OpLuc1-84)-GSSGGSGGSG-R2βB-GSSGGSGGSG-(OpLuc85-169)(SEQ ID NO：194)
		pBFB402	Met-(OpLuc1-84)-GSSGGSGGSGGGSGGSGGSG-R2βB-GSGGSGGSGGTSGGSGGSSG-(OpLuc85-169)(SEQ ID NO：195)

Residue ' 1 ' in the last table is pointed out first residue (shortage is used for the excretory signal peptide, the residue 28 among the Genbank AB030246) in the protein of mature form; The residue 266-414 (Genbank BC075800) of RII β B=people PKA modulability II β type subunit.

Protein uses TnT T7 link coupled reticulocyte lysate system to be expressed by these constructs.After the expression, make 9 μ L TNT reaction and 1 μ L 1mM cAMP stoste or H ₂O mixes, and allows to react at room temperature incubation about 15 minutes.Behind the incubation, with 10 μ L, 2 * damping fluid (300mM HEPES, pH=8.0, the 200mM thiocarbamide) add in each reaction, and after adding 100 μ L sea pansies mensuration reagent, use Turner 20/20N photometer (1 second integrating time) to measure luminous by the 20 μ L solution that produced.The result lists in following table:

Table 8

pBFB397+	918
		pBFB397-	225
pBFB398+	4,917
		pBFB398-	291
pBFB399+	38,051
		pBFB399-	356
pBFB400+	10,369
		pBFB400-	6,387
pBFB401+	6,124
		pBFB401-	2,304
pBFB402+	62,264
		pBFB402-	8,568
FL Opluc+	25,225,870
		FL Opluc-	23,231,428
No DNA+	120
		No DNA-	116

The expression of the residue 28-169 of FL Opluc=Genbank BC075800; '+'=external source cAMP is added into 50 μ M final concentrations; '-'=do not add external source cAMP.

Other carriers comprise the circular permutation mutant with the thorn shrimp luciferase (OpLuc) of being cloned into the RII β B structural domain in the pF4K-CMV plasmid, so that can express under T7 and the control of CMV promotor.Also prepared and had various circular permutation thorn shrimp luciferase (OpLuc) constructs of insertion the RII β B in the site of modifying tolerance.Referring to Figure 63.Amino-acid residue in the thorn shrimp luciferase of the corresponding mature form of the number in the bracket.The joint of respective length is pointed out in integer " 4 ", " 10 " and " 20 ".Should be understood that Met and Val residue add the N-terminal of luciferase.Therefore, two amino-acid residues are moved in the various splitted position in the circular permutation mutant.For example, the residue 52 and 53 of division mark " 50-51 " (referring to the residue order in the enzyme of natural mature form) in the actual luciferase form of using occurs between the two.

pF4K-CMV-[51-169]-4-RIIβB-4-[1-50]-OpLuc

pF4K-CMV-[51-169]-10-RIIβB-10-[1-50]-OpLuc

pF4K-CMV-[51-169]-20-RIIβB-20-[1-50]-OpLuc

pF4K-CMV-[85-169]-4-RIIβB-4-[1-84]-OpLuc

pF4K-CMV-[85-169]-10-RIIβB-10-[1-84]-OpLuc

pF4K-CMV-[85-169]-20-RIIβB-20-[1-84]-OpLuc

pF4K-CMV-[113-169]-4-RIIβB-4-[1-112]-OpLuc

pF4K-CMV-[113-169]-10-RIIβB-10-[1-112]-OpLuc

pF4K-CMV-[113-169]-20-RIIβB-20-[1-112]-OpLuc

pF4K-CMV-[135-169]-4-RIIβB-4-[1-134]-OpLuc

pF4K-CMV-[135-169]-10-RIIβB-10-[1-134]-OpLuc

pF4K-CMV-[135-169]-20-RIIβB-20-[1-134]-OpLuc

PJ15:4809-OgLuc-has the 2.7kb plasmid (passing through DNA2.0) of thorn shrimp luciferase ORF of clone's full size

PJ15:4810-has the 2.6kb plasmid (the 27aa signal peptide is removed) (passing through DNA2.0) of the ORF of sophisticated thorn shrimp luciferase ORF

pF1K-OgLucS-3.7kb。The ORF of full size is cloned into (FLOpLuc) in the pF1K

pF1K-OgLuc-3.6kb。The ORF of ripe luciferase is cloned in the pF1K

pF1K-OpLucDN-3.6kb。Be equal to pF1K-OgLuc, except preceding 4 N-terminal residues are removed

pF1K-OpLucDC-3.6kb。Be equal to pF1K-OgLuc, except last 3 C-terminal residues are removed

pF1K-OpLucDNDC-3.6kb。Be equal to pF1K-OgLuc, except preceding 4 N-terminal and last 3 C-terminal residues are removed

pFVDnK-OgLucS-4.4kb。The luciferase ORF of HaloTag and full size is merged

pFVDnK-OgLuc-4.5kb。The ORF of HaloTag and ripe luciferase is merged

pFN6K-opLuc-3.6kb。The HQ-mark is introduced in the N-terminal of ORF of ripe luciferase

Equivalent CPM OpLuc/RII β B construct (0.1 μ g plasmid/50 μ l reaction mixtures; Figure 64) in rabbit reticulocyte TnT system (Promega#L1170), express.After TnT reaction is finished, add the final concentration of cAMP to 0.1mM, and make mixture incubation 15 minutes at room temperature in addition.Reaction is diluted 10 times with the sea pansy lysis buffer, and luciferase activity is measured in sea pansy reagent as (sea pansy luciferase assay system, #E2810, the Promega Corp.) that advises.

Observe induce (Figure 64) of luciferase activity for all 4 kinds of circular permutation luciferase constructs.The luciferase splitted construct that has between residue 84 and 85 confirms the highest inducing (about 250 times).20 amino acid whose joints are supported effective folding.

The result points out that the cAMP biosensor can directly produce in any insertion site of above selecting of insertion by circular permutation or RII β B.

Embodiment XXIII

Complementary action of protein with thorn shrimp luciferase

In order to measure in the thorn shrimp luciferase for the useful site of complementary action of protein preparation N and C-terminal fusions.The carrier main chain comprises pF3A that is used for experiment in vitro and the pF5K that is used for cell experiment.Prepare following construct: " N-terminal-FRB ", that is, OpLuc (1-50 or 1-84) 10aa G/S joint-FRB, " FKBP-C end ", promptly, FKBP-(G4S) 2 joints-OpLuc (51-170 or 85-170), " FRB-N end, " are promptly, FRB-(G4S) 2 joints-OpLuc (1-50 or 1-84), " C-terminal-FKBP, " promptly, OpLuc (51-170 or 85-170)-10aa G/S joint-FKBP.See table.

Construct	Carrier	Type specification	Legend
				201518.54.06	pF5K	Total length FL-OpLuc	FL OpLuc
201518.57.E6	pF5K	The terminal FRB-OpLuc (1-50) of FRB-N	FRB-50
				201518.57.G3	pF5K	The terminal FRB-OpLuc (1-84) of FRB-N	FRB-84
201518.101.04	pF5K	The terminal FKBP-OpLuc (51-170) of FKBP-C	FKBP-51
				201518.57.H12	pF5K	The terminal FKBP-OpLuc (85-170) of FKBP-C	FKBP-85
pBFB395	pF5K	N-terminal-FRB OpLuc (1-50)-FRB	50-FRB
				pBFB396	pF5K	N-terminal-FRB OpLuc (1-84)-FRB	84-FRB
pBFB415	pF5K	C-terminal-FKBP OpLuc (51-170)-FKBP	51-FKBP
				pBFB416	pF5K	C-terminal-FKBP OpLuc (85-170)-FKBP	85-FKBP

201518.45.08	pF3A	Total length FL-OpLuc	FL OpLuc
				201518.57.A2	pF3A	The terminal FRB-OpLuc (1-50) of FRB-N	FRB-50
201518.57.A11	pF3A	The terminal F RB-OpLuc (1-84) of FRB-N	FRB-84
				201518.57.D9	pF3A	The terminal FKBP-OpLuc (51-170) of FKBP-C	FKBP-51
201518.61.H3	pF3A	The terminal FKBP-OpLuc (85-170) of FKBP-C	FKBP-85
				201518.110.4-1	pF3A	N-terminal-FRB OpLuc (1-50)-FRB	50-FRB
201518.104.04	pF3A	N-terminal-FRB OpLuc (1-84)-FRB	84-FRB
				201518.129.03	pF3A	C-terminal-FKBP OpLuc (51-170)-FKBP	51-FKBP
201518.129.06	pF3A	C-terminal-FKBP OpLuc (85-170)-FKBP	85-FKBP

Protein uses

SP6 high-throughput protein matter expression system in 30 ℃ express or coexpression 2 hours (according to the scheme of manufacturers; Promega Corp.).20 μ L lysates incubation 15 minutes at room temperature under+/-1 μ M rapamycin condition.10 μ L lysates are the 1:1 dilution in 2 * HEPES/ thiocarbamide, and 5 μ L are placed 96 orifice bores in triplicate.Luminously measure by add 100 μ L sea pansy luciferase assay reagent (R-LAR) via syringe.Be shown among Figure 56 about the splitted in vitro results on position 50/51 (50-FRB+FKBP-51), be shown among Figure 58 about those of 84/85 (84-FRB+FKBP-85).Figure 57 and 59 has shown the result who analyzes about separately SDS-PAGE.5 μ L-/+the size fractionation separation on 4-12%SDS-PAGE of rapamycin lysate.Figure 61-62 shows the in vitro results about 51-FKBP+FRB-50 and 85-FKBP+FRB-85 direction.For the data in Figure 62,7.5 μ L+/-the size fractionation separation on 4-12%SDS-PAGE of rapamycin lysate.

Figure 60 shows result in the cell that uses the HEK-293 cell.The HEK-293 cell carries out transient transfection and is incubated overnight with complementary fragment or with the individual chip of thorn shrimp luciferase in 6 orifice plates.Second day cell is subjected to trypsin treatment and with 20,000 cells/well bed board in 96 orifice plates.Simultaneously 1 μ M rapamycin or vehicle (DMSO) are added in the cell, and allow them at 37 ℃ and 5% CO ₂Following recovery is spent the night.Removed substratum in second day, and 20 μ L 1x sea pansy luciferase assay lysis buffers are added in every kind of sample, and plate was vibrated 15 minutes under 500rpm.100 μ L sea pansy luciferase assay reagent are injected in each hole, and sample follows delay in 0.5 second to measure with 3 seconds/hole.

Embodiment XXIV

CPM Lampyridea luciferase (FF Luc) and sea pansy luciferase (hRL Luc) also are used as biosensor to measure kinases/phosphatase activity.Mode with the previous biosensor that is similar to cAMP, cGMP and calcium, prepare various circular permutations (CPM) FF Luc and hRL Luc construct, to detect phosphorylation by tyrosine or serine/threonine kinase (phosphorylation on Tyr that underscore is arranged or Thr residue, hereinafter in the construct of Miao Shuing) respectively.Conformational change by the combination of the phosphorylated peptide sequence in the phospho-peptide recognition structure territory with constraint causes can cause the adjusting of the biosensor luciferase activity of fusion.This expression is with respect to existing biosensor based on FRET, and the reagent that can measure the new classification of kinase activity may have the enhanced performance characteristic.

The peptide sequence and the recognition structure territory that are used for Tyrosylprotein kinase and serine/threonine kinase are respectively: have phospho-peptide recognition structure people from territory Src SH2 structural domain (Genbank NM_005417; Aa residue 151-248) peptide GSTSGSGKPGSGEGSEI YGEF (SEQ ID NO:295) or EI YGEF (SEQ ID NO:296) and have RKRDRLG from the phospho-peptide recognition structure territory FHA2 of Rad53p TLGI (SEQ ID NO:297) (the codon optimized form of nucleotide sequence Genbank registration #AY693009, itself and the base 1717-2186 that registers #AAT93028; Aa residue 573-730 comparison).

Be used to make up the kinases biosensor about a plurality of sites that before were accredited as for the useful CPM of the biosensor among generation FF Luc and the hRL Luc.These constructs uses are connected to the interior PCR product of unique restriction site or are prepared by overlapping extension PCR (SOE-PCR) montage.FF Luc construct is prepared in the pF9A main chain, hRL Luc construct is prepared in the pF5A main chain, except the plasmid pBFB174,175,176,178,179,180,181,182,228,229 and 230 for preparing in the modified pGL4.74 main chain of describing in example II.

Prepare following construct: Met-(Luc2.0 or hRL C-terminal fragment)-(fragment X)-(by the peptide of tyrosine phosphorylation)-(fragment)-(phospho-peptide recognition structure territory)-(fragment Y)-(Luc2.0 or hRL N-terminal fragment)-Val.The preparation construct that changes of the order in the peptide by tyrosine phosphorylation and phospho-peptide recognition structure territory wherein also.In addition, prepare following construct: Met-(by the small peptide of tyrosine phosphorylation)-(fragment X)-(Luc2.0)-(fragment)-(phospho-peptide recognition structure territory)-Val for Tyrosylprotein kinase FFLuc biosensor.Referring to Figure 65.

The Tyrosylprotein kinase construct

1) Met-(Luc2.0 234-544)-GSSG-(people Src SH2 structural domain)-GSG-GSTSGSGKPGSGEGSEI YGEF-(joint Y)-(Luc2.0 4-233)-Val, wherein Y=GSGGSGGSSG (SEQ ID NO:291) or GSGGSGGSGGGSGGSGGSSG (SEQ ID NO:286).(the corresponding SEQID NO:270 of GSSG; The corresponding SEQ ID of GSGGSTSGSGKPGSGEGSEIYGEF NO:298).Clone pBFB180,181,182,365,366,367.

2) Met-EI YGEF-(joint X)-(Luc2.0 4-544)-GSSG-(people Src SH2 structural domain), wherein X=GSSG (SEQ ID NO:270), GSSGGSGGSG (SEQ IDNO:276) or GSSGGSGGSGGGSGGSGGSG (SEQ ID NO:277).(EI YThe corresponding SEQ ID of GEF NO:296).Clone pBFB174,175,176.

3) Met-(hRL 92-311)-GSG-(people Src SH2 structural domain)-GSG-GSTSGSGKPGSGEGSEI YGEF-(joint X)-GSSG-(hRL2-91)-Val, wherein X=GSSG (SEQ ID NO:270), GSGGSGGSSG (SEQ ID NO:291) or GSGGSGGSGGGSGGSGGSSG (SEQ ID NO:286).(the corresponding SEQ ID of GSGGSTSGSGKPGSGEGSEIYGEF NO:298).Clone pBFB228,229,230.

4) Met-(Luc2.0 A-544)-(joint X)-(people Src SH2 structural domain)-GSTSGSGKPGSGEGSEI YGEF-(joint Y)-(Luc2.04-B)-Val, wherein X=GSTG (SEQ ID NO:275), GSSGGSGGSG (SEQ ID NO:276) or GSSGGSGGSGGGSGGSGGSG (SEQ ID NO:277) and Y=GSSG (SEQID NO:270), GSGSGGSGGSSG (SEQ ID NO:299) or GSGGSGGSGGGSGGSGGSSG (SEQ ID NO:286) (the corresponding SEQ ID of GSTSGSGKPGSGEGSEIYGEF NO:295) CPM site [A, B]=[235,233], [359,355], [84,82], [309,307].Clone pBFB368,369,370,371,372,373,374,375,376,377,378,379.

5) Met-(hRL A-311)-(joint X)-(people Src SH2 structural domain)-GSTSGSGKPGSGEGSEI YGEF-(joint Y)-(hRL3-B)-Val, wherein X=GSSG (SEQ ID NO:270), GSSGGSGGSG (SEQ ID NO:276) or GSSGGSGGSGGGSGGSGGSG (SEQ ID NO:277) and Y=GSSG (SEQID NO:270), GSGSGGSGGSSG (SEQID NO:299) or GSGGSGGSGGGSGGSGGSSG (SEQ ID NO:286).(the corresponding SEQ ID of GSTSGSGKPGSGEGSEIYGEF NO:295).CPM site [A, B]=[92,91], [42,41], [111,110], [31,30], [69,68].Clone pBFB380,381,382,383,384,385,386,387,388,389,390,391,392,393,394.

The serine/threonine kinase construct

1) Met-(Luc2.0 A-544)-(joint X)-RKRDRLGTLGI-(GGSSGGGSGGGGSGG)-(Rad53p FHA2 structural domain)-(joint Y)-(Luc2.04-B), wherein X=GSSG (SEQ ID NO:270), GGSGGSGSSG (SEQID NO:300) or GSSGGSGGSGGGSGGSGSSG (SEQ ID NO:301), Y=GSSG (SEQ ID NO:270), GSGGSGGSGG (SEQ ID NO:281) or GSGGSGGSGGTSGGSGGSSG (SEQ ID NO:278) (the corresponding SEQ ID of RKRDRLGTLGIGGSSGGGSGGGGSGG NO:283).The CPM site is [A, B]=[235,233], [359,355], [84,82], [309,307].Clone pBFB335,336,337,338,339,340,341,342,343,344,345,346.

2) Met-(hRLA-311)-(joint X)-RKRDRLGTLGI-(GGSSGGGSGGGGSGG)-(Rad53p FHA2 structural domain)-(joint Y)-(hRL3-B), wherein X=GSSG (SEQ ID NO:270), GSSGGSGGSGGG (SEQ IDNO:302) or GSSGGSGGSGGGSGGSGGSG (SEQ ID NO:277), Y=GSSG (SEQ ID NO:270), GSGGSGGSSG (SEQ ID NO:291) or GSGGSGGSGGTSGGSGGSSG (SEQ ID NO:278). (the corresponding SEQ ID of RKRDRLGTLGIGGSSGGGSGGGGSGG NO:283).The CPM site is [A, B]=[92,91], [42,41], [111,110], [31,30], [69,68].Clone pBFB350,351,352,353,354,355,356,357,358,359,360,361,362,363,364.

3) Met-(Luc2.0A-544)-(joint X)-(Rad53p FHA2 structural domain)-GGSSG-RKRDRLG TLGI-(joint Y)-(Luc2.04-B), wherein X=GSGG (SEQ ID NO:293), GGSGGGGSGG (SEQ ID NO:294) or GSSGGSGGSGGGSGGSGGSG (SEQ ID NO:277), Y=GGSSG (SEQIDNO:304), GSSGSGGSGG (SEQ ID NO:305) or GSGGSGGSGGTSGGSGGSSG (SEQ ID NO:278) (the corresponding SEQ ID of GGSSGRKRDRLGTLGI NO:303).The CPM site is [A, B]=[235,233], [359,355].Clone pBFB417,418,419,420,421,422.

4) Met-(hRL A-311)-(joint X)-(Rad53p FHA2 structural domain)-GGSSG-RKRDRLG TLGI-(joint Y)-(hRL3-B), wherein X=GSGG (SEQID NO:293), GGSGGGGSGG (SEQ ID NO:294) or GSSGGSGGSGGGSGGSGGSG (SEQ ID NO:277), Y=GGSSG (SEQIDNO:304), GSSGSGGSGG (SEQ ID NO:305) or GSGGSGGSGGTSGGSGGSSG (SEQ ID NO:278) (the corresponding SEQ ID of GGSSGRKRDRLGTLGI NO:303).The CPM site is [A, B]=[42,41], [111,110].Clone pBFB423,424,425,426,427,428.

The vitro test of the subclass of serine/threonine kinase transmitter

Construct pBFB335,336,338,339,340,417,418,419,422,22 and 8 uses T7 link coupled reticulocyte lysate system (Promega Corp.) tests external.In brief, following component is assembled according to the scheme of manufacturer recommendation:

1 μ g plasmid DNA

25 μ L rabbit reticulocyte extracts

2 μ L TNT reaction buffers

1 μ L T7 polysaccharase

1 μ L aminoacid mixture

1μL rRNasin

DH ₂O to 50 μ L cumulative volume

After 1 hour, fusion rotein separately carries out incubation in the existence of 10ng Akt1/PKB α recombinase (Upstate Biotechnology) or not in 30 ℃ of incubations, and this incubation is by making 2 μ L

Reaction and 8 μ L water+4 μ L5x reaction buffer (40mMMOPS/NaOH pH7.0,1mM EDTA)+4 μ L 5x Mg-ATP (50mM magnesium acetate, 0.5mM ATP)+2 μ L 5ng/ μ L enzyme (by the 100ng/ul stoste dilution of dilution in PKB dilution buffer liquid [20mMMOPS (7.0), 1mM EDTA, 5% glycerine, 0.05% DTT, 1mg/ml BSA]) or only 2 μ L PKB dilution buffer liquid combination.Sample is subsequently in 30 ℃ of incubations 20 minutes.5 μ L samples are added 100 μ L luciferase assay reagent (LAR; Promega Corp.) in the solution, and draws 4X fast up and down to mix.Luminous use Turner20/20N photometer (Turner Biosystems; 1 second integrating time) measures.All samples is measured in triplicate.

The result

Compare 50% minimizing that construct pBFB340 demonstration+Akt1/PKB is luminous with no Akt1/PKB.Contrast construct pBFB22 and pBFB8 add with Akt1/PKB does not have change (Figure 66).

Scheme about other serine/threonine kinase transmitters is equal to above the sort of, except for CPM hRL Luc sample, 5 μ L samples are added 96 orifice plates+do not contain stain remover (150mM HEPES, the 100mM thiocarbamide) 5 μ L 2x sea pansy lysis buffers, and add 100 μ L sea pansies by syringe and measure reagent (Promega Corp.), wherein use Veritas microtiter plate photometer and measure luminous (Turner Biosystems; The Bright-Glo program; 3 seconds integrating times).FF Luc sample wherein uses Veritas microtiter plate photometer and measures luminous (Turner Biosystems by via syringe the 5 μ L samples that 100 μ L luciferase assay reagent add in 96 orifice plates being measured; The Bright-Glo program; 3 seconds integrating times).

The tyrosine-kinase Enzyme sensor is following to be tested: protein uses T7 link coupled reticulocyte lysate system (Promega Corp.) carries out vivoexpression.In brief, following component is assembled according to the scheme of manufacturer recommendation:

1 μ g plasmid DNA

25 μ L rabbit reticulocyte extracts

2 μ L TNT reaction buffers

1 μ L T7 polysaccharase

1 μ L aminoacid mixture

1μL rRNasin

DH ₂O to 50 μ L cumulative volume

After 1 hour, fusion rotein separately uses in 50 following μ l kinase reactions in 30 ℃ of incubations: 1X ProFlour reaction buffer (Promega Corp.)+10 μ l RR TnT reacts+100 μ M vanadic acid sodium+1mM MnCl ₂+ 1mM MgATP+0.5 μ l c-Src kinases or water.Add the back in the time of 0,30 and 60 minute at the Src kinases, obtain 10 μ l aliquots containigs and preserve in-20 ℃ until mensuration.For CPM FF Luc sample, 5 μ l are transferred to 96 orifice plates, and add 100ul luciferase assay reagent (LAR by syringe; Promega Corp.), wherein use Veritas microtiter plate photometer and measure luminous (Turner Biosystems; The Bright-Glo program; 3 seconds integrating times).For CPM hRL Luc sample, 5 μ l samples are added 96 orifice plates+do not contain stain remover (150mM HEPES, the 100mM thiocarbamide) 5 μ L 2x sea pansy lysis buffers, and add 100 μ L sea pansies by syringe and measure reagent (Promega Corp.), wherein use Veritas microtiter plate photometer and measure luminous (Turner Biosystems; The Bright-Glo program; 3 seconds integrating times).

For the kinases transmitter in the test cell, FF Luc and following test of CPM hRL Luc serine/threonine kinase biosensor: HEK293 and NIH/3T3 cell are with 1-1.5 x 10 ⁴The cell density of individual cells/well is at CO ₂Not among dependency substratum (Invitrogen)+10% FBS in 96 orifice plates bed board.They use 4.2 μ L subsequently Reagent and 1.4 μ g DNA/ holes are used

Reagent (MIRUS) carries out transfection.Allow cell at 37 ℃/10% CO ₂Following grow overnight.Changed substratum into CO in second day ₂Dependency substratum+0.2% FBS is not so that the cell serum starvation.Allow cell at 37 ℃/10%CO subsequently ₂Following grow overnight.After the transfection about 2 days, for FF Luc transmitter, cell carried out balance with luciferin-EF (Promega Corp.) of final concentration 5mM, or for CPM hRL Luc transmitter, cell carries out balance with the EnduRen (Promega Corp.) of final concentration 60 μ M.1.5 after hour, luminous baseline measures uses Mithras LB 940 photometer (BertholdTechnologies; The integrating time in 1 second/hole) under 37 ℃, measures.Next, a semicell with the kinase activator thing for example the somatomedin (PDGF, 50ng/ml final concentration) in thrombocyte source handle.Luminous continuously measured in ensuing 30 minutes under 37 ℃ subsequently.

Embodiment XXV

Under the situation that does not have three dimensional protein structure information, be used to prepare that circular permutation is proteinic closes The mensuration of suitable split point

Method

1) aminoacid sequence of acquisition target protein matter.

2) use one or more computer programs to help to determine the protein structure feature of suitable split point with prediction.Suitable split point may be exposed on the protein surface.Be positioned at conventional secondary structure element for example spiral and impossible protein structure and the function destroyed of folding outer split point.

The protein zone that the prediction surface exposes: exposed region may be hydrophilic.Hydrophilic and hydrophobic residue can use on the website that openly enters and obtains (for example from the ProtScale of the ExPASy protein science server of Switzerland bioinformation association along the distribution (hydrophobic points/score) of protein sequence based on hydrophobicity grade commonly used Http: //www.expasy.ch/cgi-bin/protscale.pl) and calculate as the program of the part of commercial sequence analysis software bag (for example from DNASTAR Lasergene).

Predicted protein matter secondary structure: this kind program can on the website that openly enters (referring to the tabulation on the ExPASy protein science instrument network address Http: //www.expasy.org/tools/#secondary) and obtain as the part of commercial sequence analysis software bag (for example from DNASTAR Lasergene).

3) select split point based on result from one or more Forecasting Methodologies.

Example

1) protein sequence: the ripe luciferase sequence (Genbank registers BAB13776, residue 28-196) of thin angle thorn shrimp.

2) the protein zone of prediction surface exposure: use window size 5 and 7, hydrophobicity score based on each residue of Kyte-Doolittle hydrophobicity rating calculation, it specifies the scope (Kyte J and Doolittle RF:A simplemethod for displaying the hydropathic character of a protein.J.Mol.Biol.157:105,1982) for the surperficial exposed region suggestion of finding supposition.

Predicted protein matter secondary structure: use 5 kinds of different prediction algorithms:

a.PSIPRED(Jones DT.(1999)Protein secondary structureprediction based on position-specific scoring matrices.J.Mol.Biol.292：195-202.McGuffin LJ，Bryson K，Jones DT)。

B.JPRED (Cuff JA, Clamp ME, Siddiqui AS, Finlay M and BartonGJ.1998.Jpred:A Consensus Secondary Structure Prediction Server, Bioinformatics 14:892-893).

c.PORTER(G Pollastri，A McLysaght.＂Porter：a new，accurateserver for protein secondary structure prediction＂.Bioinformatics，21(8)，1719-20，2005)。

d.SCRATCH(G Pollastri，D Przybylski，B Rost，P Baldi：Improving the prediction of protein secondary structure in three andeight classes using recurrent neural networks and profiles.Proteins，47，228-335，2002)。

e.PROF(M Ouali，R King：Cascaded multiple classifiers forsecondary structure prediction.Protein Science，9，1162-1176，1999)。

3) result who compiles the protein structure signatures to predict in the table is used for comparison.Be chosen in hydrophilic (low hydrophobicity score) and be arranged in the suitable split point in the outer zone of the conventional secondary structure element (spiral and folding) of prediction.Referring to table 9 (hereinafter 3 part in).

Table 9: the constitutional features of compiling about the ripe luciferase of thin angle thorn shrimp predicts the outcome.The secondary structure prediction object code is the H=spiral, and E=is folding, and C=curls, and is blank=as to curl.Hydrophobicity prediction score for hydrophobic region is〉0, be＜0 for hydrophilic region.Suitable split point example is labeled as xxx in the row of the rightmost side.

Therefore, under the situation that does not have three-dimensional structure, can select any proteinic division site, for example in PCA, use the sort of or treat as biosensor the sort of (structural domain directly insert the division site between the two or insert in the circular permutation mutant circular permutation on the division site).

Reference

People such as Altschul, J.Mol.Biol., 215: 403 (1990).

People such as Altschul, Nuc.Acids Res., 25: 3389 (1977).

Bos，Nat.Rev. Mol.Cell.Biol.， 4：733(2003)

People such as Chong, Gene, 192: 271 (1997).

People such as Corpet, Nucl.Acids Res., 16: 1088 (1988).

Dremier et.al.， FEBS Lett.， 546：163(2003).

People such as Geysen, Proc.Natl.Acad.Sci.USA, 3998 (1984).

Hanks and Hunter, FASEB J, 9: 576-595 (1995).

Henikoff&Henikoff， Proc.Natl.Acad.Sci.USA， 89：10915(1989).

People such as Higgins, Gene, 73: 237 (1988).

People such as Higgins, CABIOS, 5: 157 (1989).

People such as Huang, CABIOS, 8: 155 (1992).

People such as Kaihara, Anal.Chem., 75: 41 (2003).

Karlin&Altschul， Proc.Natl.Acad.Sci.USA， 90：5873(1993).

People such as Lee, Anal.Biochem., 316: 162 (2003).

People such as Liu, Gene, 237: 153 (1999).

Mayer and Baltimore, Trends Cell.Biol., 3: 8 (1993).

Merrifield， J.Am.Chem.Soc.，2149(1963).

People such as Murray, Nucl.Acids Res., 17:477 (1989)

Myers and Miller, LABIOS, 4: 11 (1988).

People such as Ozawa, Analytical Chemistry, 73: 2516 (2001).

People such as Needleman, J.Mol.Biol., 48: 443 (1976).

People such as Paulmurugan, Anal.Chem., 75: 1584 (2003).

People such as Paulmurugan, Proc.Natl.Acad.Sci.USA, 99:3105 (2002).

People such as Paulmurugan, PNAS, 24: 15603 (1999).

People such as Pearson, Methods Mol.Biol., 24: 307 (1994).

People such as Pearson, Proc.Natl.Acad.Sci.USA, 85: 2444 (1985.

People such as Plainkum, Nat.Struct.Biol., 10: 115 (2003).

People such as Remy, Biotechniques, 38: 763 (2005).

People such as Remy, Nat.Methods, 3: 977 (2006).

Sadowski waits the people, Mol.Cell.Bio., 6: 4396 (1986).

People such as Sala-Newby, Biochem J., 279: 727 (1991).

People such as Sala-Newby, FEBS, 307: 241 (1992).

People such as Sambrook, In: Molecular Cloning:A Laboratory Manual, ColdSpring Harbor (1989).

People such as Smith, Adv.Appl.Math., 2: 482 (1981).

People such as Stewart, Solid Phase Peptide Synthesis, 2ed., Pierce Chemical Co., Rockford, Ill., pp.11-12).

People such as Tannous, Mol.Thera., 11: 435 (2005).

People such as Wada, Nucl.Acids Res., 18: 2367 (1990).

People such as Wang, BBRC, 282: 28 (2001).

People such as Waud, BBA, 1292: 89 (1996).

People such as Zagotta, Nature, 425: 730 (2003).

All publications, patent and patent application are integrated with this paper as a reference.Although the present invention is described with regard to its some preferred embodiment in aforementioned specification, and many details are set forth for illustrational purpose, but it will be apparent to one skilled in the art that the present invention allows other embodiments, and need not to deviate from some details that ultimate principle of the present invention can change this paper considerably.

<110>Promega Corporation

Fan，Frank

Binkowski，Brock

Wigdal，Susan

Wood，Keith V.

Wood，Monika G.

＜120〉conversion and unmapped luciferase biosensor

<130>341.043W01

<150>US 60/788,608

<151>2006-04-03

<150>US 60/879,771

<151>2007-01-10

<150>US 60/901,133

<151>2007-0214

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<400>49

<210>50

<211>58

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>50

<210>51

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>51

<210>52

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>52

<210>53

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>53

<210>54

<211>64

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>54

<210>55

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>55

<210>56

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>56

<210>57

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>57

<210>58

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>58

<210>59

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>59

<210>60

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>60

<210>61

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>61

<210>62

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>62

<210>63

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>63

<210>64

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>64

<210>65

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>65

<210>66

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>66

<210>67

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>67

<210>68

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>68

<210>69

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>69

<210>70

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>70

<210>71

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>71

<210>72

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>72

<210>73

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>73

<210>74

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>74

<210>75

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>75

<210>76

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>76

<210>77

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>77

<210>78

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>78

<210>79

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>79

<210>80

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>80

<210>81

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>81

<210>82

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>82

<210>83

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>83

<210>84

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>84

<210>85

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>85

<210>86

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>86

<210>87

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>87

<210>88

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>88

<210>89

<211>43

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>89

<210>90

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>90

<210>91

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>91

<210>92

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>92

<210>93

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>93

<210>94

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>94

<210>95

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>95

<210>96

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>96

<210>97

<211>43

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>97

<210>98

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>98

<210>99

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>99

<210>100

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>100

<210>101

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>101

<210>102

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>102

<210>103

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>103

<210>104

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>104

<210>105

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>105

<210>106

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>106

<210>107

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>107

<210>108

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>108

<210>109

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>109

<210>110

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>110

<210>111

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>111

<210>112

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>112

<210>113

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>113

<210>114

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>114

<210>115

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>115

<210>116

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>116

<210>117

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>117

<210>118

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜221〉site

<222>2

＜223〉Xaa=Glu, Gln or Lys

<220>

＜221〉site

<222>3

＜223〉Xaa=Leu, Lys, Ser or Ile

<220>

＜221〉site

<222>4

＜223〉Xaa=Ala, Ile, Cys or Gly

<220>

＜221〉site

<222>5

＜223〉Xaa=Leu or Ile

<220>

＜221〉site

<222>(6)...(7)

＜223〉any amino acid of Xaa=

<220>

＜221〉site

<222>(8)...(11)

＜223〉any amino acid of Xaa=or do not exist

<220>

＜221〉site

<222>(12)...(12)

＜223〉Xaa=Pro, Val, Thr, Arg or Glu

<220>

＜221〉site

<222>(14)...(14)

＜223〉Xaa=Ala, Thr, His or Ser

<220>

＜221〉site

<222>(15)...(15)

＜223〉Xaa=Ala or Ser

<220>

＜221〉site

<222>(16)...(16)

＜223〉Xaa=Val, Thr, Ser, Asn or Trp

<400>118

<210>119

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>119

<210>120

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>120

<210>121

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>121

<210>122

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>122

<210>123

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>123

<210>124

<211>149

<212>PRT

＜213〉people

<400>124

<210>125

<211>151

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>125

<210>126

<211>153

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>126

<210>127

<211>155

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>127

<210>128

<211>157

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>128

<210>129

<211>159

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>129

<210>130

<211>151

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>130

<210>131

<211>153

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>131

<210>132

<211>155

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>132

<210>133

<211>157

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>133

<210>134

<211>159

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>134

<210>135

<211>161

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>135

<210>136

<211>153

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>136

<210>137

<211>155

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>137

<210>138

<211>157

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>138

<210>139

<211>159

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>139

<210>140

<211>161

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>140

<210>141

<211>163

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>141

<210>142

<211>155

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>142

<210>143

<211>157

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>143

<210>144

<211>159

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>144

<210>145

<211>161

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>145

<210>146

<211>163

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>146

<210>147

<211>165

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>147

<210>148

<211>157

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>148

<210>149

<211>159

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>149

<210>150

<211>161

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>150

<210>151

<211>163

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>151

<210>152

<211>165

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>152

<210>153

<211>167

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>153

<210>154

<211>159

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>154

<210>155

<211>161

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>155

<210>156

<211>163

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>156

<210>157

<211>165

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>157

<210>158

<211>167

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>158

<210>159

<211>169

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>159

<210>160

<211>157

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>160

<210>161

<211>155

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>161

<210>162

<211>153

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>162

<210>163

<211>151

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>163

<210>164

<211>149

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>164

<210>165

<211>147

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>165

<210>166

<211>145

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>166

<210>167

<211>161

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>167

<210>168

<211>163

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>168

<210>169

<211>165

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>169

<210>170

<211>167

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>170

<210>171

<211>169

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>171

<210>172

<211>699

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>172

<210>173

<211>711

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>173

<210>174

<211>731

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>174

<210>175

<211>696

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>175

<210>176

<211>708

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>176

<210>177

<211>728

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>177

<210>178

<211>699

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>178

<210>179

<211>711

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>179

<210>180

<211>731

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>180

<210>181

<211>699

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>181

<210>182

<211>711

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>182

<210>183

<211>731

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>183

<210>184

<211>704

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>184

<210>185

<211>487

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>185

<210>186

<211>485

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>186

<210>187

<211>157

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>187

<210>188

<211>169

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>188

<210>189

<211>189

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>189

<210>190

<211>327

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>190

<210>191

<211>339

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>191

<210>192

<211>359

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>192

<210>193

<211>327

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>193

<210>194

<211>339

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>194

<210>195

<211>359

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>195

<210>196

<211>42

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>196

<210>197

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>197

<210>198

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>198

<210>199

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>199

<210>200

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>200

<210>201

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>201

<210>202

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>202

<210>203

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>203

<210>204

<211>185

<212>PRT

<213>Gaussia princeps

<400>204

<210>205

<211>517

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>205

<210>206

<211>465

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>206

<210>207

<211>567

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>207

<210>208

<211>729

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>208

<210>209

<211>627

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>209

<210>210

<211>550

<212>PRT

<213>Photinus pyralis

<400>210

<210>211

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic oligonucleotide

<400>211

<210>212

<211>733

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>212

<210>213

<211>745

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>213

<210>214

<211>731

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>214

<210>215

<211>743

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>215

<210>216

<211>763

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>216

<210>217

<211>733

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>217

<210>218

<211>745

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>218

<210>219

<211>733

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>219

<210>220

<211>745

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>220

<210>221

<211>502

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>221

<210>222

<211>516

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>222

<210>223

<211>534

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>223

<210>224

<211>502

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>224

<210>225

<211>516

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>225

<210>226

<211>534

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>226

<210>227

<211>502

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>227

<210>228

<211>516

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>228

<210>229

<211>534

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>229

<210>230

<211>502

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>230

<210>231

<211>516

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>231

<210>232

<211>534

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>232

<210>233

<211>502

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>233

<210>234

<211>516

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>234

<210>235

<211>534

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>235

<210>236

<211>724

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>236

<210>237

<211>735

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>237

<210>238

<211>755

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>238

<210>239

<211>750

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>239

<210>240

<211>493

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>240

<210>241

<211>504

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>241

<210>242

<211>524

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>242

<210>243

<211>493

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>243

<210>244

<211>504

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>244

<210>245

<211>521

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>245

<210>246

<211>676

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>246

<210>247

<211>684

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>247

<210>248

<211>668

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>248

<210>249

<211>682

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>249

<210>250

<211>700

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>250

<210>251

<211>666

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>251

<210>252

<211>680

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>252

<210>253

<211>698

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>253

<210>254

<211>668

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>254

<210>255

<211>680

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>255

<210>256

<211>668

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>256

<210>257

<211>682

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>257

<210>258

<211>700

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>258

<210>259

<211>437

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>259

<210>260

<211>451

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>260

<210>261

<211>437

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>261

<210>262

<211>451

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>262

<210>263

<211>437

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>263

<210>264

<211>451

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>264

<210>265

<211>437

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>265

<210>266

<211>451

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<400>266

<210>267

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>267

<210>268

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>268

<210>269

<211>26

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>269

<210>270

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>270

<210>271

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>271

<210>272

<211>31

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>272

<210>273

<211>41

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>273

<210>274

<211>33

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>274

<210>275

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>275

<210>276

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>276

<210>277

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>277

<210>278

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>278

<210>279

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>279

<210>280

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>280

<210>281

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>281

<210>282

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>282

<210>283

<211>26

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>283

<210>284

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>284

<210>285

<211>26

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>285

<210>286

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>286

<210>287

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>287

<210>288

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>288

<210>289

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>289

<210>290

<211>38

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>290

<210>291

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>291

<210>292

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>292

<210>293

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>293

<210>294

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>294

<210>295

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>295

<210>296

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>296

<210>297

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>297

<210>298

<211>24

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>298

<210>299

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>299

<210>300

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>300

<210>301

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>301

<210>302

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>302

<210>303

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>303

<210>304

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>304

<210>305

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>305

<210>306

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>306

<210>307

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>307

Claims (122)

1. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of circular permutation luciferase, described circular permutation luciferase comprises the ring-type nucleotide binding site, the corresponding therein not conversion luciferase of described luciferase is on the site of modifying tolerance or carry out conversion in the zone, and the activity of wherein said circular permutation luciferase is detectable.

2. the polynucleotide of claim 1, wherein said circular permutation luciferase further comprises at least one amino acid whose mark on N-terminal, C-terminal or both.

3. the polynucleotide of claim 2, wherein said mark is PEST sequence, GST sequence or polyhistidyl sequence.

4. the polynucleotide of claim 1, wherein said circular permutation luciferase further comprise the disappearance of luciferase sequence on the sequence of the N-terminal of the described non-conversion luciferase of correspondence and/or C-terminal.

5. the polynucleotide of claim 4, the disappearance on wherein said N-terminal or the described C-terminal is no more than 15 residues of luciferase sequence.

6. claim 1 or 5 polynucleotide, wherein said ring-type nucleotide binding site is on the sequence of the described N-terminal of the described non-conversion luciferase of correspondence and/or C-terminal.

7. the polynucleotide of claim 1, wherein said ring-type nucleotide binding site length is about 200 amino-acid residues of about 4-.

8. the polynucleotide of claim 1, wherein said luciferase is the Coleoptera luciferase.

9. the polynucleotide of claim 1, wherein said luciferase is Pleonomus luciferase or Lampyridea luciferase.

10. the polynucleotide of claim 1, wherein said luciferase is the coral polyp luciferase.

11. the polynucleotide of claim 10, wherein said luciferase are the sea pansy luciferases.

12. the polynucleotide of claim 1, wherein said ring-type nucleotide binding site is in the zone of residue 2-12, residue 32-53, residue 70-88, residue 102-126, residue 139-165, residue 183-203, residue 220-247, residue 262-273, residue 303-313, residue 353-408, residue 485-495 or the residue 535-546 of corresponding Lampyridea luciferase.

13. the polynucleotide of claim 1, wherein said ring-type nucleotide binding site is in the zone of residue 15-30, residue 112-122, residue 352-362, residue 371-384, residue 393-414 or the residue 485-495 of corresponding Pleonomus luciferase.

14. the polynucleotide of claim 1, wherein said ring-type nucleotide binding site is in the zone of residue 2-12, residue 26-47, residue 64-74, residue 86-116, residue 147-157, residue 223-234 or the residue 301-311 of corresponding sea pansy luciferase.

15. the polynucleotide of claim 1, wherein said conversion is in the zone of residue 2-12, residue 32-53, residue 70-88, residue 102-126, residue 139-165, residue 203-193, residue 220-247, residue 262-273, residue 303-313, residue 353-408, residue 485-495 or the residue 535-546 of corresponding Lampyridea luciferase.

16. the polynucleotide of claim 1, wherein said conversion is in the zone of residue 15-30, residue 112-122, residue 352-362, residue 371-384, residue 393-414 or the residue 485-495 of corresponding Pleonomus luciferase.

17. the polynucleotide of claim 1, wherein said conversion is in the zone of residue 26-47, residue 64-74, residue 85-116, residue 147-157, residue 223-234 or the residue 301-311 of corresponding sea pansy luciferase.

18. the polynucleotide of claim 1, wherein said ring-type nucleotide binding site combines with cAMP.

19. the polynucleotide of claim 1, wherein said ring-type nucleotide binding site combines with cGMP.

20. a carrier, it comprises among the claim 1-19 each polynucleotide.

21. a host cell, it comprises the polynucleotide in the carrier of claim 20.

22. a circular permutation luciferase, it is by each polynucleotide encoding among the claim 1-19.

23. a method that detects or measure the ring nucleus thuja acid in the cell, it comprises:

A) provide host cell or its lysate that comprises claim 21 and the mixture that is used for the reagent of luminous reaction; With

B) detect or measure luminous in the described mixture, thereby detect or measure the existence or the amount of the ring nucleus thuja acid in the described cell.

24. one kind detect or working sample in the method for ring nucleus thuja acid, it comprises:

A) provide and comprise the circular permutation luciferase of suspecting sample with ring nucleus thuja acid, claim 22 and the mixture that is used for the reagent of luminous reaction; With

B) detect or measure luminous in the described mixture.

25. the method for claim 23 or 24, wherein said luciferase are the Lampyridea luciferases.

26. the method for claim 23 or 24, wherein said luciferase are the Pleonomus luciferases.

27. the method for claim 23 or 24, wherein said luciferase are the sea pansy luciferases.

28. the method for claim 23 or 24, wherein the ring nucleus thuja acid causes luminous increase with combining of ring-type nucleotide binding site.

29. the method for claim 24, wherein said sample comprises cell.

30. the method for claim 24, wherein said sample comprise cell lysate or cell fraction.

31. a method that detects one or more conditioning agents of g protein coupled receptor, it comprises:

A) sampling, the reagent that it comprises host cell or its lysate of one or more test agent, claim 21 and is used for luminous reaction; With

B) detect or measure luminous in the described sample.

32. a method that detects one or more conditioning agents of g protein coupled receptor, it comprises:

A) sampling, the reagent that it comprises the circular permutation luciferase of one or more test agent, claim 22 and is used for luminous reaction; With

B) detect or measure luminous in the described sample.

33. a method that detects one or more conditioning agents of g protein coupled receptor, it comprises:

A) relatively from the luminous of first kind of luminous reaction mixture luminous with from corresponding luminous reaction mixture, described first kind of luminous reaction mixture comprises host cell or its lysate of one or more test agent, claim 21 and is used for the reagent of luminous reaction, corresponding luminous reaction mixture does not comprise described one or more test agent, but comprises host cell or its lysate of claim 21 and be used for the reagent of luminous reaction; With

B) detect or measure with respect to corresponding luminous reaction mixture, whether described one or more test agent in described first kind of luminous reaction mixture change luminous in described first kind of luminous reaction mixture.

34. a method that detects one or more conditioning agents of g protein coupled receptor, it comprises:

A) relatively from the luminous of first kind of luminous reaction mixture luminous with from corresponding luminous reaction mixture, described first kind of luminous reaction mixture comprises the circular permutation luciferase of one or more test agent, claim 22 and is used for the reagent of luminous reaction, corresponding luminous reaction mixture does not comprise described one or more test agent, but comprises the circular permutation luciferase and the described reagent of claim 22; With

B) detect or measure with respect to corresponding luminous reaction mixture, whether the described reagent in described first kind of luminous reaction mixture changes luminous in described first kind of luminous reaction mixture.

35. each method among the claim 31-34, wherein said one or more reagent strengthen the g protein coupled receptor activity.

36. each method among the claim 31-34, wherein said one or more reagent suppress the g protein coupled receptor activity.

37. the method for claim 31 or 34, wherein said one or more reagent contacted with solid substrate before adding described host cell or its lysate and reagent.

38. the method for claim 32 or 34, wherein said one or more reagent contacted with solid substrate before adding described circular permutation luciferase and reagent.

39. the method for claim 31 or 33, wherein said host cell expression recombinant G protein coupled receptor.

40. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the optional open reading-frame (ORF) that comprises the circular permutation coral polyp luciferase of insertion, described insertion comprises and the direct or indirect interactional aminoacid sequence of molecules of interest, on the residue that wherein said conversion tolerates modification in coral polyp luciferase sequence or in the zone, and wherein said insertion is arranged in coral polyp luciferase sequence on the different residue of modifying tolerance or zone.

41. the polynucleotide of claim 40, wherein said nucleic acid sequence encoding fusion rotein, described fusion rotein comprise described circular permutation coral polyp luciferase and at described N-terminal, C-terminal or at least one the amino acid whose mark on both.

42. the polynucleotide of claim 40, wherein said circular permutation coral polyp luciferase further comprise the disappearance corresponding to the coral polyp luciferase sequence of the N-terminal of corresponding non-annularity conversion coral polyp luciferase and/or C-terminal sequence.

43. the polynucleotide of claim 42, wherein said disappearance are no more than 15 residues of coral polyp luciferase sequence.

44. the polynucleotide of claim 40, wherein said circular permutation coral polyp luciferase further comprises the disappearance of the coral polyp luciferase sequence that is positioned at described insertion N-terminal and/or C-terminal.

45. the polynucleotide of claim 44, wherein said disappearance are no more than 15 residues of coral polyp luciferase sequence.

46. the polynucleotide of claim 40 are on the wherein said sequence that is inserted in corresponding to the N-terminal of corresponding non-annularity conversion coral polyp luciferase and/or C-terminal.

47. the polynucleotide of claim 40, wherein said about 200 amino-acid residues of about 4-that are inserted as.

48. the polynucleotide of claim 40 are in the zone of wherein said residue 2-12, residue 26-47, residue 64-74, residue 86-116, residue 147-157, residue 223-234 or the residue 301-311 that is inserted in corresponding non-annularity conversion sea pansy luciferase.

49. the polynucleotide of claim 40, wherein said conversion is in the zone of residue 2-12, residue 26-47, residue 64-74, residue 86-116, residue 147-157, residue 223-234 or the residue 301-311 of corresponding non-annularity conversion sea pansy luciferase.

50. a carrier, it comprises among the claim 40-49 each polynucleotide.

51. a host cell, it comprises the described polynucleotide in the carrier of claim 50.

52. a modified coral polyp luciferase, it is by each polynucleotide encoding among the claim 40-49.

53. a method that detects or measure the molecules of interest in the cell, it comprises:

A) provide host cell or its lysate that comprises claim 51 and the mixture that is used for the reagent of luminous reaction, wherein said circular permutation luciferase comprises insertion; With

B) detect or measure luminous in the described mixture, thereby detect or measure the existence or the amount of the described molecule in the described cell.

54. one kind detect or working sample in the method for molecules of interest, it comprises:

A) provide modified luciferase that comprises the claim 52 of suspecting sample, comprising insertion and the mixture that is used for the reagent of luminous reaction with ring nucleus thuja acid; With

B) detect or measure luminous in the described mixture.

55. the method for one or more conditioning agents of a testing goal molecule, it comprises:

A) provide host cell or its lysate that comprises one or more test agent, claim 51 and the mixture that is used for the reagent of luminous reaction; With

B) detect or measure luminous in the described sample.

56. the method for one or more conditioning agents of a testing goal molecule, it comprises:

A) provide and comprise one or more test agent, comprise insertion claim 52 luciferase and be used for the sample of the reagent of luminous reaction; With

B) detect or measure luminous in the described sample.

57. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of modified beetle luciferase, wherein said modified beetle luciferase comprises the ring-type nucleotide binding site with respect to the beetle luciferase of corresponding unmodified, wherein said ring nucleus thuja acid is combined in the beetle luciferase sequence on the residue of modifying tolerance or in the zone, and the activity of wherein said modified beetle luciferase is detectable.

58. the polynucleotide of claim 57, wherein said ring-type nucleotide binding site is on the C-terminal of modified beetle luciferase.

59. the polynucleotide of claim 57, wherein said ring-type nucleotide binding site is in N-terminal and C-terminal inside.

60. the polynucleotide of claim 57, wherein said nucleotide sequence further are coded in N-terminal, C-terminal or at least one the amino acid whose mark on both of modified beetle luciferase.

61. the polynucleotide of claim 57, wherein said modified beetle luciferase further comprises the disappearance of the beetle luciferase sequence that is positioned at described ring-type nucleotide binding site N-terminal and/or C-terminal.

62. the polynucleotide of claim 61, wherein said disappearance are no more than 15 residues of beetle luciferase sequence.

63. the polynucleotide of claim 57, wherein said modified beetle luciferase further comprise the disappearance of beetle luciferase sequence on the sequence of the N-terminal of the beetle luciferase of the described unmodified of correspondence and/or C-terminal.

64. the polynucleotide of claim 63, wherein the disappearance on described N-terminal and/or described C-terminal is no more than 15 residues of beetle luciferase sequence.

65. the polynucleotide of claim 57, wherein said beetle luciferase is the Pleonomus luciferase.

66. the polynucleotide of claim 57, wherein said beetle luciferase is the Lampyridea luciferase.

67. the polynucleotide of claim 57, wherein said ring-type nucleotide binding site is in the zone of residue 2-12, residue 32-53, residue 70-88, residue 102-126, residue 139-165, residue 183-203, residue 220-247, residue 262-273, residue 303-313, residue 353-408, residue 485-495 or the residue 535-546 of corresponding Lampyridea luciferase.

68. the polynucleotide of claim 57, wherein said ring-type nucleotide binding site is in the zone of residue 15-30, residue 112-122, residue 352-362, residue 371-384, residue 393-414 or the residue 485-495 of corresponding Pleonomus luciferase.

69. the polynucleotide of claim 57, wherein said ring-type nucleotide binding site combines with cAMP.

70. the polynucleotide of claim 57, wherein said ring-type nucleotide binding site combines with cGMP.

71. a carrier, it comprises among the claim 57-70 each polynucleotide.

72. a host cell, it comprises the carrier of claim 71.

73. a modified beetle luciferase, it is by each polynucleotide encoding among the claim 57-70.

74. a method that detects the ring nucleus thuja acid in the cell, it comprises: cell is contacted with the carrier of claim 71; And b) detection or mensuration are by the activity of the modified beetle luciferase of described vector encoded.

75. the method for the ring nucleus thuja acid in the test sample, it comprises: sample is contacted with the modified beetle luciferase of claim 73; And b) detection or mensuration are by the activity of the modified beetle luciferase of described vector encoded.

76. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of modified coral polyp luciferase, wherein said modified coral polyp luciferase comprises inner the insertion with respect to the coral polyp luciferase of corresponding unmodified, the described corresponding wild type coral polyp luciferase wherein of being inserted in is on the residue of modifying tolerance or in the zone, wherein said insertion comprises and the direct or indirect interactional amino acid of molecules of interest, and the activity of wherein said modified coral polyp luciferase is detectable.

77. the polynucleotide of claim 76 are in the zone of wherein said residue 2-12, residue 26-47, residue 64-74, residue 86-116, residue 147-157, residue 223-234 or the residue 301-311 that is inserted in corresponding sea pansy luciferase.

78. a carrier, it comprises the polynucleotide of claim 76 or 77.

79. a host cell, it comprises the carrier of claim 78.

80. a modified coral polyp luciferase, it is by the polynucleotide encoding of claim 76 or 77.

81. a method that detects the molecules of interest in the cell, it comprises: a) make to have cell or the in-vitro transcription/sample of translation mixture contacts with the carrier of claim 78, wherein said insertion is discerned by described molecules of interest; And b) detects or measure activity, thereby detect or measure the existence or the amount of the described molecule in the described sample by the modified coral polyp luciferase of described vector encoded.

82. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of modified Lampyridea luciferase, wherein said modified Lampyridea luciferase comprises inner the insertion with respect to the Lampyridea luciferase of corresponding unmodified, described being inserted in the Lampyridea luciferase sequence on the residue of modifying tolerance or in the zone, wherein said insertion comprises and the direct or indirect interactional aminoacid sequence of molecules of interest with respect to the Lampyridea luciferase of corresponding unmodified, and the activity of wherein said modified Lampyridea luciferase is detectable, and wherein said insertion is not at the residue 2-12 of corresponding Lampyridea luciferase, residue 116-126, residue 228-238, residue 262-272, residue 289-308, residue 356-366, residue 432-442, or in the zone of residue 535-546.

83. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of modified Lampyridea luciferase, wherein said modified Lampyridea luciferase comprises the fragment of insertion and Lampyridea luciferase sequence with respect to the Lampyridea luciferase of corresponding unmodified, wherein said fragment has at least 50 of Lampyridea luciferase of described corresponding unmodified in abutting connection with amino-acid residue, wherein said being inserted in the Lampyridea luciferase sequence on the residue of modifying tolerance or in the zone, the activity of wherein said modified Lampyridea luciferase is increased by second fragment of Lampyridea luciferase sequence, described second fragment corresponding to the Lampyridea luciferase of corresponding unmodified different at least 50 in abutting connection with amino-acid residue, wherein said insertion comprises and the direct or indirect interactional aminoacid sequence of molecules of interest, and the residue in wherein said segmental described C-terminal or the not corresponding a kind of zone of N-terminal, the residue 116-126 of the corresponding Lampyridea luciferase in described zone, residue 228-238, residue 262-272, residue 289-308, residue 356-366, or residue 432-442.

84. the polynucleotide of claim 82 or 83, wherein said modified Lampyridea luciferase further comprises the disappearance of the Lampyridea luciferase sequence that is positioned at described insertion N-terminal and/or C-terminal.

85. the polynucleotide of claim 84, wherein said disappearance are no more than 15 residues of Lampyridea luciferase sequence.

86. the polynucleotide of claim 82 or 83, wherein said modified Lampyridea luciferase further comprise the disappearance of Lampyridea luciferase sequence on the sequence of the N-terminal of the Lampyridea luciferase of the described unmodified of correspondence and/or C-terminal.

87. the polynucleotide of claim 86, wherein the disappearance on described N-terminal or described C-terminal is no more than 15 residues of Lampyridea luciferase sequence.

88. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the optional open reading-frame (ORF) that comprises the circular permutation oar foot animal luciferase of insertion, described insertion comprises and the direct or indirect interactional aminoacid sequence of molecules of interest, on the residue that wherein said conversion tolerates modification in oar foot animal luciferase sequence or in the zone, and wherein said being inserted in the oar foot animal luciferase sequence on the different residue of modifying tolerance or in the zone.

89. the polynucleotide of claim 88, wherein said conversion is in the zone of residue 43-53, residue 63-73, residue 79-89, residue 95-105, residue 105-115, residue 109-119, residue 121-131 or the residue 157-168 of the bent firefly luciferase of correspondence.

90. the method for claim 23 or 33, wherein said mixture, host cell, luminous detection or mensuration or its any combination are at about 30 ℃-Yue 47 ℃.

91. the method for claim 31, wherein said host cell, sample, luminous detection or mensuration or its any combination are at about 30 ℃-Yue 47 ℃.

92. the method for claim 74, wherein said cell, active detection or mensuration or its any combination are at about 30 ℃-Yue 47 ℃.

93. claim 23,31 or 33 method, wherein said host cell is with described polynucleotide stable transfection.

94. the method for claim 74, wherein said cell is with described carrier stable transfection.

A 95. existence or active method that detects or measure the molecules of interest in the cell, it comprises: the luminous reaction that comprises the cell with carrier mixture a) is provided, described carrier has the nucleotide sequence of the open reading-frame (ORF) that comprises modified luciferase, wherein said modified luciferase comprises insertion with respect to the luciferase of corresponding unmodified, described being inserted in the luciferase sequence on the residue of modifying tolerance or in the zone, wherein said insertion comprises and the direct or indirect interactional aminoacid sequence of molecules of interest with respect to the luciferase of corresponding unmodified, the activity of wherein said modified luciferase is detectable, and wherein said mixture is at about 30 ℃-Yue 47 ℃; With

B) detect or measure luminous in the described mixture, thereby detect or measure existence, amount or the activity of the described molecule in the described cell.

96. the method for claim 95, wherein said modified luciferase is the beetle luciferase.

97. the method for claim 96, wherein said modified beetle luciferase is the Lampyridea luciferase.

98. the method for claim 96, wherein said modified beetle luciferase is the Pleonomus luciferase.

99. the method for claim 95, wherein said modified luciferase is the circular permutation luciferase.

100. the method for claim 95, wherein said insertion are the ring-type nucleotide binding sites.

101. the method for claim 95, wherein said mixture further comprises one or more test agent.

102. the method for claim 101 is wherein with the luminous comparison in the described luminous and respective mixtures that lacks described one or more test agent in the described mixture.

103. the method for claim 95 wherein makes described mixture be in about 5% CO ₂Condition.

104. identify the active reagent of heterologous sequence that directly or indirectly changes in the modified beetle luciferase for one kind, it comprises:

A) the luminous reaction mixture that comprises the cell of expressing modified luciferase is contacted with one or more reagent, wherein said modified luciferase is by the vector encoded with nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of modified luciferase, wherein said modified luciferase comprises heterologous sequence with respect to the luciferase of corresponding unmodified, on the residue that described heterologous sequence tolerates modification in the luciferase sequence or in the zone, wherein said heterologous sequence comprises and the direct or indirect interactional aminoacid sequence of molecules of interest with respect to the luciferase of corresponding unmodified, the activity of wherein said modified luciferase is detectable, and wherein said mixture is at about 30 ℃-Yue 47 ℃; With

B) identify whether described one or more reagent change the activity of described modified beetle luciferase.

105. the method for claim 104, wherein said modified luciferase is the beetle luciferase.

106. the method for claim 105, wherein said beetle luciferase is the Lampyridea luciferase.

107. the method for claim 105, wherein said beetle luciferase is the Pleonomus luciferase.

108. the method for claim 95 or 105, wherein said cell is with described carrier stable transfection.

109. the method for claim 104, wherein said heterologous sequence are the ring-type nucleotide binding sites.

110. the method for claim 104 wherein is in the condition of about 5%CO2 to described mixture.

111. the method for claim 95 or 104, wherein said modified luciferase are modified coral polyp luciferases.

112. the polynucleotide of claim 40, wherein said insertion comprises the peptide substrates of Serine, Threonine or Tyrosylprotein kinase.

113. the polynucleotide of claim 40, wherein said insertion comprise phosphoserine peptide binding domains, phosphothreonine peptide binding domains or Tyrosine O-phosphate peptide binding domains.

114. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of circular permutation luciferase, described circular permutation luciferase comprises the allogeneic amino acid sequence, described allogeneic amino acid sequence comprises the peptide substrates of Serine, Threonine or Tyrosylprotein kinase, the corresponding therein not conversion luciferase of described luciferase is on the site of modifying tolerance or carry out conversion in the zone, and the activity of wherein said circular permutation luciferase is detectable.

115. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of circular permutation luciferase, described circular permutation luciferase comprises the allogeneic amino acid sequence, described allogeneic amino acid sequence comprises phosphoserine peptide binding domains, phosphothreonine peptide binding domains or Tyrosine O-phosphate peptide binding domains, the corresponding therein not conversion luciferase of described luciferase is on the site of modifying tolerance or carry out conversion in the zone, and the activity of wherein said circular permutation luciferase is detectable.

116. the polynucleotide of claim 114 or 115, wherein said luciferase are the coral polyp luciferases.

117. the polynucleotide of claim 114 or 115, wherein said luciferase are the beetle luciferases.

118. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of modified decapod luciferase, described modified decapod luciferase comprises first fragment of insertion and decapod luciferase sequence with respect to the decapod luciferase of corresponding unmodified, wherein said first fragment has at least 35 of sophisticated decapod luciferase of described corresponding unmodified in abutting connection with amino-acid residue, wherein said being inserted in the sophisticated decapod luciferase sequence on the residue of modifying tolerance or in the zone, the activity of wherein said modified decapod luciferase is increased by second fragment of decapod luciferase sequence, described second fragment corresponding to the sophisticated decapod luciferase of corresponding unmodified different at least 35 in abutting connection with amino-acid residue, wherein said insertion comprises and the direct or indirect interactional aminoacid sequence of molecules of interest.

119. polynucleotide that comprise nucleotide sequence, described nucleotide sequence comprises the open reading-frame (ORF) of modified decapod luciferase, wherein said modified decapod luciferase comprises inner the insertion with respect to the decapod luciferase of corresponding unmodified, described being inserted in the sophisticated decapod luciferase sequence on the residue of modifying tolerance or in the zone, wherein said insertion comprises and the direct or indirect interactional aminoacid sequence of molecules of interest with respect to the sophisticated decapod luciferase of corresponding unmodified, and the activity of wherein said modified decapod luciferase is detectable.

120. the polynucleotide of claim 118, wherein said first fragment has at least 85 residues of 45-at least.

121. the polynucleotide of claim 119 are in the zone of wherein said residue 45-55 that is inserted in corresponding thorn shrimp luciferase or residue 79-89.

122. a method of measuring the one or more sites that in the natural protein modification tolerated, it comprises:

In the protein that may have the natural structure that comprises secondary structure, identify the one or more residues in the described proteinic described aminoacid sequence, described residue is in the zone with a plurality of wetting ability residues, and described zone unlikely forms secondary structure in described natural protein structure.

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