CN103184200A - Expression, purification and crystal structure of protein-tyrosine-phosphatase PTPN12 - Google Patents

Abstract

The invention provides a crystal three-dimensional structure of a protein-tyrosine-phosphatase PTPN12 protein, wherein the PTPN12 is an N-terminal domain of the protein-tyrosine-phosphatase PTPN12 and encoding genes of 301 amino acids from a 1-site amino acid to a 301-site amino acid; atoms in the crystal three-dimensional structure have at least part of atomic coordinates listed in a table 1, or any structure having an average root mean square deviation smaller than or equal to 1.7 angstroms with the three-dimensional coordinates of main chain carbon skeleton atoms of at least 40% amino acid residues in the coordinates. The invention also provides a method for expression, purification and crystallization of the protein-tyrosine-phosphatase PTPN12 protein, and theoretical guiding significance and applications of the crystal structure in aspects such as function researches, potential drug screening and drug design.

Description

Protein-tyrosine-phosphatase PTPN12 expression and purification and crystalline structure

Technical field

The present invention relates to Protein-tyrosine-phosphatase PTPN12 is carried out genetic engineering modified obtaining the method for efficiently expressing in intestinal bacteria, purifying and crystallization, and the 1st three-dimensional crystalline structure to the 301st amino acids of Protein-tyrosine-phosphatase PTPN12.

Background technology

Having delivered medical college of Harvard University and the expert of Harbin Medical University on 2011 " CELL " is the result of study of novel cancer suppressor gene about Protein-tyrosine-phosphatase PTPN12.Discover, the reduction of PTPN12 expression level can cause the normal cell malignancy, when the PTPN12 expression is suppressed, the network propylhomoserin phosphorylation of the promotion sensitivity gene EGFR receptor family of mammary cancer significantly strengthens, and the EGFR receptor family is activated through a series of network propylhomoserin phosphorylations just, thereby produces short cell carcinogenesis effect; The PTPN12 overexpression then can have certain inhibition to the malignant cell growth.The researchist finds that after further research PTPN12 has the afunction transgenation at multiple three cloudy type breast cancer cell lines.In clinical three cloudy type patient with breast cancer's sample pathological sections of 60%, the PTPN12 protein expression is low.These clinical case results of study further support PTPN12 to occupy critical role in the generation progression of three cloudy type mammary cancer.PTPN12 albumen is the common concern that the newest research results of novel cancer suppressor gene has caused international tumour circle.

PTPN12 belongs to Protein-tyrosine-phosphatase family.Protein-tyrosine-phosphatase mainly by 90 genetic expressions, is divided into 4 families in human genome.The phosphorylation of protein-tyrosine residue plays an important role for regulating the physiologic response journey, for example cell proliferation and differentiation, cell migration, activated immune cell and apoptosis etc.Therefore, if the tyrosine acidification imbalance in the cell can cause serious consequence, comprise to cause that body immunity, improper metabolism reply and cell transformation etc.

Protein-tyrosine-phosphatase PTPN12 comprises 780 amino-acid residues by the PTPN12 genes encoding, and the molecular weight size is about 88kDa, and its N end is the Phosphoric acid esterase structural domain, and the mediation catalytic activity has the conservative property of height; The C end is non-enzymic activity structural domain, and proline rich, L-glutamic acid, Serine and threonine residues are called the PEST sequence again, mainly mediate mutually combining of PTPN12 and substrate protein white matter.PTPN12 has had many investigators that it has been carried out the research on the function as a kind of important intracellular protein tyrosine phosphatase zymoprotein.At first, PTPN12 by with FAK, P130Cas, the adhesion of substrate interaction regulating cells such as paxillin, the migration be connected with intercellular.Secondly, the PTPN12 negativity is regulated and control lymphocytic activation.PTPN12 is wide expression in multiple histocyte, after one's own heart, lung, kidney, spleen, thymus gland and placenta, and expresses very highly in thymus gland and spleen, and the expression amount difference of PTPN12 in different tissues indicates that it should participate in immunologic cellular activity and regulate.

Summary of the invention

One aspect of the present invention, provide a kind of Protein-tyrosine-phosphatase PTPN12 is carried out genetic engineering modified, with improve its in intestinal bacteria, can access efficiently express, down can the stable method that keeps the albumen of phosphatase activity of long period at normal temperature (37 ± 5 ℃ Celsius).The present invention preferably uses colibacillary procaryotic cell expression system (but not get rid of other expression system, as in other bacteriums or other eukaryotic cells, expressing), the above gene integration is expressed in the pET-28a carrier, mode with the HIS tag fusion protein is expressed, and is used for the method for crystallization of protein.The present invention addresses the efficient expression method that obtains Protein-tyrosine-phosphatase PTPN12 in intestinal bacteria.

Preferably, the present invention has carried out Protein-tyrosine-phosphatase PTPN12 genetic engineering modified, the PTPN12 full-length gene has been carried out the research of brachymemma, a method that can efficiently express in intestinal bacteria is provided, this method has comprised about 1～15 amino acids of aminoterminal to the encoding gene of about 301～315 amino acids, more preferably comprise about 1 to 301 amino acids.

Second aspect the invention provides the method for a kind of purifying protein tyrosine phosphatase PTPN12.The PTPN12 gene that the gene engineering method transformation is later excessively is connected in the expressivity plasmid vector that has label, and conversion enters in the Bacillus coli cells and expresses, purifying.

Preferably, wherein said label is selected from GST, Flag-tag, Myc-tag, MBP-tag, His-tag, specific antibody; Described carrier contains the selected marker.Method described and special tag recognition is to be undertaken by affinity column, and the method for described separation and purification albumen is further to use method separation and purification PTPN12 albumen such as gel-filtration and ion exchange chromatography; Determine the purity of protein with the method for gel electrophoresis.

The 3rd aspect the invention provides the method for the above-mentioned PTPN12 albumen that obtains of a kind of crystallization, and described method comprises: PTPN12 albumen is concentrated into 5-10mg/ml, at 4-30 degree centigrade down with gas phase diffusion method screening crystal growth condition.

The 4th aspect the invention provides the good PTPN12 protein crystal of diffraction property.

The 5th aspect the invention provides the three-dimensional structure of PTPN12 albumen, this structrual description PTPN12 albumen secondary structure composition, peptide chain trend, enterprise schema and three-dimensional molecular structure.Wherein carry out the X ray crystalline diffraction at the PTPN12 albumin crystal, obtain above-mentioned crystalline diffraction data, further by structure elucidation, make up the three-dimensional structure of PTPN12 albumen with the diffraction data of described protein crystal.

In a concrete embodiment, PTPN12 albumin crystal three-dimensional structure is provided, wherein PTPN12 albumen is albumen aminoterminal part (1-301 amino acid), and the average root variance (average root mean square deviation (RMSD)) that the atom in the wherein said crystal three-dimensional structure has in the atomic coordinate of at least a portion listed in the table one or any and this coordinate a main chain carbon skeletal atom three-dimensional coordinate of at least 40% amino-acid residue is less than or equal to the structure of 1.7 dusts.

Preferably, wherein said PTPN12 albumin crystal has the spacer of C2, and unit cell parameters is for approximately: a=136 dust, b=41 dust, c=75 dust, α=γ=90 °, β=116 °.

In a specific embodiments, the protein structure of whole PTPN12 presents the structural pattern of typical alpha+beta.Its core constitutes the β lamella by 8 antiparallel βZhe Dies, and 7 α spirals are centered around the both sides of β lamella.Particularly, core β lamella comprises βZhe Die 1, namely comprise the amino acid section that comprises from the Ile of 94 of the 91st Ala to the, βZhe Die 2, namely comprise the amino acid section that comprises from the Thr of 106 of the 102nd Ala to the, βZhe Die 3, namely comprise the amino acid section that comprises from the Met of 131 of the 128th Ile to the, βZhe Die 4, namely comprise the amino acid section that comprises from the Phe of 157 of the 155th Ile to the, βZhe Die 5, namely comprise the amino acid section that comprises from the Arg of 170 of the 160th Phe to the, βZhe Die 6, namely comprise the amino acid section that comprises from the Phe of 182 of the 173rd Tyr to the, βZhe Die 7 namely comprises the amino acid section that comprises from the Tyr of 194 of the 185th Glu to the, βZhe Die 8 namely comprises the amino acid section that comprises from the His of 230 of the 227th Ile to the.The α spiral that is distributed in β lamella both sides comprises α spiral 1, namely comprise the amino acid section that comprises from the Lys of 18 of the 3rd Gln to the, α spiral 2, namely comprise the amino acid section that comprises from the Arg of 43 of the 27th Aln to the, α spiral 4, namely comprise the amino acid section that comprises from the Tyr of 219 of the 212nd Met to the, α spiral 5, namely comprise the amino acid section that comprises from the Ala of 253 of the 236th Gly to the, α spiral 6, namely comprise the amino acid section that comprises from the Gln of 272 of the 262nd Val to the, α spiral 7, namely comprise the amino acid section that comprises from the Leu of 300 of the 280th Lys to the, and α spiral 3, namely comprise the amino acid section that comprises from the Tyr of 124 of the 114th Val to the.Wherein, α spiral 1, α spiral 2, α spiral 4, α spiral 5, α spiral 6 and α spiral 7 are distributed in a side of center β lamella, and α spiral 3 is distributed in the opposite side of center β lamella.

The 6th aspect, the crystal three-dimensional structure that the invention provides Protein-tyrosine-phosphatase PTPN12 is used for the treatment of each peptide species of the disease that causes with the PTPN12 abnormal expression in design and screening, application in protein, inorganic or organic compound, antibody or the immune conjugate comprises:

According to the protein three-dimensional structure coordinate, be combined in polypeptide, protein, inorganic or organic compound, antibody or the immune conjugate molecule of Protein-tyrosine-phosphatase PTPN12 active pocket by computer simulation design.

The 7th aspect, the invention provides the protein three-dimensional structure based on Protein-tyrosine-phosphatase PTPN12, explore the method for its function, comprise: the crystal that obtains Protein-tyrosine-phosphatase PTPN12 by the albumen crystallization method, perhaps obtain the three-dimensional structure coordinate of Protein-tyrosine-phosphatase PTPN12 protein crystal, wherein said three-dimensional structure comprises that the average root variance of the main chain carbon skeletal atom three-dimensional coordinate that contains 40% amino-acid residue in any and this coordinate at least is smaller or equal to the structure of 1.7 dusts; Carry out the biochemical test analysis verification based on the prediction theory of structure.

Description of drawings

Fig. 1 is other the amino acid whose sequence alignment of catalytic site of PTPN12 and PTP family, comprises the homologous protein of 3 kinds of PTPN12 protein families.This comparing result shows that PTPN12 has the high conservative amino-acid residue similar to the catalytic site of other PTP albumen.The amino acid that comes out with mark among the figure is represented conservative property.Concrete amino acid position all is the orders with PTPN12 in specification sheets and claims Illustrate.

Fig. 2: be the tomograph of Protein-tyrosine-phosphatase PTPN12.Be specially the vertical view of the structure ribbon figure of Protein-tyrosine-phosphatase PTPN12, wherein βZhe Die shows with yellow, and the α spiral must show with red, and the atom in the active centre of PTPN12 albumen is with blue demonstration, and phosphate radical is with pink spherical demonstration.Concrete amino acid position all is the example explanation with Fig. 2 in specification sheets and claims.

Fig. 3: the vertical view of the structure ribbon figure of Protein-tyrosine-phosphatase PTPN12 is specially Fig. 1 along Y-axis Rotate 180 °.Wherein βZhe Die shows with yellow, and the α spiral must show with red, and the atom in the active centre of PTPN12 albumen is with blue demonstration, and phosphate radical is with pink spherical demonstration.

Fig. 4: the active centre of PTPN12 albumen exists the stability of a phosphate radical in conjunction with diagram.Particularly, the active centre of PTPN12 comprises P type flexible region (P-loop zone), namely comprise 237 Arg of His to the from the 230th, it is the H230CSAGCGR237 zone, and a phosphate radical interacts by N nitrogen-atoms on hydrogen bond and these amino acid backbone, and stability is combined in here.Concrete embodiment

The inventor provide a kind of Protein-tyrosine-phosphatase PTPN12 is carried out genetic engineering modified, with improve its in intestinal bacteria, can access efficiently express, down can the stable method that keeps the albumen of lactamase activity of long period at normal temperature (37 ± 5 ℃ Celsius).Resolved Protein-tyrosine-phosphatase PTPN12 three-dimensional crystalline structure by X ray diffractive crystal method.

The expression and purification method of PTPN12 albumen:

Derive from people source PTPN12 gene, its encoded protein matter sequence is:

The PTPN12 protein sequence

MEQVEILRKFIQRVQAMKSPDHNGEDNFARDFMRLRRLSTKYRTEKIYPTATGEKEENVKKNRYKDILPFDHSRVKLTLKTPSQDSDYINANFIKGVYGPKAYVATQGPLANTVIDFWRMIWEYNVVIIVMACREFEMGRKKCERYWPLYGEDPITFAPFKISCEDEQARTDYFIRTLLLEFQNESRRLYQFHYVNWPDHDVPSSFDSILDMISLMRKYQEHEDVPICIHCSAGCGRTGAICAIDYTWNLLKAGKIPEEFNVFNLIQEMRTQRHSAVQTKEQYELVHRAIAQLFEKQLQLY-

Namely

Met Glu Gln Val Glu Iie Leu Arg Lys Phe Iie Gln Arg Val Gln Ala Met Lys Ser Pro Asp His Asn Gly Glu Asp Asn Phe Ala Arg Asp Phe Met Arg Leu Arg Arg Lue Ger Thr Lys Tyr Arg Thr Glu Lys Ile Tyr Pro Thr Ala Thr Gly Glu Lys Glu Glu Asn Val Lys Lys Asn Arg Tyr Lys Asp Ile Leu Pro Phe Asp His Ser Arg Val Lys Leu Thr Leu Lys Thr Pro Ser Gln Asp Ser Asp Thr Iie Asn Ala Asn Phe Iie lys Gly Val Thr Gly Pro Lys Gla Tyr Val Gla Thr Gln Gly Pro Leu Gla Asn Thr Val Iie Asp Phe Trp Arg Met Iie Trp Glu Tyr Asn Val Val Iie Iie Val Met Gla Cys Arg Glu Phe Glu Met Gly Arg Lys Lys Cys Glu Arg Tyr Trp Pro Leu Gly Glu Asp Pro Iie Thr Phe Lys Gla Pro Phe Lys Iie Ser Cys Glu Asp Glu Gln Gla Arg Thr Asp Tyr Phe Iie Arg Thr Leu Leu Leu Glu Phe Gln Asn Glu Ser Arg Arg Leu Tyr Gln Phe His Tyr Val Asn Trp Pro Asp His Asp Val Pro Ser Ser Phe Asp Ser Iie Leu Asp Met Iie Ser Leu Met Arg Lys Tyr Gln Glu His Glu Asp Val Pro Iie Cys Iie His Cys Ser Gla Gly Cys Gly Arg Thr Gly Gla Iie Cys Gla Iie Asp Tyr Thr Trp Asn Leu Leu Lys Gla Gly Lys Iie Pro Glu Glu Phe Asn Val Phe Asn Leu Iie Gln Glu Met Arg Thr Gln Arg His Ser Gla Val Gln Thr Lys Glu Gln Tyr Glu Leu Val His Arg Gla Iie Gla Gln Leu Phe Glu Lys Gln Leu Gln Leu Tyr

By molecule clone technology the PTPN12 gene is cloned, be cloned on the pET-28a carrier (GE), so that further expressing protein.The fusion rotein (His-PTPN12) of expressing N-terminal fusion 6 * His tag is gone up in improved PTPN12 gene clone to pET-28a carrier (GE), clone's plasmid is transformed in the e. coli bl21 (DE3), working concentration is that the IPTG (sec.-propyl-β-D thiogalactoside) of 0.1mM induces escherichia coli expression albumen in BL21 (DE3), specifically referring to embodiment 1

Embodiment 1

Engineered protein tyrosine phosphatase PTPN12 is for the method for escherichia coli expression

Inventor's initial stage attempts PTPN12 albumen is carried out total length expressed, goes through at different expression vector pGEX, and pET etc., and different expression strain, as BL21 (DE3), BL21plys, the trial on the C41 etc. all can not obtain high purity protein.Hinting the transformation that to carry out the gene of NDM-1 on the gene level.Through design pcr amplification primer, made up from the carboxyl terminal of PTPN12 albumen, delete amino acid whose a large amount of mono-clonal one by one, having obtained one can the great expression purifying and the structure of crystallization, is PTPN12Met ₁-Tyr ₃₀₁

The expression and purification of PTPN12 in intestinal bacteria

By molecule clone technology the N end (the 1st to 301 amino acid) of PTPN12 is cloned on the pET-28a carrier (GE), its cloning site is NheI and XhoI.Clone's the PTPN12 expression of gene plasmid that contains is transformed among the colibacillary BL21 (DE3), carries out the expression of albumen, thereby make bacterium hold (aminoterminal) to be connected with the fusion rotein of 6 * His label by expressing protein N.After being transformed into the fusion protein expression plasmid that builds among the BL21 (DE3), use the LB substratum incubated overnight of 5mL earlier at 37 degree, after about 12 hours, be transferred to enlarged culturing in the LB substratum of 800mL, shaking under 37 degree and being cultured to OD in the bottle is about 0.4-0.6, be approximately four hours, reduce culture temperature to 16 degree subsequently, the IPTG (sec.-propyl-β-D thiogalactoside) that adds 0.1mM then carries out abduction delivering, approximately pass through centrifugal collection bacterium after 16-20 hour, be used for purifying.With the expression bacterium of centrifugal collection with containing the 50mM Mes (pH6.5) that has an appointment, 300Mm NaCl, suspend in the damping fluid of 1mM DTT, use low-temperature ultrahigh-pressure cytoclasis instrument (Guangdong Juneng Biology ﹠ Technology Co., Ltd.) lysing cell, go out infusible precipitate by centrifugation, (about 16,000rmp) supernatant that obtains of back comes initial gross separation purifying PTPN12 albumen by Ni Sepharose affinity column (GE) with high speed centrifugation.Target protein just can be incorporated on the affinity column, but there is the foreign protein also can be owing to non-specific binding is attached on the NI-NTA affinity column simultaneously, can remove the part foreign protein by gradient imidazoles wash-out, after groping through experiment repeatedly, respectively with containing 50mM imidazoles albumen buffer and 100mM imidazoles albumen buffer flush away part foreign protein, the target protein purity that affinity interaction is attached on the NI medium can reach 80%, with the 300mM imidazoles target protein is eluted, be concentrated into about 1ml, change albumen buffer into and use gel exclusion chromatography (GE) and ion exchange chromatography (GE) to be further purified out PTPN12 albumen, purity generally can reach more than 99%.Albumen by above step purifying uses evaporating pipe to be concentrated into 7mg/mL for crystallization experiment.

Embodiment 2

The crystallization of Protein-tyrosine-phosphatase PTPN12 and optimization:

To be concentrated into concentration in order to the good Protein-tyrosine-phosphatase PTPN12 of last method expression and purification and be about 5-10mg/mL, use the gas phase sessile drop method, use crystallization reagent (from the test kits such as Screen KitI/II, Index of companies such as Hampton Research) screening crystal growth condition.Through preliminary screening, the inventor has obtained initial crystal under crystallization reagent condition.By further optimization, wherein, using 40mM KH2PO4,20% (v/w) Glycerine has obtained the crystal that resolving power is about the 2.5 Izod right sides under the condition of and 30% (v/w) PEG 8000, and and then collects corresponding X ray diffracting data.

Embodiment 3

The crystalline structure of Protein-tyrosine-phosphatase PTPN12

The crystal of PTPN12 is under the liquid nitrogen protection of 100K in temperature; wavelength is that to collect a cover highest resolution under the X-ray of 0.97929 dust be the crystal data of 2.6 dusts; utilize HK2000 (Otwinowski 1997) processing data; find that crystal is the C2 spacer; a monomer is arranged in each asymmetric cell, and Matthews's coefficient VM is Corresponding solvent is 55.8%, and the structure of mixture, is resolved by PHASER as the initial ranging model with the structure of PTPN22 (PDB ID:3H2X), manually builds model in COOT again, carries out preliminary correction with PHENIX.In position correction subsequently, restricted condition is relaxed, the solvent that carries out according to Rfree is proofreaied and correct, proved its geometric model by PROCHECK, in the Fo-Fc electron density map that σ A calculates, solvent molecule is positioned on the rational peak, and the model of last correction data comes out by the PYMOL calculations show.

Embodiment 4

The three-dimensional structure of PTPN12

We use method, software among the embodiment 3, have finished the structure elucidation work to PTPN12, use software COOT, pymol, further analyze secondary structure composition, peptide chain trend, enterprise schema and the three-dimensional molecular structure of PTPN12.We find that PTPN12 albumen presents the structural pattern of typical alpha+beta on the whole.Those of ordinary skill in the art as can be known, has in the table 1 listed at least 40% atomic coordinate with atom in the wherein said crystal three-dimensional structure, perhaps the average root variance of the atomic structure coordinate of at least 40% amino acid whose main chain carbon skeleton and the coordinate in the table 1 is less than or equal to the atomic coordinate of 1.5 dusts in the crystal three-dimensional structure of PTPN12 albumen, all can be considered to have identical structure with 3D albumen.

Particularly, the PTPN12 core constitutes the β lamella by 8 antiparallel βZhe Dies, and 7 α spirals are centered around the both sides of β lamella.Particularly, core β lamella comprises βZhe Die 1, namely comprise the amino acid section that comprises from the Ile of 94 of the 91st Ala to the, βZhe Die 2, namely comprise the amino acid section that comprises from the Thr of 106 of the 102nd Ala to the, βZhe Die 3, namely comprise the amino acid section that comprises from the Met of 131 of the 128th Ile to the, βZhe Die 4, namely comprise the amino acid section that comprises from the Phe of 157 of the 155th Ile to the, βZhe Die 5, namely comprise the amino acid section that comprises from the Arg of 170 of the 160th Phe to the, βZhe Die 6, namely comprise the amino acid section that comprises from the Phe of 182 of the 173rd Tyr to the, βZhe Die 7 namely comprises the amino acid section that comprises from the Tyr of 194 of the 185th Glu to the, βZhe Die 8 namely comprises the amino acid section that comprises from the His of 230 of the 227th Ile to the.The α spiral that is distributed in β lamella both sides comprises α spiral 1, namely comprise the amino acid section that comprises from the Lys of 18 of the 3rd Gln to the, α spiral 2, namely comprise the amino acid section that comprises from the Arg of 43 of the 27th Aln to the, α spiral 4, namely comprise the amino acid section that comprises from the Tyr of 219 of the 212nd Met to the, α spiral 5, namely comprise the amino acid section that comprises from the Ala of 253 of the 236th Gly to the, α spiral 6, namely comprise the amino acid section that comprises from the Gln of 272 of the 262nd Val to the, α spiral 7, namely comprise the amino acid section that comprises from the Leu of 300 of the 280th Lys to the, and α spiral 3, namely comprise the amino acid section that comprises from the Tyr of 124 of the 114th Val to the.Wherein, α spiral 1, α spiral 2, α spiral 4, α spiral 5, α spiral 6 and α spiral 7 are distributed in a side of center β lamella, and α spiral 3 is distributed in the opposite side of center β lamella.

In the structure of this external whole PTPN12, we find to exist in the active centre of albumen the stability combination of a phosphate radical.Particularly, the active centre of PTPN 12 comprises P type flexible region (P-loop zone), namely comprise 237 Arg of His to the from the 230th, it is the H230CSAGCGR237 zone, and a phosphate radical interacts by N nitrogen-atoms on hydrogen bond and these amino acid backbone, and stability is combined in here.

Experimental result

The N end structure territory atomic coordinate group of the monomolecular PTPN12 of table 1 is as follows:

Note coordinate date created: on November 22nd, 2011, date edited: on December 5th, 2011.

Note high resolving power scope (dust): 2.4

Note low resolution scope (dust): 50

Claims (11)

1. Protein-tyrosine-phosphatase PTPN12 crystalline structure, wherein, described PTPN12 is the N end structure territory of described Protein-tyrosine-phosphatase PTPN12, from the 1st to the 301 amino acids encoding gene of totally 301 amino acids.Atom in the wherein said crystal three-dimensional structure has the average root variance of the main chain carbon skeletal atom three-dimensional coordinate of at least 40% amino-acid residue in the atomic coordinate of at least a portion listed in the table 1 or any and this coordinate smaller or equal to the structure of 1.7 dusts.

2. the crystalline structure of Protein-tyrosine-phosphatase PTPN12 according to claim 1, wherein, described Protein-tyrosine-phosphatase PTPN12 is the encoding gene from 1 amino acids to 301 amino acids of described Protein-tyrosine-phosphatase PTPN12 full-length proteins sequence, be Met1-Tyr301, below used numbering all be dependent on this.

3. the crystalline structure of PTPN12 albumen according to claim 1, wherein said crystal has preferably, and the crystal of wherein said PTPN12 has the spacer of C2, unit cell parameters is about: a=136 dust, b=41 dust, c=75 dust, α=γ=90 °, β=116 °.

4. the protein structure of whole PTPN12 according to claim 1 presents the structural pattern of typical alpha+beta.Its core constitutes the β lamella by 8 antiparallel βZhe Dies, and 7 α spirals are centered around the both sides of β lamella.Particularly, core β lamella comprises βZhe Die 1, namely comprise the amino acid section that comprises from the Ile of 94 of the 91st Ala to the, βZhe Die 2, namely comprise the amino acid section that comprises from the Thr of 106 of the 102nd Ala to the, βZhe Die 3, namely comprise the amino acid section that comprises from the Met of 131 of the 128th Ile to the, βZhe Die 4, namely comprise the amino acid section that comprises from the Phe of 157 of the 155th Ile to the, βZhe Die 5, namely comprise the amino acid section that comprises from the Arg of 170 of the 160th Phe to the, βZhe Die 6, namely comprise the amino acid section that comprises from the Phe of 182 of the 173rd Tyr to the, βZhe Die 7 namely comprises the amino acid section that comprises from the Tyr of 194 of the 185th Glu to the, βZhe Die 8 namely comprises the amino acid section that comprises from the His of 230 of the 227th Ile to the.The α spiral that is distributed in β lamella both sides comprises α spiral 1, namely comprise the amino acid section that comprises from the Lys of 18 of the 3rd Gln to the, α spiral 2, namely comprise the amino acid section that comprises from the Arg of 43 of the 27th Aln to the, α spiral 4, namely comprise the amino acid section that comprises from the Tyr of 219 of the 212nd Met to the, α spiral 5, namely comprise the amino acid section that comprises from the Ala of 253 of the 236th Gly to the, α spiral 6, namely comprise the amino acid section that comprises from the Gln of 272 of the 262nd Val to the, α spiral 7, namely comprise the amino acid section that comprises from the Leu of 300 of the 280th Lys to the, and α spiral 3, namely comprise the amino acid section that comprises from the Tyr of 124 of the 114th Val to the.Wherein, α spiral 1, α spiral 2, α spiral 4, α spiral 5, α spiral 6 and α spiral 7 are distributed in a side of center β lamella, and α spiral 3 is distributed in the opposite side of center β lamella.

5. according to the crystalline structure of the described PTPN12 albumen of claim 1-3, there is the stability combination of a phosphate radical in the active centre of albumen.Particularly, the active centre of PTPN12 comprises P type flexible region (P-loop zone), namely comprises 237 Arg of His to the from the 230th, i.e. the H230CSAGCGR237 zone.

6. according to the crystalline structure of the described PTPN12 albumen of claim 1-5, wherein there is the stability combination of a phosphate radical in the active centre of albumen, the excavation that is found to be its function and the explaination in PTPN12 crystalline structure and active centre provide the theoretical direction foundation, also for designing and screening possible inhibitor and established theoretical basis.

According to the crystal three-dimensional structure of each described Protein-tyrosine-phosphatase PTPN12 among the claim 1-6 in the application aspect the possible inhibitor of its functional study, design and screening, comprising:

According to the three-dimensional structure of protein, seek possible specific avtive spot or binding pocket by computer simulation;

Three-dimensional structure according to protein, by possible specific avtive spot or the binding pocket that finds, look for possible substrate type, carry out biochemical test, and with possible specific avtive spot or the amino acid mutation in the binding pocket, the effect of checking sudden change;

Will be according to the three-dimensional structure of protein, in conjunction with above-mentioned experimental result, carry out the excavation of natural substrate type in the body, and it is expanded to described Protein-tyrosine-phosphatase PTPN12 sequence similarity in other viral kinds of at least 30%, and then analyze and sum up.

8. the method for a purifying PTPN12 albumen, in the expressivity plasmid vector that right 1 is required the gene of described PTPN12 to be connected in to have label, transform enter in the Bacillus coli cells express, purifying.

9. method according to claim 8, wherein said label is selected from GST, Flag-tag, Myc-tag, MBP-tag, His-tag, specific antibody, and described carrier contains the selected marker.

10. the method for any one described PTPN12 albumen in the aforementioned claim of crystallization, described method comprises: PTPN12 albumen is concentrated into 5-10mg/mL, at 4-30 degree centigrade down with gas phase diffusion method screening crystal growth condition.

11. a diffraction property good according to each described Protein-tyrosine-phosphatase PTPN12 protein crystal in the aforementioned claim.

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