CN106632687A - Fusion protein for screening weak MdmX inhibitor or testing inhibition activity of weak MdmX inhibitor - Google Patents
Fusion protein for screening weak MdmX inhibitor or testing inhibition activity of weak MdmX inhibitor Download PDFInfo
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- CN106632687A CN106632687A CN201611143042.6A CN201611143042A CN106632687A CN 106632687 A CN106632687 A CN 106632687A CN 201611143042 A CN201611143042 A CN 201611143042A CN 106632687 A CN106632687 A CN 106632687A
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- mdmx
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- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Abstract
The invention discloses a fusion protein for screening a weak MdmX inhibitor or testing the inhibition activity of a weak MdmX inhibitor. The testing sequence is an MdmX sequence or a homologous sequence thereof. The combination sequence is the mutant of p53 segment of the MdmX binding domain of p53 sequence. The linker arm sequence is a peptide segment, and the upstream and lower stream of the linker arm sequence are connected to one of the testing sequence and the combination sequence. The emission spectrums of tryptophan in the fusion protein are different when the combination structural domain and the testing structural domain are connected/disconnected; and for the mutant of p53 segment, compared with a wild p53 segment, the interaction between the p53 combination structural domain and the MdmX combination structural domain is weakened, so that the MdmX inhibitor with a weak binding force can compete with the combination sequence to combine the testing sequence, and thus the inhibition degree of the weak MdmX inhibitor on MdmX can be effectively measured.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of suppression for screening MdmX inhibitor or test Mdmx inhibitor
The preparation and application of the recombinant protein of system activity, particularly plants for screening weak MdmX inhibitor or testing weak Mdmx inhibitor
The preparation and application of the recombinant protein of inhibitory activity, be more particularly to a kind of p53 polypeptides and MdmX aminoterminals fusion protein and its
The preparation of analog and its application in the inhibitory activity of screening MdmX inhibitor or test MdmX inhibitor.
Background technology
P53 is a kind of cancer suppressor protein, is sent out in the bioprocess such as cell-cycle arrest, DNA damage reparation and Apoptosis
The effect of waving, and become important cancer target.The suppression canceration activity of p53 is subject to intracellular overexpression MdmX and Mdm2
Suppression, it is related to more than half cancers.It is the target for developing novel cancer medicine to release suppression of the MdmX and Mdm2 to p53
Point.
MdmX and Mdm2 are the important controlling gene albumen in p53 downstreams, and MdmX and Mdm2 is homologous protein, and both structures are high
Degree is similar, but suppresses the mechanism of p53 different.The screening of Mdm2 inhibitor at present and design are more ripe, and many has been entered
Enter clinical investigation phase, such as nutlin-3a modifieds molecule RG7112 will enter II phase clinical research (Ray-Coquard
I,Blay J Y,Italiano A,et al.Effect of the MDM2antagonist RG7112 on the P53
pathway in patients with MDM2-amplified,well-differentiated or
dedifferentiated liposarcoma:an exploratory proof-of-mechanism study[J].The
lancet oncology,2012,13(11):1133-1140).But, the Effective selection and design progress of MdmX inhibitor delay
Slowly.Due to resistance problems (Lane D P, Cheok C F, the Lain S.p53-based cancer of Mdm2 inhibitor
therapy[J].Cold Spring Harbor perspectives in biology,2010,2(9):A001222.), cancer
MdmX in cell can further overexpression, aggravate cancer cell deterioration.Therefore, MdmX inhibitor is found, and with MdmX as base
Plinth and the MdmX/Mdm2 double inhibitors that design are the active drugs for treating nearly half cancer.
There is the binding domain of p53 in the N-terminal of MdmX, the p53 binding domain of MdmX can be with the transcriptional activation structure of p53 albumen
Domain combines, and forms p53/MdmX complexs, and the complex can suppress the transcriptional activity of p53, but the degraded of mediation p53,
Make p53 protein inactivations and induced tumor.
If a kind of compound, the transcriptional activity of p53 with p53-MdmX compound competitive bindings, can be discharged, or with competing
Striving property or irreversibility combine free MdmX, then this compound can be released or suppression of the past release MdmX to p53, be made
The effect that p53 plays suppression cancer is obtained, therefore, this compound can be considered the medicine for the treatment of or the alleviation of cancer.
Some are disclosed in prior art by testing the technical side of MdmX inhibitor with p53 competitive bindings MdmX
Case.
Chinese patent literature CN105936646 (publication date：2016.09.14) disclose the mini albumen of a class and its application.
The mini albumen with the 3 α helical bundles in albumin combination domain as skelemin, and in the three-dimensional structure table of the skelemin
Introducing in face, N-terminal, C-terminal at least one has cationic amino acid, and cationic amino acid is at least connected with p53 described in Jing
The key amino acid site combined with MDM2/MDMX.The mini albumen has concurrently and is combined into born of the same parents and with albumin, MDM2/MDMX
Ability, and can well suppress the interaction between p53 and Mdm2/MdmX, can be used to prepare cancer therapy drug etc., can be used to advise
Modelling is produced and applied.The fluorescence polarization detection of the combination of little albumen and Mdm2/MdmX.
Chinese patent literature CN103923067 (publication date：2014.07.16 a kind of small molecule of MdmX/Mdm2) is disclosed
Inhibitor, also relates to a kind of preparation method of the micromolecular inhibitor of MdmX/Mdm2, the molecule inhibitor compounds energy
Suppress the interaction of MdmX protein and p53 albumen, can also suppress the interaction of Mdm2 protein and p53 albumen, the party
MdmX inhibitor screenings are determined using fluorescence polarization method in method.
Come with some shortcomings in the model of the inhibitor of above-mentioned detection MdmX：
1.p53 fragments are separately added into test system with MdmX fragments, and the mol ratio of the two is difficult to be accurately determined, will
Bring larger systematic error or random error.
2. p53 fragments are marked using fluorescein in scheme, this will be made in each marking operation, fluorescein molecule and p53
The mol ratio of fragments molecules is not quite identical, can bring about larger random error, is just more difficult to carry out accurately quantitative study.
3. p53 fragments are marked using fluorescein in scheme, and fluorescein holds easy decomposition and degeneration and loses glimmering under visible light
Light function, this requires that corresponding operating lucifuge, photic decomposition can reduce the accuracy of detection method, it is desirable to which lucifuge can increase operation
Difficulty and experimental cost.
4., due to the hydrophobicity of fluorescein, the accuracy of detection method and data is reduced.
5.p53 fragments and MdmX need to prepare respectively, can increase the complexity of operation.
Although 6. MdmX and Mdm2 is homologous protein, both three-dimensional structures are highly similar, existing Mdm2 little molecules in inhibiting
Agent is very weak to the inhibition of MdmX.
7. MdmX inhibitor visibly different for amount of force is tested with same model, it is impossible to distinguish strong
MdmX inhibitor is in weak MdmX inhibitor, it is often more important that, the weaker MdmX inhibitor of active force is because of can not be effectively
Competitive binding p53 domains are without detecting.
The content of the invention
Protein has a light absorbs phenomenon in the range of 270～300nm absorbing wavelengths, this optical absorption property mainly from
Tryptophan, tyrosine and phenylalanine in protein, so the protein containing the amino acid has natural fluoresence property.
When not having tryptophan in protein, its maximum emission wavelength is about at 304nm, and the position of its fluorescence spectrum is not divided greatly
The impact of sub- conformation；Hydrophobic ring when there is tryptophan in protein, residing for its fluorescent emission maximum wavelength and tryptophan
Border degree has obvious relation, its fluorescence maximum emission wavelength in the range of 308～355nm (Vivian, J.T.Callis, P.R.,
Mechanisms of tryptophan fluorescence shifts in proteins.HYPERLINK"https://
Www.ncbi.nlm.nih.gov/pmc/articles/PMC1301402/ " Biophys J.2001,80 (5):2093–
2109), when tryptophan is imbedded in hydrophobic pocket completely, fluorescence emission wavelengths in 321nm or so, when being completely exposed to water phase,
Fluorescence emission wavelengths are in 350nm or so.
The complex crystal structure that the MdmX (23～111 sequence) for having parsed and p53 (15～29 sequence) is formed
(PDB ID:3DAB, referring to document：Popowicz G,Czarna A,Holak T.Structure of the human Mdmx
protein bound to the p53tumor suppressor transactivation domain[J].Cell
Cycle,2008,7(15):2441-3.) show, the transcriptional activation domain (15～29 sequence) of p53 passes through 3 in alpha-helix form
Key amino acid (Phe19, Trp23 and Leu26) interacts with the N- ends hydrophobic region of MdmX.3 crucial works on MdmX
Three binding pockets, i.e. F19 pockets, W23 pockets and L26 pockets are constituted with site together with its neighbouring amino acid.
N- terminal domains (23～111 sequence) the corresponding domain of people source MdmX is referred to as into p53 integrated structures herein
Domain (is defined as N-MdmX), is called that MdmX binding structural domains (are defined as by the corresponding domain of 15～29 sequences of people source p53
p53p)。
To solve at least the above technical problem, the present invention provides one kind and suppresses for screening MdmX inhibitor or test MdmX
The fusion protein of the inhibitory activity of agent.Its core idea is as mentioned below.
P53p is connected with N-MdmX by one section of linking arm amino acid sequence (being defined as XX), fusion protein is formed, should
(23 herein represent the tryptophan from the 23rd of wild-type p 53 protein to the tryptophan Trp23 of p53p fragments in fusion protein
Position, and the position in fusion protein is not represented, similarly hereinafter), it is both the key amino acid combined with N-MdmX, it is again that endogenous is glimmering
Light probe.(, its fluorescent emission maximum wavelength will reflect the binding ability of p53p fragments and N-MdmX.Due to the tryptophan it is buried
(Popowicz G, Czarna A, the Holak T.Structure of the human in N-MdmX binding pocket hydrophobic pockets
Mdmx protein bound to the p53tumor suppressor transactivation domain[J].Cell
Cycle,2008,7(15):2441-3.), its fluorescent emission maximum wavelength is in 321nm.When MdmX inhibitor competitively with this
After the p53 binding structural domains of the MdmX parts in fusion protein combine, part or all of p53p will be separated with N-MdmX, this
When tryptophan Trp23 be completely exposed to water phase, the fluorescence signal intensity at 321nm will increase with inhibitor concentration and occur aobvious
Change is write, and there is certain mathematical, while fluorescence emission wavelengths are also to the red shift of 350nm directions.
Therefore, in MdmX inhibitor fore-and-aft survey fusion protein system to be measured is added 321nm launching lights Strength Changes,
Size of the MdmX inhibitor to the inhibitory activity/inhibition level of MdmX can be calculated, and then, the fusion protein serves screening
The effect of MdmX inhibitor or the effect of the inhibitory activity of test MdmX inhibitor.
Against existing technologies, the fusion protein at least has the following advantage：
MdmX binding structural domains in the p53 binding structural domains of MdmX and p53 due to being present on same peptide chain, they
Molal quantity be equal, be fixed with the relative amount of MdmX equivalent to the p53 parts competed with MdmX inhibitor, this will
Reduce the random error between batch operation.This 1：1 pattern also can more accurately reflect the part of screening and p53p parts
The ability of competitive binding MdmX protein surface hydrophobic pockets.
The fusion protein does not need mark fluorescent probe, the error that would not come because of fluorescently-labeled degree different band.
Tryptophan in fusion protein does not meet with light decomposition yet, and tryptophan and the mol ratio of fusion protein are changeless, and
The emission spectrum of corresponding tryptophan is different when p53p is in combination or free state from the tryptophan that MdmX is combined.Therefore, no
In needing lucifuge equipment, and above-mentioned fusion protein, free tryptophan Trp23 is tied with free p53 parts and with MdmX parts
The MdmX inhibitor of conjunction (assumes initially that the mol ratio that MdmX inhibitor is combined with MdmX is 1：1) it is equimolar, the color of combination
Propylhomoserin Trp23 and p53 parts are equimolar with the compound of MdmX parts, and corresponding launching light is 321nm or so, that is,
Say that the situation of change for adding the intensity of 321nm launching lights before and after MdmX inhibitor is with the degree of MdmX inhibitor competitive bindings
Corresponding, hinge structure has less systematic error or random error.
To achieve these goals, this is used for the fusion of the inhibitory activity for screening MdmX inhibitor or test MdmX inhibitor
The core technology scheme of albumen is：
The fusion protein includes cycle tests, binding sequence and linking arm sequence；
The cycle tests is MdmX sequences or the MdmX fragments comprising the p53 binding structural domains in MdmX sequences；
The binding sequence be p53p sequences, the p53 fragments comprising the MdmX binding structural domains in p53 sequences or keep with
MdmX has the mutant of the p53 fragments comprising the MdmX binding structural domains in p53 sequences of binding activity；
The linking arm sequence is a peptide fragment, the upstream and downstream of the linking arm respectively with the cycle tests and the knot
Close the connection of one of sequence；The amino acid sequence of the linking arm is not involved in the formation of the secondary structure of the fusion protein or dimension
Hold；The length and flexibility of the linking arm be enough to any surface part of the space structure for making the cycle tests and the combination
Any surface part of the space structure of sequence spatially has an opportunity to contact.
Alternatively, the upstream or downstream of the fusion protein has the sequence label for Protein Separation and/or purifying.
Further, due to people source p53 (GenBank:AB082923.1 there are four tryptophans, only one of which color in)
Propylhomoserin falls into its MdmX binding structural domain；People source MdmX gene (GeneBank:AF007111.1 there are seven tryptophans in), not
Fall into p53 binding structural domains.Further technical scheme is, by the p53 domains of the MdmX domains of p53 and MdmX by connecting
Arm connection is connect, and tryptophan is not contained in linking arm.So, only has unique trp residue, the color ammonia in whole fusion protein
The fluorescence spectrum of acid changes with the environment of tryptophan.It is exactly specifically when p53 binding structural domains and MdmX binding structural domains
When combining, the unique trp residue in fusion protein is ensconced in hydrophobic pocket, and fluorescence emission wavelengths are in 321nm or so.
After MdmX inhibitor is combined with the p53 binding structural domains in MdmX, just by the unique color ammonia in part or all of fusion protein
Sour residue is exposed to water phase, and fluorescence emission wavelengths are in 350nm or so.In such cases, before and after MdmX inhibitor to be measured is added
The intensity of 321nm launching lights in measurement fusion protein system, avoids not combine with MdmX inhibitor and change the characteristics of luminescence
Tryptophan tryptophan luminescent spectrum impact, also can just make experimental results sensitiveer.
Specifically, for above-mentioned purpose, the technical scheme is：
Fusion protein formula p53p-XX-N-MdmX is represented, it is characterised in that
P53p represents the binding sequence SEQ ID NO.1, specially：SQETFSDLWKLLPEN；
XX represents the linking arm sequence SEQ ID NO.2, specially：GSGSSENLYFQ；
N-MdmX represents the cycle tests SEQ ID NO.3, specially：
GSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSP
LYDMLRKNLVTLAT；
In p53p-XX-N-MdmX fusion proteins, the primary structure of amino acid shows, a color ammonia is only existed in its sequence
Acid, and it also corresponds to the key amino acid that p53 polypeptides and N-MdmX interact, can be according to the endogenous fluorescence of tryptophan
Feature come screen with p53p-XX-N-MdmX fusion proteins with the micromolecular compound of p53 polypeptide competitive binding MdmX.
For the convenience purified after expressing fusion protein, can merge in fusion protein fused upstream His-Tag sequence
Histidine-tagged fusion protein sequence can be：
GSSHHHHHHGSSQETFSDLWKLLPENGSGSSENLYFQGSQINQVRPKLPLLKILHAAGAQGEMFTVKEV
MHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT
Different MdmX inhibitor has different, stronger for binding ability MdmX from the binding ability of MdmX and suppresses
Agent, it fully can be combined MdmX parts with p53 partial competitions.And the MdmX inhibitor weaker for binding ability, it can
The degree that detectable competitive binding occurs can be not enough to.Therefore above-mentioned fusion protein cannot effectively detect such MdmX
Inhibitor.
In order to further enable above-mentioned fusion protein detect the MdmX inhibitor of weak binding power, a kind of scheme is by prominent
Become MdmX binding structural domains, make the adhesion between MdmX binding structural domains and p53 binding structural domains die down, for example will be with MdmX
With reference to one of p53p key amino acids Phe19 or Leu26 (this 19 or 26 represents in corresponding p53p amino acid in wild type
Position, similarly hereinafter) mutation, make MdmX binding structural domains and the combination of p53 binding structural domains die down, to make adhesion weaker
MdmX inhibitor is able to and MdmX binding structural domain competitive binding p53 binding structural domains, makes fusion protein can be used in screening
The inhibitory activity of the weaker MdmX inhibitor of the weaker MdmX inhibitor of adhesion or test adhesion.Weakened using the two
Model can further screen micromolecular compound of the affinity in the range of micro-molar concentration.
Specifically, the technical scheme is：
The binding sequence that p53p is represented is sported：SQETASDLWKLLPEN.
The binding sequence that p53p is represented is sported：SQETFSDLWKLAPEN.
Present invention also offers a kind of nucleotide sequence, the nucleotide sequence can be expressed described in as above each scheme
For screening the fusion protein of the inhibitory activity of MdmX inhibitor or test MdmX inhibitor.
Alternatively, above-mentioned nucleotides sequence is classified as SEQ ID NO.04 (for expressing the base sequence of p53p-XX-N-MdmX
Row), SEQ ID NO.05 are (for expressing p53pF19AThe base sequence of-XX-N-MdmX) or SEQ ID NO.06 (for expressing
p53pL26AThe base sequence of-XX-N-MdmX) shown in sequence.
Present invention also offers a kind of fusion for screening the inhibitory activity of MdmX inhibitor or test MdmX inhibitor
The expression vector of albumen, the expression vector can suppress by described in host expresses as above each scheme for screening MdmX
The fusion protein of the inhibitory activity of agent or test MdmX inhibitor.
Alternatively, the carrier is pET28a carriers, and the host is e. coli bl21 (DE3).
Present invention also offers pressing down for screening MdmX inhibitor or test Mdmx described in a kind of as above each scheme
The fusion protein of the inhibitory activity of preparation screening MdmX inhibitor or test Mdmx inhibitor inhibitory activity purposes, its
In, the MdmX inhibitor is can be with the inhibitor of P53 competitive binding MdmX.
More specifically, nutlin-3a also has certain as a kind of generally acknowledged stronger Mdm2 inhibitor to MdmX
Inhibitory action, the K of nutlin-3a and MdmX and Mdm2dValue be respectively 28 μM, 0.7 μM (Laurie N A, Donovan S L,
Shih C S,et al.Inactivation of the p53pathway in retinoblastoma.[J].Nature,
2006,444(7115):61-6).Using nutlin-3a as the generation for identifying p53p-XX-N-MdmX fusion protein model validations
Table small molecule is rational.
In addition, the 32 Mdm2 micromolecular inhibitors that can be obtained with business are further reflected to this model
It is fixed.
In order to realize above-mentioned purpose, following technical measures are present invention employs：
The present invention constructs p53p-XX-N-MdmX, p53p using molecular cloningF19A- XX-N-MdmX and p53pL26A-XX-
N-MdmX fusion protein expression plasmids, and prepared using colibacillus engineering BL21 (DE3) expression, purifying.Using F-7000
Sepectrophotofluorometer analyzes the competitive knot of nutlin-3a and 32 Mdm2 inhibitor and p53p-XX-N-MdmX fusion proteins
Conjunction ability is verified with this model to screen the feasibility and validity of inhibitor with this.Afterwards by key amino acid Phe19 and
Leu26 sports respectively Ala and constructs p53pF19A- XX-N-MdmX and p53pL26ATwo fusion proteins of-XX-N-MdmX screen mould
Type, and the stability of mutation rear fusion protein is demonstrated using protein denaturant guanidine hydrochloride, as a result also demonstrate that crucial amino
The N-terminal binding abilities of p53p and MdmX weaken after acid mutation, afterwards, by the LS45/55 types fluorescence/phosphorus of PerkinElmer
Light/luminescence spectrophotometer screens nutlin-3a and 32 Mdm2 inhibitor simultaneously with three kinds of models, and the selection result shows,
The model for improving is significantly more efficient to screening the low inhibitor of affinity.
Description of the drawings
Fig. 1 is p53p-XX-N-MdmX fusion protein tertiary structure figures.
Fig. 2 is that p53p-XX-N-MdmX fusion protein expression plasmids build three step PCR reaction conditions.
Fig. 3 is sample SDS- after Ni-NTA chromatographies during p53p-XX-N-MdmX fusion protein purifications
PAGE glue figure (a), by gel chromatographic columnses (Supredex 16/60075pg) rear AKTA collection of illustrative plates (b) and SDS- are further purified
PAGE glue figure (c).
Fig. 4 is p53p-XX-N-MdmX fusion proteins in λEXFor 278nm when, λEMIt is glimmering in the range of 290nm～500nm
The light curve of spectrum (a) and in λEMFor 321nm when, λEXIn excitation light spectral curve (b) of 245nm～300nm.
It is OD that Fig. 5 is nutlin-3a titration concentrations280nmDuring=0.1 p53p-XX-N-MdmX fusion proteins, λEX
278nm, as nutlin-3a concentration increases, λEMFor fluorescence intensity change curve (a) and λ in the range of 290nm～500nmEM
The matched curve (b) of peak fluorescence intensity trend at 321nm.
Fig. 6 is competitive binding ideographs of the nutlin-3a to p53p-XX-N-MdmX fusion proteins.
Fig. 7 is λEXIn 278nm, nutlin-3a (a) and p53p (b) are in λEMFluorescence in the range of 290nm～500nm
Spectral radiation curves.
It is OD that Fig. 8 is DMSO (without nutlin-3a) titration concentration280nmDuring=0.1 p53p-XX-N-MdmX fusion proteins,
λEXDuring=278nm, as DMSO concentration increases, λEMFor fluorescence intensity change curve (a) and λ in the range of 290nm～500nmEM
The matched curve (b) of peak fluorescence intensity at 321nm.
Fig. 9 is that nutlin-3a and 32 Mdm2 micromolecular inhibitor of contrast is to p53p- using nutlin-3a as control
The Competitive assays constant Ki values of XX-N-MdmX fusion proteins.
Figure 10 is p53pF19A- XX-N-MdmX (a) and p53pL26A- XX-N-MdmX (b) fusion proteins tertiary structure is simulated
Figure.
Figure 11 is that p53p-XX-N-MdmX fusion protein mutant expression plasmid builds three step PCR reaction conditions.
Figure 12 is p53pF19ADNA glue figures in-XX-N-MdmX fusion protein molecule cloning procedures.
Figure 13 is λEXIn 278nm, with the increase of concentration of guanidine hydrochloride, p53p-XX-N-MdmX (a), p53pF19A-XX-
N-MdmX (b) and p53pL26AThe fluorescence emission spectral curve of-XX-N-MdmX (c) fusion proteins is in λEM290nm～500nm's
Variation tendency.
Figure 14 is that three kinds of fusion proteins are respectively in λ as concentration of guanidine hydrochloride increasesmaxAt 321nm, 331nm and 330nm
Peak fluorescence intensity changing trend diagram.
Figure 15 is, with p53p-XX-N-MdmX fusion proteins as control, to use p53pF19A- XX-N-MdmX and p53pL26A-XX-
32 Mdm2 micromolecular inhibitors of N-MdmX fusion proteins model discrimination and nutlin-3a are showing to be mutated rear fusion protein to little
The change of molecule binding ability.
Specific embodiment
In order to preferably explain technical scheme, each enforcement of the present invention is discussed in detail below in conjunction with the accompanying drawings
Example.Following examples are used to further illustrate the present invention, but should not be construed as fixation or restriction to the present invention.If not referring in particular to
It is bright, in embodiment technical characteristic used could alternatively be with the premise of without departing substantially from inventive concept equivalent or identity function or
Other techniques known in the art features of effect, any combination of the disparate modules of the present invention each falls within the protection model of the present invention
Enclose.
Embodiment 1：P53p-XX-N-MdmX fusion protein expression plasmids build
The crystal of the N-terminal p53 binding structural domains of MdmX binding structural domains p53p and MdmX of p53 to have announced
Structure (PDB ID：(Structure of the human Mdmx protein bound to the are referred to based on 3DAB)
p53tumor suppressor transactivation domain,Popowicz,G.M.,Czarna,A.,Holak,T.A.
(2008)CellCycle 7:2441-2443), we establish following p53p-XX-N-MdmX fusion proteins model.
The amino acid sequence of p53p-XX-N-MdmX fusion proteins is as follows (to remove albumen after label G SSHHHHHHGS
The tertiary structure of matter with PymolWin simulate simulation drawing is as shown in Figure 1)：
GSSHHHHHHGSSQETFSDLWKLLPENGSGSSENLYFQGSQINQVRPKLPLLKILHAAGAQGEMFTVKEV
MHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT
Wherein, SQETFSDLWKLLPEN from p53 MdmX binding structural domain sequences,
GSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSP
From the p53 binding structural domain sequences of MdmX, GSGSSENLYFQ is linking arm amino acid sequence to LYDMLRKNLVTLAT.
GSSHHHHHHGS contributes to the sequence label that fusion protein is isolated and purified, and the GSS or GS of both end of which are flexible amino acid
Fragment.
It is inclined according to e. coli bl21 (DE3) codon in order to build above amino acid sequence by molecular cloning
Good property has synthesized the DNA sequence dna of N-MdmX, and its nucleotide coding sequence is as shown in SEQ ID NO.4.
By the nucleotides by Nco I and EcoR I double digestions, pET28a plasmids (the double enzymes of Nco I and EcoR I are connected to
Cut) on, by molecular cloning flow process, the recombinant plasmid of the coded sequence containing N-MdmX is obtained, it is named as pET28a-N-
MdmX。
In order to build and expressing above fusion protein, according to the pET28a-N-MdmX plasmids for having built, following drawing is devised
Thing sequence：
Primer1:5'-CAT GCC ATG GGC AGC AGC CAT CAC CAT CAT CAC CAC GGC AGC-3'
Primer2:5'-GTT TCC ACA GAT CGC TAA AGG TTT CCT GGC TGC TGC CGT GGT
GAT GAT G-3'
Primer3:5'-TTA GCG ATC TGT GGA AAC TGC TGC CGG AAA ATG GCA GCG GCA
GCA GCG A-3'
Primer4:5'-CGG GAT CCC TGA AAA TAC AGG TTT TCG CTG CTG CCG CTG CCA-3'
Fragment amplification is carried out according to following system with the primer for designing.
PCR system 1:
PCR1 reaction conditions are as shown in Fig. 2-a；
PCR system 2:
PCR2 reaction conditions are as shown in Fig. 2-b；
PCR system 3:
PCR3 reaction conditions are as shown in fig. 2-c；
PCR3 product agents box is reclaimed, recovery product Nco I and the double digestions of BamH I, as Insert Fragment.
PCR recovery product digestion systems：
37 DEG C, 3h, digestion products kit (OMEGA, Plasmid Mini Kit I) is reclaimed.
In the reaction of above three PCR, masterplate, two primer base complementary pairings are not needed in PCR1 and PCR2 reaction systems
Extend, two sections of nucleotide sequences are formed, without recovery product.With PCR1 and PCR2 products as masterplate, fusion DNA vaccine experiment is carried out,
For PCR3 experiments.And then obtained, containing histidine-tagged, p53p and XX fusion nucleotide sequences, after digestion, being connected to
On pET28a-N-MdmX plasmids.
By pET28a-N-MdmX plasmids Nco I and the double digestions of BamH I, as carrier.
Plasmid vector digestion system：
37 DEG C, 3h, digestion products are cut glue kit (health be century, Gel Extraction Kit) and are reclaimed.
It is 1 that coupled reaction presses carrier DNA with Insert Fragment DNA mol ratios:3 ratios mix, and add high with system identical
Effect DNA ligase (TOYOBO, Ligation high Ver.2), connects 3h by 16 DEG C.
Whole connection products are proceeded in 50 μ l DH5 α (top10) Escherichia coli, ice bath 30min, after 42 DEG C of heat shock 90s
Ice bath is put back to immediately, and 2min adds the activation of 200 μ l LB fluid nutrient mediums, 37 DEG C, applies again after the recovery of 200rpm shaken cultivations 1h
LB solid medium flat boards containing kanamycins, 37 DEG C are inverted 12～16h of culture, observe colony growth situation.
With the presence of single bacterium colony, the identification of bacterium solution PCR is carried out, band is correct, upgrading grain pET28a-p53p-XX-N-MdmX, matter
Granzyme cuts identification, and band is correct, send plasmid order-checking, and sequencing result is correct, and preservation of bacteria strain is stand-by.
PET28a-p53p-XX-N-MdmX sequencing results nucleotide sequence is as shown in SEQ ID NO.4.
Embodiment 2：P53p-XX-N-MdmX expressing fusion proteins and purifying
PET28a-p53p-XX-N-MdmX plasmids are proceeded to into e. coli bl21 (DE3)；Single bacterium colony is chosen to 2ml LB K+(Kanamycin) culture medium, 37 DEG C, 200rpm incubated overnights；Take 500 μ l bacterium solutions and proceed to 50ml LB K+Culture medium, 37 DEG C,
200rpm cultivates 3h；50ml bacterium solutions are proceeded to into 1L LB K entirely+Culture medium, 37 DEG C, 200rpm cultures, until cell concentration reaches
OD280nm=0.8 or so, IPTG (final concentration 0.4mM) inductions are added, (centrifuge parameters set about 20h collects thallines during collects thalline
Be set to 3500rpm, 30min, 4 DEG C).
Ultrasonic cell-break crusher machine cell.According to thalline volume：Buffer solution volume=1:5 ratio, adds
BuffeA buffer solutions (50mM Na2HPO4, 200mM NaCl, 10mM imidazoles, 1mM BME (beta -mercaptoethanol), pH8.0), parameter
It is set to：Total time 2min；Ultrasonic time 2s；Off time 4s；24 DEG C of temperature alarming；(the Ningbo Xin Zhisheng of ultrasonic power 40%
The JY92-IIN ultrasonic cell disruptors of thing Science and Technology Co., Ltd.).Repeat 2～3 times, until bacterial cell disruption it is complete.Will
Clasmatosis liquid is balanced with the packing of 50ml centrifuge tubes, centrifugation, 30min, 4 DEG C, 18000rpm, and supernatant, precipitation are collected in respectively
In reagent bottle (pipe).
Broken supernatant is crossed into Ni-NTA chromatographic columns (Fig. 3 a) and gel chromatographic columnses (Supredex 16/ by AKTA pure
60075pg or Superdex 26/60075pg, Fig. 3 b, c), collect the albumen of purifying, concentration, packing, per a 200 μ l, liquid nitrogen
It is quick-frozen, it is frozen in -80 DEG C of refrigerators.
Embodiment 3：P53p-XX-N-MdmX fusion protein model spectrofluorimetries
The p53p-XX-N-MdmX albumen of a 200 frozen μ l is taken, quick-thawing uses phosphate buffer diluted protein
Concentration is to OD280nm=0.1.400 μ l are taken into quartz colorimetric utensil, fluorescent scanning is carried out on F-7000 sepectrophotofluorometers.
According to the endogenous fluorescence feature of tryptophan, λ is takenEXFor 278nm, λ is scannedEMFluorescence intensity in the range of 290nm～500nm, sends out
Existing maximum fluorescence intensity is at 321nm (Fig. 4 a).Therefore determine λEMFor 321nm, λ is scannedEXFor the fluorescence excitation spectrum of 245nm～300nm
Curve, as a result as shown in Figure 4 b, will also realize that in λEXIn 278nm, fluorescence intensity is maximum, therefore determines a length of 278nm of excitation light wave.
Embodiment 4：Using p53p-XX-N-MdmX fusion protein fluorescence spectrometry nutlin-3a and MdmX affinity
Take frozen in -80 DEG C of p53p-XX-N-MdmX albumen, quick-thawing, be diluted to albumen with phosphate buffer dense
Spend for OD280nm=0.1,400 μ l are taken into quartz colorimetric utensil, take λ on F-7000 sepectrophotofluorometersEXFor 278nm, λEM
Fluorescent scanning is carried out in 290nm～500nm scopes.According to nutlin-3a in such as table 1 below (mother liquor 2.5mM, be dissolved in DMSO) concentration
Gradient is titrated (often add a nutlin-3a to be well mixed).
The nutlin-3a of table 1 titrates p53p-XX-N-MdmX fusion protein concentration gradients
Competitive binding ideographs of the Nutlin-3a to p53p-XX-N-MdmX fusion proteins, as shown in Figure 6.
It is as shown in Figure 5 a impact of the difference nutlin-3a concentration to fusion protein fluorescence spectrum, with nutlin-3a drops
Determine the increase of concentration, the fluorescence intensity at maximum emission wavelength 321nm occurs significant changes.In figure 5b with nutlin-3a
It is standard for 0 μM of peak fluorescence intensity, with nutlin-3a change in concentration, in the fluorescence intensity level variation tendency of 321nm
Figure, is fitted to trend curve according to equation below, and fitting result can obtain inhibition constants of the nutlin-3a to N-MdmX
(KiValue, competition binding dissociation constant), fitting result is as shown in Figure 5 b.
Fitting formula is：fi=I/ [I+Ki(1+LT/Kd)] (refer to Chen Y, Prusoff W H.Relationship
between the inhibition constant and the concentration of an inhibitor that
Cause a 50%inhibition of an enzyme reaction [J] .Biochem Pharmacol, 1973,22:
3099-3108.)；Wherein, fiThe part inhibition level competed N-MdmX for inhibitor (is change in fluorescence relative intensity, specifically
Unit is percentage, sees below and illustrates).I is the inhibitor concentration (μM) increased in Reverse transcriptase；Ki is competitive dissociation
The affine dissociation constant of constant, i.e. inhibitor and p53p competitive binding N-MdmX；LTFor the concentration (μ of p53p in fusion protein
M)；KdFor dissociation constant, i.e. p53p and the affine dissociation constant of N-MdmX.
And inhibitor is to N-MdmX Competitive assays degree fiCan be reflected by the pad value of fluorescence, i.e. fi=(A1-y)/
(A1-A2), wherein A1 is fluorescence intensity maximum in matched curve, and A2 is fluorescence intensity minimum of a value in matched curve, and y is fitting
Any point on curve, the formula after integration is：Y=A1-X (A1-A2)/[X+Ki (1+LT/Kd)]。
Can significantly judge, as the concentration of nutlin-3a increases, there are two obvious peak shape ripples in figure from Fig. 5 a
It is dynamic, λEMFor 321nm and λEMFor 350nm～450nm, wherein λEMAt 321nm to be peak that fluorescence intensity is gradually lowered, the above
Analyze with being clarified above, the change at the peak is the Strength Changes of the transmitting fluorescence of tryptophan.And in the range of 350nm～450nm
Presence is the side peak that a fluorescence intensity gradually increases, by the way that individually respectively to small molecule nutlin-3a and p53p, (synthesis contains
Ten five amino acid polypeptides：SQETFSDLWKLLPEN) phosphate system (without p53p-XX-N-MdmX, other are consistent)
The checking of middle peak fluorescence intensity, it is by nutlin-3a itself to judge that reason occurs in the side peak located in the range of 350nm～450nm
Fluorescence cause or with p53p competitive bindings after p53p is free causes.
P53p-XX-N-MdmX identical test conditions are titrated with nutlin-3a, λ is takenEXFor 278nm, λEMFor 290～
500nm, as a result as shown in Fig. 7 a (nutlin-3a) and 7b (p53p).Shown in Fig. 7 a, λmaxFor 375nm, shown in Fig. 7 b, λmaxFor
350nm, it may be determined that fluorescence emission wavelengths be 350nm～450nm at side peak be with the titration of nutlin-3a,
The fluorescence intensity that nutlin-3a is introduced, therefore can be to ignore irrespective in this experiment test and result of calculation.
Take 400 μ l phosphate buffers and be diluted to OD280nm=0.1 p53p-XX-N-MdmX albumen is done without nutlin-
The blank experiment of 3a, except without nutlin-3a, other experiment conditions are completely the same with above-mentioned.Such as Fig. 8 a are (glimmering for experimental result
Intensity variation) and Fig. 8 b (peak fluorescence intensity is with change in concentration) shown in.
Nutlin-3a titration results illustrate that nutlin-3a and p53p has competitive binding pass to the binding site of MdmX
System, just causes transmitting peak fluorescence intensity of the tryptophan at 321nm to reduce obvious.The present invention will be tied further with known to 32
The micromolecular compound of conjunction ability makees further checking.
Embodiment 5：Using p53p-XX-N-MdmX fusion proteins fluorescence method and polarized fluorescence method (FP) screening small molecule
Compound storehouse
(1) inhibitor used is tested：
The Mdm2 micromolecular inhibitors that micromolecular compound storehouse can obtain comprising 32 business.Its structure can be found in table 2.
Compound structure and its two kinds of K in the micromolecular compound storehouse of table 2iMeasurement result
(2) p53p-XX-N-MdmX fusion proteins Fluorimetric screening test：
With reference to nutlin-3a titration schedules (details is referring to embodiment 4), with phosphate buffer by p53p-XX-N-MdmX
Albumen is diluted to OD280nm=0.1, take 400 μ l and be placed in quartz colorimetric utensil.By 32 small molecules each according to concentration in such as table 3 below
Gradient titration (32 small molecule mother liquid concentrations are 2.5mM, are dissolved in DMSO).
3 32 small molecules of table titrate p53p-XX-N-MdmX fusion protein concentration gradients
Obtain after titration with little molecular concentration increase, in λEMIt is peak fluorescence intensity at 321nm with change in concentration
Ki values (small molecule dissociation constant) carry out linear fit according to formula.The Ki values of concrete each molecule are arranged referring to table 2 the 3rd.
The fitting Ki values of the fitting Ki values of 32 small molecules and nutlin-3a are carried out collecting comparing, then compares them
Competition binding ability and nutlin-3a contrast.Comparing result is as shown in Figure 9.
(2) p53p and N-MdmX polarized fluorescences (FP) screening method：
P53p polypeptides (15～29 amino acids) are marked into (fluorescein- by fluorescein
GSGSSQETFSDLWKLLPEN,Flu-p53p)。
PET28a-N-MdmX plasmids are proceeded to into BL21 (DE3), 0.4mM IPTG abduction deliverings simultaneously purify (expression and purification
Method is with reference to embodiment 2), liquid nitrogen flash freezer is frozen in -80 DEG C of refrigerators.
The buffer solution of FP experiments is PBS (137mM NaCl；2.7mM KCl；10mM Na2HPO4；2mM KH2PO4；
PH7.4), 0.025%Tween-20；0.5%BSA；pH7.5.
32 micromolecular compounds and nutlin-3a are diluted to into final concentration of 2mM with DMSO.
Micromolecular compound and nutlin-3a that 3 μ l are diluted with DMSO are taken, the buffer solution dilution small molecule of 47 μ l is added
Final concentration of 0.12mM.20 μ l micromolecular compounds are taken with liquid-transfering gun be placed in 96 orifice plates according to the order for having marked, each little point
Son does and once repeats, totally 66 hole.Using not plus micromolecular compound similarity condition DMSO as control, prepare 4 holes.
N-MdmX albumen quick-thawings are taken, 14000rpm, 10min, are centrifuged by 4 DEG C.
Prep solution 1 includes 15nM Flu-p53p polypeptides and OD280nm=0.1 N-MdmX protein solutions, solution 2 is included
The Flu-p53p polypeptide solutions of 15nM.
Solution 1 is added wherein holes in 66 holes for having micromolecular compound and the control wells for not adding micromolecular compound
(as negative control), per the μ l of hole 40；Solution 2 is added in 2 holes in control wells (as positive control), per the μ l of hole 40.
96 orifice plate 200g are centrifuged into 2min, in the dark mixed at room temperature 30 minutes.
Detected using the EnVision multiple labeling micropores board detector of PerkinElmer, take exciting light for 555nm, fluorescence
For 632nm detections.Analysis of test results formula is：(negative control-sample value)/(negative control-positive is right for inhibiting rate %=
According to) (Zhang Q, Lu H.Identification of small molecules affecting p53-MDM2/MDMX
interaction by fluorescence polarization[J].p53Protocols,2013:95-111.), as a result such as
Shown in the row of table 2 the 4th.
The following is the analysis of the result reflected to Fig. 9 and Biao 2.By taking Cpd20, Cpd23, Cpd28, Cpd31 as an example, this four
Individual compound is that, with Isosorbide-5-Nitrae-benzene phenodiazine -2,5- (1H, 4H)-diketone is a class compound of frame design (with the frame design
One class Mdm2 inhibitor, Novel Isosorbide-5-Nitrae-benzodiazepine-2,5-diones as Hdm2antagonists with
Improved cellular activity), and pass through their Ki values respectively 6.32 μM, 10.33 μ with MdmX that FP is measured
M, 3.58 μM, 10.63 μM, its result and the K that this four compounds fitting is screened with p53p-XX-N-MdmX fusion proteinsiValue
Respectively 5.90 μM, 9.79 μM, 2.07 μM, 8.29 μM it is basically identical.
By taking Cpd4 and CPd6 as an example, from figure it can be seen that the K of Cpd4 and Cpd6iValue difference is not larger, is measured by FP
They are respectively 1.85 μM and 67.3 μM with the Ki values of MdmX, and pass through the fluorimetry of p53p-XX-N-MdmX fusion proteins
Ki is respectively 1.49 μM and 51.73 μM, the fluorescent method and the K measured by FP methods of the prior art of this explanation present inventioni
Value is also what is be consistent.
Although the comparative descriptions of result shown in table 2, existed with the titration experiments result of nutlin-3a and 32 small molecule
Certain error, but screened with this p53p-XX-N-MdmX fusion proteins model set up suitable micromolecular inhibitor with
FP methods measured result of the prior art is also what is substantially conformed to, illustrates that the fusion protein screening system is effective.
Embodiment 6：The plasmid construction of p53p-XX-N-MdmX fusion protein mutant
p53pF19A- XX-N-MdmX and p53pL26A- XX-N-MdmX is the mutant mould of p53p-XX-N-MdmX fusion proteins
Type, they be on the basis of p53p-XX-N-MdmX fusion proteins respectively the Phe19 on p53p peptide fragments sport Ala19,
Leu26 sports Ala26 (19 and 26 refer to position of the corresponding amino acid in wild type p53 polypeptide).It is incorporated into three combinations
Two amino acid in three critical amino acid residues of pocket are mutated respectively, to weaken p53p polypeptide segments and MdmX respectively
The binding ability of N-terminal domain, to filter out the weaker new small molecule drug matrices of binding ability basis is provided.
Two kinds of Mutant models set up as follows according to p53p-XX-N-MdmX fusion proteins model：
p53pF19A- XX-N-MdmX amino acid sequences are as follows, and (tertiary structure simulates to obtain simulation drawing with PymolWin
As shown in Figure 10 a, not including GSSHHHHHHGS)：
GSSHHHHHHGSSQETASDLWKLLPENGSGSSENLYFQGSQINQVRPKLPLLKILHAAGAQGEMFTVKEV
MHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT
p53pL26A- XX-N-MdmX amino acid sequences are as follows, and (tertiary structure simulates to obtain simulation drawing with PymolWin
As shown in fig. lob, not including GSSHHHHHHGS)：
GSSHHHHHHGSSQETFSDLWKLAPENGSGSSENLYFQGSQINQVRPKLPLLKILHAAGAQGEMFTVKEV
MHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLAT
In order to build above fusion protein model, according to existing plasmid pET28a-p53p-XX-N-MdmX, devise following
Primer sequence：
F19A-forward：5’-GGC AGC AGC CAG GAA ACC GCT AGC GAT CTG TGG AAA-3’
F19A-reverse：5’-TTT CCA CAG ATC GCT AGC GGT TTC CTG GCT GCT GCC-3’
L26A-forward:5’-AGC GAT CTG TGG AAA CTG GCG CCG GAA AAT GGC AGC-3’
L26A-reverse:5’-GCT GCC ATT TTC CGG CGC CAG TTT CCA CAG ATC GCT-3’
p53pF19A- XX-N-MdmX fusion protein constructions：
PCR system 1:
PCR1 reaction conditions are as shown in fig. 11a；
DNA glue figure is as figure 12 a shows；
PCR system 2:
PCR2 reaction conditions are as shown in figure 11b；
DNA glue figure is as shown in Figure 12b；
PCR system 3:
PCR3 reaction conditions are as shown in fig. 11c；
DNA glue figure is as shown in fig. 12 c；
PCR primer is reclaimed, recovery product Nco I and EcoR I double digestions, as Insert Fragment.
Here it is fusion DNA vaccine.PCR1 and PCR2 obtains two sections of nucleotide sequences, and PCR3 is fusion DNA vaccine, only to single amino
Acid mutation.
PCR recovery product digestion systems：
37 DEG C, 3h, digestion products kit is reclaimed.
By pET28a-N-MdmX plasmids Nco I and the double digestions of EcoR I, glue reclaim is cut, as carrier.
It is 1 that coupled reaction presses carrier DNA with Insert Fragment DNA mol ratios：3 ratios mix, and add high with system identical
Effect ligase (TOYOBO, Ligation high Ver.2), connects 3h by 16 DEG C.
Whole connection products are proceeded in 50 μ l DH5 α (top10) Escherichia coli, ice bath 30min, after 42 DEG C of heat shock 90s
Ice bath is put back to immediately, and 2min adds the activation of 200 μ l LB fluid nutrient mediums, 37 DEG C, applies again after the recovery of 200rpm shaken cultivations 1h
LB solid medium flat boards containing kanamycins, 37 DEG C are inverted 12～16h of culture, observe colony growth situation.Extracting
pET28a-p53pF19A- XX-N-MdmX plasmids, sequencing, sequencing result is correct, preservation of bacteria strain.
p53pF19A- XX--N-MdmX and p53pL26AThe molecular cloning process of-XX-N-MdmX removes primer sequence during PCR
Outward, other conditions are completely the same.
p53pF19AThe nucleic acid coding sequence of-XX-N-MdmX as shown in SEQ ID NO.5, p53pL26AThe core of-XX-N-MdmX
Coding sequences are as shown in SEQ ID NO.6.
Embodiment 7：p53pF19A- XX-N-MdmX fusion proteins and p53pL26AThe expression of-XX-N-MdmX fusion proteins with it is pure
Change
By pET28a-p53pF19A- XX-N-MdmX and pET28a-p53pL26ARespectively chemical conversion enters-XX-N-MdmX plasmids
E. coli bl21 (DE3)；Single bacterium colony is chosen to 2ml LB K+Culture medium, 37 DEG C, 200rpm incubated overnights；Take 500 μ l bacterium solutions to turn
Enter 50ml LB K+Culture medium, 37 DEG C, 200rpm culture 3h；50ml bacterium solutions are proceeded to into 1L LB K entirely+Culture medium, 37 DEG C,
200rpm is cultivated, until cell concentration reaches OD280=0.8 or so, add IPTG (final concentration 0.4mM) inductions, about 20h to receive bacterium
(during microorganism collection centrifuge parameters be set to 3500rpm, 30min, 4 DEG C).
Ultrasonic cell-break crusher machine cell.According to thalline volume：Buffer solution volume=1:5 ratio, adds
BuffeA buffer solutions (50mM Na2HPO4, 200mM NaCl, 10mM imidazoles, 1mM BME, PH 8.0), parameter is set to：When total
Between 2min；Ultrasonic time 2s；Off time 4s；24 DEG C of temperature alarming；Ultrasonic power 40%.Repeat 2～3 times and (observe thalline
Sliminess after broken is determining broken number of times), until bacterial cell disruption it is complete.Clasmatosis liquid is dispensed with 50ml centrifuge tubes
Balance, centrifugation, 30min, 4 DEG C, 18000rpm are respectively collected in supernatant, precipitation in reagent bottle (pipe).
Supernatant is crossed into Ni-NTA chromatographic columns and Gel-filtration (sSupredex 16/ by AKTA pure
60075pg or Superdex 26/60075pg), the albumen of purifying is collected, concentration is dispensed, and per a 200 μ l, liquid nitrogen flash freezer freezes
It is stored in -80 DEG C of refrigerators.
Embodiment 8：Checking p53p-XX-N-MdmX, p53pF19A- XX-N-MdmX and p53pL26A- XX-N-MdmX threes'
Stability difference
At room temperature, 3～4mol/L guanidine hydrochlorides can make that protein can be made to be converted to denatured state from native state, generally increase
Plus denaturant concentration can improve denaturation degrees, usual 6mol/L guanidine hydrochlorides can make protein be completely transformed into denatured state.
Compared to p53p-XX-N-MdmX fusion proteins, p53pF19A- XX-N-MdmX fusion proteins and p53pL26A-XX-N-
MdmX fusion proteins be on the basis of p53p-XX-N-MdmX respectively the Phe19 on p53p polypeptide segments change into Ala19,
Leu26 changes into Ala26.Two amino acid being incorporated in three critical amino acid residues of three binding pockets enter respectively
Row mutation, to weaken the binding ability of p53p and MdmX N-terminal domains respectively, the stability for making fusion protein weakens, and is come with this
The micromolecular inhibitor of the weak new framework structure of screening competition binding ability.
With the guanidine hydrochloride solution 100ml that ultra-pure water compound concentration is 8M.
Take the p53p-XX-N-MdmX (OD of 4 200 μ l/280=1.0), the p53p of 3 200 μ l/F19A-XX-N-
MdmX(OD280=1.38), the p53p of 2 200 μ l/L26A-XX-N-MdmX(OD280=2.0) fusion protein quick-thawing, mixes
Close, 14000rpm, 10min, centrifugation.
According to table 4 below, table 5 and the ratio of table 6, be respectively configured p53p-XX-N-MdmX under different concentration of guanidine hydrochloride,
p53pF19A- XX-N-MdmX and p53pL26A- XX-N-MdmX fusion protein solution.
Solution of the p53p-XX-N-MdmX fusion proteins of table 4 under 0～4M concentration of guanidine hydrochloride
The p53p of table 5F19ASolution of-N-MdmX the fusion proteins under 0～4M concentration of guanidine hydrochloride
The p53p of table 6L26ASolution of-N-MdmX the fusion proteins under 0～4M concentration of guanidine hydrochloride
According to concentration of guanidine hydrochloride gradient, 400 μ l samples are taken in quartz colorimetric utensil, cuvette is put into into F-7000 fluorescence point
Light photometer, takes λEXFor 278nm, λEM290nm～500nm is taken, is changed according to concentration of guanidine hydrochloride, p53p-XX-N-MdmX,
p53pF19A- XX-N-MdmX and p53pL26A- XX-N-MdmX obtains figure of fluorescence intensity changes for example shown in Figure 13 a, b and c.
According to the change of peak fluorescence intensity, by the peak fluorescence intensity of three kinds of fusion proteins as concentration of guanidine hydrochloride changes
Result arrange as shown in figure 14：
According to Figure 13 a, 13b and 13c, the peak value of fluorescence emission wavelengths is respectively p53p-XX-N-MdmX：λEM=
321nm、p53pF19A-XX-N-MdmX：λEM=331nm and p53pL26A-XX-N-MdmX：λEM=330nm, according to fluorescent emission ripple
Long peak value may determine that the tryptophan on p53p combines the depth order in the hydrophobic pocket of N-MdmX and is followed successively by p53p-XX-
N-MdmX、p53pL26A-XX-N-MdmX、p53pF19A- XX-N-MdmX, you can with tentatively judge fusion protein stability it is strong
It is weak to be followed successively by：p53p-XX-N-MdmX、p53pL26A- XX-N-MdmX and p53pF19A-XX-N-MdmX。
Shown in Figure 14, the peak fluorescence intensity of three kinds of fusion proteins is with the change in concentration figure of protein denaturant guanidine hydrochloride
As can be seen that analysis of fluorescence intensity peak IC50It was found that, when peak fluorescence intensity reduces half, the concentration of required guanidine hydrochloride
It is followed successively by：p53p-XX-N-MdmX、p53pL26A-XX-N-MdmX、p53pF19A- XX-N-MdmX, you can further to judge
The power of the stability of fusion protein is followed successively by：p53p-XX-N-MdmX、p53pL26A- XX-N-MdmX and p53pF19A-XX-N-
MdmX。
As a result p53p is illustratedL26A- XX-N-MdmX and p53pF19A- XX-N-MdmX is more unstable than p53p-XX-N-MdmX structure
It is fixed, illustrate that the affinity between the former two domains is weaker, and then the former is easier by the weaker MdmX inhibitor of affinity
Institute's competitive binding.
This shows to use p53pL26A- XX-N-MdmX and p53pF19A- XX-N-MdmX is weak as screening model screening affinity
Small molecule is theoretically feasible.
Embodiment 9：With above-mentioned 32 small molecules and nutlin-3a to p53p-XX-N-MdmX, p53pF19A-XX-N-MdmX
And p53pL26A- XX-N-MdmX is titrated to verify the change of binding ability.
Fusion protein quick-thawing, 14000rpm, 10min, centrifugation discards the albumen precipitated because of denaturation.
Small molecule mother liquid concentration 10mM, takes 1 μ l small molecules mother liquors plus 9 μ l DMSO are diluted to 1mM.
According to final concentration of 10 μM of small molecule, protein concentration is OD280nm=0.5, solution is added according to this concentration
96 hole black microwell plates (to add the DMSO of same volume as blank), in 4 DEG C of refrigerator mixing 2h.With
PerkinElmer LS-45/55 fluorescent/phosphorescents/luminescence spectrophotometer reads 96 orifice plate fluorescence datas, first takes λEXFor 278nm,
p53p-XX-N-MdmXλEMFor 321nm, p53pF19A-XX-N-MdmXλEMFor 331nm, p53pL26A-XX-N-MdmXλEMFor
330nm, excites grating 15nm, fluorescence grating 20nm, threshold value (cut off) 290nm, can read exciting light for 278nm when,
The fluorescence intensity level of three kinds of fusion proteins.Again with λEXFor 278nm, p53p-XX-N-MdmX, p53pF19A-XXN-MdmX、
p53pL26A-N-MdmXλEMFor 375nm, threshold value (cut off) 350nm can respectively read exciting light for 278nm when, three kinds
The fluorescence intensity level of fusion protein.Survey once every 4h, repeatedly twice every time.
96 orifice plates at most once can simultaneously test the suppression effect of 32 inhibitor and nutlin-3a to three kinds of fusion proteins
Really (one group of parallel laboratory test can be done).
By taking p53p-XX-N-MdmX fusion proteins as an example, in λEMFor the fluorescence that 321nm, cut-off wavelength read when being 290nm
Intensity is to add the fluorescence intensity level after small molecule, in λEMFor 375nm, the fluorescence intensity that cut-off wavelength reads when being 350nm is
Nutlin-3a (or other compounds) introduced fluorescence intensity level, the difference of twi-read fluorescence intensity level is little point
The fluorescence intensity change value of the tryptophan after son addition, the fluorescence intensity level using after the DMSO for adding same volume is right as blank
According to the difference between the fluorescence intensity change value and blank of the tryptophan after addition small molecule as adds small molecule chemical combination
The free degree of the tryptophan caused with p53p competitive bindings after thing, negative value is bigger, illustrates that the free degree of tryptophan is bigger, i.e.,
The binding ability of the small molecule is higher, and fixed this difference is ordinate, if as on the occasion of then illustrating that tryptophan is combined strengthens, you can
Interaction can be there is makes N-MdmX domains more stable, and the affinity of p53p and N-MdmX strengthens.With small molecule numbering
For abscissa, this experiment is sequentially 1#-32# and nutlin-3a, arranges result as shown in figure 15.
Figure 15 results show that the result negative value of p53p-XX-N-MdmX is less than p53p for single small moleculeF19A-
XX-N-MdmX and p53pL26AThe negative value of-XX-N-MdmX, this same small molecule of explanation is competed in p53p-XX-N-MdmX
The ability of p53 parts is weaker than p53pF19A- XX-N-MdmX and p53pL26A- XX-N-MdmX, in this explanation p53p-XX-N-MdmX
P53 binding structural domains are bigger with the bond strength of MdmX binding structural domains, as a result meet the purpose of design of the present invention.
Meanwhile, same inhibitor molecules are to p53pF19A- XX-N-MdmX and p53pL26AN-MdmX portions in-XX-N-MdmX
The competitive binding ability divided is higher, it is meant that p53pF19A- XX-N-MdmX and p53pL26A- XX-N-MdmX go for
The screening or test of the weaker micromolecular inhibitor of N-MdmX parts adhesion.This further illustrates result and meets setting for the present invention
Meter purpose.And the parent with 32 micromolecular inhibitors that p53p-XX-N-MdmX fusion proteins screening model is showed in Figure 15
What is reflected with power and Fig. 8 is also to match substantially, the further description validity of the model.
Above each embodiment is only intended to further illustrate the present invention, is not the protection model for limiting the present invention
Enclose, it is every based on the present invention design done by equivalents and to the present invention each technical scheme obviously change
It is dynamic, each fall within protection scope of the present invention.
SEQUENCE LISTING
<110>Hubei University Of Technology
<120>A kind of fusion protein of the inhibitory activity for screening weak MdmX inhibitor or testing weak MdmX inhibitor
<130> 2016-12-07
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> PRT
<213>Artificial sequence
<400> 1
Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn
1 5 10 15
<210> 2
<211> 11
<212> PRT
<213>Artificial sequence
<400> 2
Gly Ser Gly Ser Ser Glu Asn Leu Tyr Phe Gln
1 5 10
<210> 3
<211> 91
<212> PRT
<213>Artificial sequence
<400> 3
Gly Ser Gln Ile Asn Gln Val Arg Pro Lys Leu Pro Leu Leu Lys Ile
1 5 10 15
Leu His Ala Ala Gly Ala Gln Gly Glu Met Phe Thr Val Lys Glu Val
20 25 30
Met His Tyr Leu Gly Gln Tyr Ile Met Val Lys Gln Leu Tyr Asp Gln
35 40 45
Gln Glu Gln His Met Val Tyr Cys Gly Gly Asp Leu Leu Gly Glu Leu
50 55 60
Leu Gly Arg Gln Ser Phe Ser Val Lys Asp Pro Ser Pro Leu Tyr Asp
65 70 75 80
Met Leu Arg Lys Asn Leu Val Thr Leu Ala Thr
85 90
<210> 4
<211> 384
<212> DNA
<213>Artificial sequence
<400> 4
ggcagcagcc atcaccatca tcaccacggc agcagccagg aaacctttag cgatctgtgg 60
aaactgctgc cggaaaatgg cagcggcagc agcgaaaacc tgtattttca gggatcccag 120
attaaccagg tgcgtccgaa actgccgctg ctgaaaattc tgcatgcggc gggcgcgcag 180
ggcgaaatgt ttaccgtgaa agaagtgatg cattatctgg gccagtatat tatggtgaaa 240
cagctgtatg atcagcagga acagcacatg gtgtattgcg gcggcgatct gctgggcgaa 300
ctgctgggcc gtcagagctt tagcgtgaaa gatccgagcc cgctgtatga tatgctgcgt 360
aaaaacctgg tgaccctggc gacc 384
<210> 5
<211> 384
<212> DNA
<213>Artificial sequence
<400> 5
ggcagcagcc atcaccatca tcaccacggc agcagccagg aaaccgctag cgatctgtgg 60
aaactgctgc cggaaaatgg cagcggcagc agcgaaaacc tgtattttca gggatcccag 120
attaaccagg tgcgtccgaa actgccgctg ctgaaaattc tgcatgcggc gggcgcgcag 180
ggcgaaatgt ttaccgtgaa agaagtgatg cattatctgg gccagtatat tatggtgaaa 240
cagctgtatg atcagcagga acagcacatg gtgtattgcg gcggcgatct gctgggcgaa 300
ctgctgggcc gtcagagctt tagcgtgaaa gatccgagcc cgctgtatga tatgctgcgt 360
aaaaacctgg tgaccctggc gacc 384
<210> 6
<211> 384
<212> DNA
<213>Artificial sequence
<400> 6
ggcagcagcc atcaccatca tcaccacggc agcagccagg aaacctttag cgatctgtgg 60
aaactggcgc cggaaaatgg cagcggcagc agcgaaaacc tgtattttca gggatcccag 120
attaaccagg tgcgtccgaa actgccgctg ctgaaaattc tgcatgcggc gggcgcgcag 180
ggcgaaatgt ttaccgtgaa agaagtgatg cattatctgg gccagtatat tatggtgaaa 240
cagctgtatg atcagcagga acagcacatg gtgtattgcg gcggcgatct gctgggcgaa 300
ctgctgggcc gtcagagctt tagcgtgaaa gatccgagcc cgctgtatga tatgctgcgt 360
aaaaacctgg tgaccctggc gacc 384
Claims (10)
1. a kind of fusion protein of the inhibitory activity for screening weak MdmX inhibitor or testing weak MdmX inhibitor, its feature exists
In the fusion protein includes cycle tests, binding sequence and linking arm sequence；
The cycle tests is MdmX sequences or the MdmX fragments comprising the p53 binding structural domains in MdmX sequences；
The binding sequence is the p53 pieces comprising the MdmX binding structural domains in p53 sequences for keeping having binding activity with MdmX
The mutant of section；
The linking arm sequence is a peptide fragment, the upstream and downstream of the linking arm respectively with the cycle tests and the combination sequence
The connection of one of row；The amino acid sequence of the linking arm is not involved in the formation or maintenance of the secondary structure of the fusion protein；Institute
The length and flexibility for stating linking arm be enough to any surface part of the space structure for making the cycle tests and the binding sequence
Any surface part of space structure spatially have an opportunity to contact.
2. the fusion of the inhibitory activity for being used to screen weak MdmX inhibitor or test weak MdmX inhibitor as claimed in claim 1
Albumen, it is characterised in that the upstream or downstream of the fusion protein has the sequence label for Protein Separation and/or purifying.
3. it is as claimed in claim 1 or 2 to be used to screen the inhibitory activity of weak MdmX inhibitor or the weak MdmX inhibitor of test
Fusion protein, fusion protein formula p53pF19A- XX-N-MdmX is represented, it is characterised in that
p53pF19AThe binding sequence is represented, specially：SQETASDLWKLLPEN.
XX represents the linking arm sequence SEQ ID NO.2, specially：GSGSSENLYFQ；
N-MdmX represents the cycle tests SEQ ID NO.3, specially：
GSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSP
LYDMLRKNLVTLAT。
4. the fusion of the inhibitory activity for being used to screen MdmX inhibitor or test MdmX inhibitor as claimed in claim 1 or 2
Albumen, fusion protein formula p53pL26A- XX-N-MdmX is represented, it is characterised in that
p53pL26AThe binding sequence is represented, specially：SQETFSDLWKLAPEN.
XX represents the linking arm sequence SEQ ID NO.2, specially：GSGSSENLYFQ；
N-MdmX represents the cycle tests SEQ ID NO.3, specially：
GSQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSP
LYDMLRKNLVTLAT。
5. a kind of nucleotide sequence, it is characterised in that the nucleotide sequence can be expressed such as any one of claim 1-4 institute
The fusion protein for screening the inhibitory activity of weak MdmX inhibitor or the weak MdmX inhibitor of test stated.
6. nucleotide sequence as claimed in claim 5, it is characterised in that the nucleotide sequence is as shown in SEQ ID NO.5.
7. nucleotide sequence as claimed in claim 5, it is characterised in that the nucleotide sequence is as shown in SEQ ID NO.6.
8. a kind of expression of the fusion protein for the inhibitory activity screened weak MdmX inhibitor or test weak MdmX inhibitor is carried
Body, it is characterised in that the expression vector can by host expresses as any one of claim 1-4 for screening
The fusion protein of the inhibitory activity of weak MdmX inhibitor or the weak Mdmx inhibitor of test.
9. the fusion of the inhibitory activity for being used to screen weak MdmX inhibitor or test weak MdmX inhibitor as claimed in claim 8
The expression vector of albumen, it is characterised in that the carrier is pET-28a carriers, the host is e. coli bl21 (DE3).
10. the suppression for screening weak MdmX inhibitor or the weak MdmX inhibitor of test as any one of claim 1-4
The fusion protein of system activity exists in the weak MdmX inhibitor of screening or the purposes of the inhibitory activity for testing weak MdmX inhibitor, its feature
In the weak MdmX inhibitor is can be with the inhibitor of p53 competitive binding MdmX.
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Cited By (1)
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|---|---|---|---|---|
| CN108864113A (en) * | 2018-08-03 | 2018-11-23 | 南方科技大学 | MDM2-HDAC double-target inhibitor, pharmaceutical composition, preparation and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105936646A (en) * | 2015-02-09 | 2016-09-14 | 中国科学院苏州纳米技术与纳米仿生研究所 | Mini protein and application thereof |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105936646A (en) * | 2015-02-09 | 2016-09-14 | 中国科学院苏州纳米技术与纳米仿生研究所 | Mini protein and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| RONG CHEN ET AL.: "A Fusion Protein of p53 Peptide and MdmX as an Efficient Model for Screening of Anticancer Prodrugs with Fluorescence Spectroscopy", 《BIOPHYSICAL JOURNAL》 * |
| 盛荣等: "p53-MDMZ结合抑制剂药效团模型的构建", 《物理化学学报》 * |
| 覃凌云等: "Mdm2/MdmX抑制剂", 《中国生物工程杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108864113A (en) * | 2018-08-03 | 2018-11-23 | 南方科技大学 | MDM2-HDAC double-target inhibitor, pharmaceutical composition, preparation and application thereof |
| CN108864113B (en) * | 2018-08-03 | 2021-08-13 | 南方科技大学 | MDM2-HDAC double-target inhibitor, pharmaceutical composition, preparation and application thereof |
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