CN101845098B - Crystal of rat RANK/RANKL extracellular compound, preparation method and application thereof - Google Patents
Crystal of rat RANK/RANKL extracellular compound, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a method for purifying and expressing RANK extracellular area protein, and also provides a method for preparing RANK/RANKL compound crystals, a three-dimensional crystal structure of the RANK/RANKL compound, and applications of the crystal in the medical field.
Description
Technical field
The present invention relates to the method for purifying and expression RANK extracellular protein, additionally provide a kind of crystal of RANK/RANKL complex body, particularly provide the crystal that a kind of mouse RANK/RANKL extracellular protein is formed.The invention provides a kind of method preparing the outer complex body crystal of mouse RANK/RANKL born of the same parents, and the application of this complex structure in field of medicaments.
Background technology
Cytokine in tumour necrosis factor (TNF) family plays important role in the multiple biological functions such as such as inflammatory reaction, organ generation, host defense, autoimmunization and apoptosis.These biological regulation factors are by receptor-ligand binding, thus the change of signal transmission in trigger cell and cell phenotype and play corresponding physiological action.Important role is play as in the RANKL of TNF family member and its acceptor RANK various activities in vivo, its remodeling process for bone serves important regulating effect, and the interaction between RANK and RANKL molecule is the adjustable intracellular signaling of T cell/dendritic cell, the survival of dendritic cell and adenoid growth also, receives in recent years and pays close attention to widely.
In the remodeling process of human bone, osteoclast is bone remodeling " promotor ", scleroblast is bone remodeling " attemperator ", the common pathological characters of bone resorption class disease is that osteoclast dysregulation causes balance between bone forming and bone resorption, and the activation mechanism of therefore understanding osteoclast in depth will have vital meaning for what successfully prevent and treat bone resorption class disease.1997, several different research group found Tumor Necrosis Factor Receptors-part superfamily newcomer: OPG (osteoprotegerin, OPG), RANKL and RANK; Subsequently, multinomial research shows in succession: RANKL, OPG have and regulate differentiation of osteoclast, growth, affect the effect of its function, and RANK is the key that RANKL, OPG play a role, and they form a bone regulating shaft, regulate the remodeling process of bone.
RANKL is a kind of II type transmembrane protein, it mostly is the complex body that three monomer polymerizations become, mainly be distributed in the cells such as activating T cell, marrow stromal cell and scleroblast, think to there are three kinds of RANKL albumen in Mammals at present: RANKL1, RANKL2, RANKL3, wherein first two is embrane-associated protein, rear a kind of soluble proteins for being secreted into outside born of the same parents.The RANKL molecule of people and rat comprises 317,316 amino-acid residues respectively, has the homology of 87%; Mankind RANKL gene is positioned at 13q14, and its promoter region comprises the activity of the key transcription factor cbaf-1 of osteoblast differentiation.RANKLmRNA can express at Various Tissues, and with the highest in bone and Lymphoid tissue, in bone, RANKL is mainly expressed in bone trabecula and marrow; All RANKL mRNA can be detected at marrow stromal cell and scleroblast.The people such as Ito S in 2004 and international patent application NO.WO 03/014077 both disclose the three-dimensional structure of RANKL, and its full content is hereby expressly incorporated by reference.Every research shows: each RANKL monomer comprises a sandwich structure, and it is made up of two plane anti-parallel ss-sheets layers.First lamella is by beta chain A ", A, H, C and F form; second lamella is made up of B ', B, G, D and E; wherein the first lamella is internal layer β-lamella; this lamella is rich in hydrophobic amino acid; and be combined with each other by hydrophobic interaction and two other RANKL; and the outer β-lamella of B ', B, G, D and E chain formation, and this lamella mostly is charged and polare Aminosaeren, is responsible for being combined with acceptor RANK; An edge of the β-sandwich structure in each RANKL monomer, chain E and F wrap up the inner hydrophobic layer of the AHCF β-lamella of adjacent monomer thus form RANKL Trimeric structures.The trimerical crystal structure characteristic of RANKL is: have P2
12
12
1spacer, unit cell parameters is a=65.3
± 0.2
, b=82.0
± 0.2
, c=99.5
± 0.2
, and spacer R3, born of the same parents' crystalline substance is a=b=150.6
± 0.2
, c=139.5
± 0.2
.Its shape just as by truncated taper, near the summit place of one end of film wider than far-end.RANKL trimerization height 55
, base diameter is about 55
, the diameter at summit place is about 35
RANK is the acceptor of RANKL, belongs to TNF receptor family, and be a kind of I type transmembrane protein, people RANK molecule expression amount in osteoclast precursor cells and mature osteoclast is higher.The RANK assignment of genes gene mapping is in 18q22.1, the protein of its coding exists with transmembrane protein type in vivo, it is expressed in monocytes/macrophages system, osteoclast precursor cells, T lymphocyte, bone-marrow-derived lymphocyte, dendritic cell etc., the RANK being expressed in osteoclast precursor cells film surface mediates the signal of RANKL, plays the function regulating differentiation of osteoclast.
After part RANKL is combined with the extracellular regions of acceptor RANK, result in the activation of RANK, RANK after activation by the TRAF binding domain of its intracellular region and TRAF1,2,3,5, the protein binding such as 6, and pass through intracellular two the important signal paths of these TRAF protein activation: JNK/SAPK path and NF κ B signal path.The expression of the gene that the activation-inducing of this two signal paths is relevant to differentiation of osteoclast, finally facilitates the differentiation of osteoclast with ripe.And various bone metabolism regulatory factor, hormone participate in the adjustment of RANKL-RANK-OPG axis signal.The same with other signal pathway, also there is positive regeeration and negative-feedback regu-lation in RANKL-RANK-OPG axis signal.1 α, 25-(OH)
2d
3, parathyroid hormone, prostaglandin E2, interleukin-11, the bone resorbing factor such as IL-6 mediate path respectively by Vitamin D Receptor, protein kinase A mediation path, gp130/STAT mediate path and raise scleroblast/marrow stromal cell RANKL mrna expression; IL-1, TNF-α strengthens RANKL signal respectively by IL-1R and TNFR1 activating osteoclast surface; The RANK that TGF-β then can increase osteoclast surface raises RANKL signal.
As the key link of RANKL-RANK-OPG signalling system regulation and control differentiation of osteoclast, the interaction between RANKL and RANK and the micromechanism impelling RANK to activate thereof are the hot issues in bone resorption class disease research field all the time.One of its focal issue is the crystalline structure of RANKL, rank protein and RANKL-RANK complex body.Although obtained the crystalline structure of RANKL albumen at present, but still there is no the crystalline structure of rank protein and RANK/RANKL complex body, just because of not enough to the understanding of this structure, so how scholars are regulated and controled for RANKL-RANK-OPG signal scheme, and the micromechanism of modulated rear RANKL-RANK-OPG executive signal transmission there is no clear and definite understanding.This has had a strong impact on the research of exploration about bone resorption class disease mechanism of causing a disease and anti-bone resorption medicine.
Analyze the reason also not obtaining the crystalline structure of rank protein and RANKL-RANK complex body at present and mainly contain following 2 points: 1.RANK albumen is comparatively difficult to preparation and purifying.Be found in the time so far at RANK, express and the relevant report of purifying without rank protein E.coli always.2. relative to the structure compared with rigidity that the TNF protein family members comprising RANKL has, its structure of TNFR protein family member comprising RANK has larger mutability, is thus more difficult to obtain crystallization.
Summary of the invention
The invention provides a kind of protein complexes crystal, wherein said complex body comprises the aminoacid sequence of mouse RANK/RANKL extracellular regions albumen.The extracellular regions of described mouse RANKL refers to that described RANKL monomer C-end is from 161 amino acids to 316 amino acids, and the extracellular regions of described RANK refers to that described RANK monomer N-end is from 26 amino acids to 210 amino acids.
According in mouse RANK/RANKL complex body crystal provided by the invention, described crystal belongs to P6
3hexagonal space group, unit cell dimension is a=b=121.23
and c=94.67
In crystal provided by the invention, this complex body comprises the tripolymer kernel by three RANKL monomer chain formation, and another three RANK monomer chains.RANK monomer chain is combined on RANKL tripolymer kernel respectively, thus forms six aggressiveness.Each RANK receptors bind is in the crack formed by adjacent two RANKL part subunits; This six aggressiveness complex body has 10,161
2solvent accessible surface, by RANK contribute 5,340
2, RANKL contribution 4,821
2.
In crystal provided by the invention, the ring 120S (i.e. residue 122-127) of RANK monomer chain is combined with part RANKL completely, in two regions of RANK monomer chain, i.e. residue 182-185, and residue 194-196 forms 2 short β-sheet conformation, thus form comparatively orderly structural domain further.
In crystal provided by the invention, in described complex body, the extracellular regions of the contiguous trans-membrane region of RANK, namely residue 154-199 is very orderly, and this residue 154-199 region comprises CRD4 structural domain.
In crystal provided by the invention, there is a sodium ion in each RANK monomer chain, and this sodium ion can with the Cys-134 in RANK/RANKL, the carbonyl group chelating of the Ser-161 side chain on Ala-135, Phe-138, Val-163 and skeleton.
In crystal provided by the invention, Lys-281 and Arg-283 in Asp-85 and the RANKL of each RANK monomer forms polar interaction, the skeleton of the Gly-191 in Glu-76 and the RANKL in RANK forms hydrogen bond, Asp-124 in RANK and His-179 and Lys-180 in Glu-126 and RANKL forms polar interaction, Arg-129 and Arg-130 in RANK respectively with the Glu-225 in RANKL, Asn-266 and Asp-268 interacts, Ser-123 skeleton (trunk on RANK ring 100S (residue 102-112), backbone) Lys-180 of carbonyl group and RANKL interacts and forms hydrogen bond, Arg-222, Asp-299 and Asp-301 in the ring 90S residue A sp-94 and Lys-97 and RANKL of RANK form polar interaction.
The invention provides a kind of method of expression and purification RANK extracellular protein, comprise: (1) builds the carrier of the cDNA with coding RANK extracellular regions, by this vector in prokaryotic cell prokaryocyte, thus obtain with the restructuring RANK of inclusion bodies expression; (2) supersound process is carried out to this inclusion body, be then again dissolved in the Guanidinium hydrochloride of 6M, obtain the restructuring RANK of dissociated state; (3) again described restructuring RANK is folded; (4) agarose 75 chromatographic column is utilized to isolate restructuring RANK; (5) collected by SDS-PAGE (SDS-PAGE) and analyze correct folding RANK.Method provided by the invention is particularly useful for the expression and purification of mouse RANK extracellular regions albumen.
In expression and purification method provided by the invention, the product that the cDNA of the coding rank protein mRNA that is mouse RAW264.7 cell obtains through reverse transcription PCR.
In expression and purification method provided by the invention, the prokaryotic cell prokaryocyte of employing is E.coli bacterial strain BL21-Gold.
Present invention also offers a kind of method preparing RANK/RANKL extracellular regions complex body crystal, comprise: the expression and purification of (1) RANKL extracellular regions: utilize Triptide thioltransferase fusion rotein mode to express solubility RANKL, and utilize Glutathione Sepharose 4B (Glutathione-Sepharose fast flow 4B beads) by this fusion monomer of affinity purification purifying, to cut with PreScission proteolytic enzyme.Cut RANKL agarose 75 chromatography, thus obtain the extracellular regions monomer of the RANKL after purifying.(2) the RANK monomer method according to above-mentioned expression and purification RANK obtained and this RANKL, be condensed into 10mg/mL at the 1M TRIS of pH7.00 in buffered soln, and with 1: 1 ratio mixing; (3) in temperature be 294K condition under, utilize high-throughput nano to rise to sit and drip vapor diffusion method and form the crystal of RANK-RANKL extracellular regions; (4) described crystal is in 0.1M SODIUM PHOSPHATE, MONOBASIC, 2M sodium-chlor, grows under the condition of 0.1M Kdp and 0.1M MES (pH6.5).The method is particularly useful for the preparation of mouse RANK/RANKL extracellular regions complex body crystal.
In the method for formation RANK/RANKL complex body provided by the invention crystallization, the crystal obtained can also 20% PEG 3350 and 0.2M ammonium formiate in grow.This growth conditions is particularly useful for the growth of the outer complex body crystal of mouse RANK/RANKL born of the same parents.
According to the application of RANK/RANKL complex body crystal provided by the invention in screening treatment bone resorption class disease or anti-bone resorption medicine, comprising: according to the crystalline structure of complex body, designed arbitrary polypeptide that can block complex body and be formed by computer simulation, protein, antibody, immune conjugate, inorganics or organism, wherein said polypeptide, protein, antibody, immune conjugate, it is one or more that the site that inorganics or organism can act on is selected from the group of following action site composition: the action site of Lys-281 and Arg-283 in Asp-85 and the RANKL of RANK formation, the action site of the skeleton formation of the Gly-191 in Glu-76 and the RANKL in RANK, the action site that Asp-124 in RANK and His-179 and Lys-180 in Glu-126 and RANKL is formed, Arg-129 and Arg-130 in RANK respectively with the Glu-225 in RANKL, the action site that Asn-266 and Asp-268 is formed, the action site that Lys-180 in the backbone carbonyl group of the Ser-123 on RANK ring 100S and RANKL is formed, Arg-222 in the ring 90S residue A sp-94 and Lys-97 and RANKL of RANK, the action site that Asp-299 and Asp-301 is formed, and the residue of the ring 120S of described RANK monomer chain, i.e. residue 122-127, wherein, the polypeptide of the bonding force of RANK and RANKL decline 30%, protein, antibody, immune conjugate, inorganics or organism is made to be candidate compound, the application of described candidate compound in treatment bone resorption class disease or anti-bone resorption medicine is determined by biological experiment.
Accompanying drawing explanation
Fig. 1 shows the three-dimensional structure of RANK/RANKL complex body;
Fig. 2 A shows the result of three RANK monomer overlaps in complex body;
Fig. 2 B shows the result that RANK monomer is overlapping with TNFR1, RANK and DR5 overlap obtains;
Fig. 3 shows the overlapping result obtained of the configuration of RANKL and bonding state and the RANKL of unbound state;
Fig. 4 shows upper contact area in acceptor and ligand interaction and lower contact area;
Fig. 5 shows TNF β-TNFR1, Trail-DR5, and the hydrophobic bond in the upper contact layer of RANKL-RANK tri-kinds of mixtures;
Fig. 6 A shows the important residue in RANK/RANKL interacts;
Fig. 6 B is added with dark-background on the basis of Fig. 6 A, the important residue in interacting with clearer identification RANK/RANKL;
Fig. 7 shows the equivalent amino acid pair between mRANK and DR5 and TNFR1.
Embodiment
One, Expression and purification
1. the Expression and purification of mouse RANK extracellular regions albumen
The aminoacid sequence of mouse RANK is as follows:
MAPRARRRRQLPAPLLALCVLLVPL
QVTLQVTPPCTQERHYEHLGRCCSR
CEPGKYLSSKCTPTSDSVCLPCGPDEYLDTWNEEDKCLLHKVCDAGKALV
AVDPGNHTAPRRCACTAGYHWNSDCECCRRNTECAPGFGAQHPLQLNKDT
VCTPCLLGFFSDVFSSTDKCKPWTNCTLLGKLEAHQGTTESDVVCSSSMT
LRRPPKEAQAYLPSLIVLLLFISVVVVAAIIFGVYYRKGGKALTANLWNW
VNDACSSLSGNKESSGDRCAGSHSATSSQQEVCEGILLMTREEKMVPEDG
AGVCGPVCAAGGPWAEVRDSRTFTLVSEVETQGDLSRKIPTEDEYTDRPS
QPSTGSLLLIQQGSKSIPPFQEPLEVGENDSLSQCFTGTESTVDSEGCDF
TEPPSRTDSMPVSPEKHLTKEIEGDSCLPWVVSSNSTDGYTGSGNTPGED
HEPFPGSLKCGPLPQCAYSMGFPSEAAASMAEAGVRPQDRADERGASGSG
SSPSDQPPASGNVTGNSNSTFISSGQVMNFKGDIIVVYVSQTSQEGPGSA
EPESEPVGRPVQEETLAHRDSFAGTAPRFPDVCATGAGLQEQGAPRQKDG
TSRPVQEQGGAQTSLHTQGSGQCAE
Dashed part is the aminoacid sequence of mouse RANK extracellular regions
Material
Oligonucleotide is prepared from by Sangon Biotech (China); Restriction enzyme, T4DNA ligase enzyme and the first chain cDNA synthetic agent box is buied from Fermentas; Pfu archaeal dna polymerase is buied from Tiangen Biotech (China); Triptide (reduced form and oxidized form) is buied from Sigma; Glutathione Sepharose 4B is available from General Electric's Medical Group (GE Healthcare); TRIZOL reagent is purchased from Invitrogen.Inc.; All other obtains chemical substance and is analytical pure.
Plasmid and bacterial strain
The cDNA of encoding mature mouse RANK extracellular regions (residue 26-210) is obtained through reverse transcription PCR by the mRNA of mouse RAW264.7 cell, and is cloned in pET28 carrier (Novagen).The plasmid (residue 159-361) of expressing for GST-RANKL is received in Daved H.Fremont professor (Department of Pathology andImmunology, Washington University School of Medicine, USA.), recombinant protein is expressed with E.coli bacterial strain BL21-Gold (buying in Stratagene company).
Expression and purification
In E.coli bacterial strain BL21-Gold (DE3), restructuring RANK mainly expresses with inclusion bodies.This inclusion body is purified by supersound process and is again dissolved in the Guanidinium hydrochloride of 6M.The again folding of restructuring RANK realizes as follows: this inclusion body is diluted in the Na comprising 20mM
2hPO
4(pH7.3), in renaturation (IB) solution of 1M L-arginine, 20% glycerine, 10mM reduced glutathione and 1mM oxidized glutathione, then at the Na containing 20mM
2hPO
4(pH7.3), dialysis 12 hours at 4 DEG C in the liquid of folding buffered again 1 of 0.5M L-arginine, 10% glycerine, then at the Na containing 20mM
2hPO
4(pH7.3), dialysis 12 hours at 4 DEG C in the liquid of folding buffered again 2 of 0.2M L-arginine, 5% glycerine, finally at the Na of 20mM
2hPO
4(pH7.3), dialyse 12 hours at 4 DEG C in 0.2M L-arginine, after 20000g is centrifugal 10 minutes, supernatant liquor 75 (Superdex75) chromatographic column (purchased from Amersham pharmacia company) is carried out purifying, and collect correct folding mouse RANK, and analyze with SDS-PAGE.
The expression and purification of 2.RANKL
The aminoacid sequence of mouse RANKL is as follows:
MRRASRDYGKYLRSSEEMGSGPGVPHEGPLHPAPSAPAPAPPPAASRSMF
LALLGLGLGQVVCSIALFLYFRAQMDPNRISEDSTHCFYRILRLHENADL
QDSTLESEDTLPDSCRRMKQAFQGAVQKELQHIVGPQRFSGAPAMMEGSW
LDVAQRGKPE
AQPFAHLTINAASIPSGSHKVTLSSWYHDRGWAKISNMTL
SNGKLRVNQDGFYYLYANICFRHHETSGSVPTDYLQLMVYVVKTSIKIPS
SHNLMKGGSTKNWSGNSEFHFYSINVGGFFKLRAGEEISIQVSNPSLLDP
DQDATYFGAFKVQDID
Dashed part is the aminoacid sequence of mouse RANKL extracellular regions
The extracellular soluble exterior domain of mouse RANKL is with Triptide thioltransferase fusion protein form expression; Fusion rotein utilizes Glutathione Sepharose 4B (purchased from Amersham pharmacia company) to carry out affinity purification, cuts with PreScission proteolytic enzyme; Enzyme cuts RANKL agarose 75 (Superdex 75) chromatographic column (purchased from the Amersham pharmacia company) purifying of gained.
Two, the crystallization of RANK/RANKL complex body
Ratio with 1: 1 mixes the RANK monomeric protein and RANKL monomeric protein and obtained RANK/RANKL complex protein solution that two kinds of purifying obtain.Be under the condition of 294K in temperature, utilize high-throughput nano to rise and sit and drip the crystal grid (crystallization screen) that vapor diffusion method (sitting dropvapour diffusion method) forms RANK/RANKL extracellular regions, and utilize the automatic crystallographic image system in the protein product instrument of Oxford to carry out monitoring (Mayo etc. 2005, Walter etc. 2005).9 Secondary Shocks (hit) are given, the condition adopted in this 9 Secondary Shocks: the inorganic salt (comprising ammonium acetate, ammonium formiate or ammonium nitrate) of PEG3350 and lower concentration, all can have one at every turn and change to initial crystal grid.The best crystal of growing state is obtained: 1) 0.1M SODIUM PHOSPHATE, MONOBASIC, 2M sodium-chlor, 0.1M Kdp and 0.1M MES (pH6.5) under following two optimum conditions; 2) PEG 3350 of 20% and the ammonium formiate of 0.2M, containing 100nL protein solution and 100nL pond liquid in crystallization drop.The crystal grown under these two conditions has identical morphology, is generally the length with hexagonal transverse cross section bar-shaped.These crystal occurred usually within one week, and can in two months continued growth until overall dimension.
Three, the mensuration of mouse RANK/RANKL extracellular regions complex body crystal
The X-ray data obtaining RANK/RANKL complex body crystal in condition for 1 time is at ESRF, utilizes light beam line ID23-EH2 to obtain.0.873
under, 180 images that diffraction concussion is 1.0 ° are collected from 2 positions of monocrystalline.
At wavelength 1.060
under, utilize the light beam line 103 of diamond to obtain the X-ray data of the complex body crystal of condition 2 times growths.25% glycerine joins in crystallization drop as cryoprotectant, and crystal is frozen and in whole data acquisition phase, adopts the freezing stream of nitrogen to keep crystal to be in 100K.Utilize HKL2000 (Ref.) to carry out index to data image, anomalous integral merges.The statistic data to X-ray data is given in table 1.
Four, result is resolved:
1. crystal test result
Table 1 X-ray diffraction result
Data in bracket are high-resolution results.R
workand R
freebe defined as R=∑
hkl|| F
obs|-| F
calc||/∑
hkl| F
obs|, wherein h, k, l refer to that reflection index is (for R
workbecome more meticulous; 5%, be not used in R
freemeticulous), F
obsand F
calcbe structure factor, from the intensity measured, inference out and calculated by model.
Average B-factor is used for albumen, water and other atoms
RANK/RANKL complex body crystal belongs to P6
3hexagonal space group, unit cell dimension is a=b=121.23
and c=94.67
The structure elucidation of 2.RANK/RANKL complex body
The RANK structure of 2.1 bonding states
In RANK/RANKL complex body crystal, do not contact with each other in extracellular regions between RANK monomer.They are attached to three major grooves (groove) of RANKL homotrimer respectively and show a three fold symmetry.In complex body, three monomers of RANK have very high similarity in configuration, and arrange (Fig. 1 and Fig. 2 A) in an orderly way, have 0.14
root-mean-square deviation (RMSD).But in one-piece construction, the RANK in complex body takes the configuration similar to non-binding partner RANK, utilize VegaZZ 2.1 software analysis known, based on the paired overlap of the corresponding CRD of C alpha atom, the scope presenting RMSD is from 0.59 CRD3
to 1.63 in CRD2
; The RMSD that whole molecule obtains as the overlap of a compact unit is 4.69
The preamble section that has of the RANK in complex body comprises four CRD, and this point and other the TNF receptor family member resolved, comprise TNFR1 and DR5 and there is significant difference, the CRD4 of the TNF receptor family member resolved is unordered or lacks completely.RANK and with overlap (superposition) and the RANK of the TNFR1 that TNF β combines with demonstrate with the overlapping same of the DR5 that TRAIL combines, in all CRD, the structural similarity of CRD2 structural domain is maximum (see Fig. 2 B), RANK and TNFR1 compares, and the RMSD based on the CRD2 of C alpha atom is 0.54
, RANK and DR5 compares, and RMSD is 0.88
, and the CRD1 structural domain of RANK and TNFR1 also have the structural similarity of height, RANK and TNFR1 compares, and RMSD is 2.65
, but the CRD1 of RANK and DR5 has poor structural similarity, because front 20 residues of DR5 are very unordered.Two overlaps all illustrate, the CRD3 comprising DE ring (DE loop) at the N-terminal of CRD3 has lower structural similarity (see Fig. 2 B), and one of CRD3 important feature basis being considered to receptor-ligand interaction (between TNF family complex body member and their acceptor).As compared to the combination of TNFR1 with TNF β, the combination of RANK and RANKL makes the junction (Ala-114) between CRD2 and the CRD3 of RANK, windup-degree there is the change of about 30 degree, compared with the combination of DR5 and TRAIL, there is the change of about 25 degree in the N-terminal windup-degree of the CRD3 of RANK, like this, due to the difference of the change of windup-degree in the difference between CRD3 and cohesive process, the difference of different geometric configurations can be realized on the receptors bind surface of different TNF superfamily member.
It is reported in TNF β-TNFR1 complex body, the territory, C-terminal subprovince (CRD4) (it is adjacent to the trans-membrane region of acceptor) in receptor extracellular region territory is unordered.In TRAIL-DR5, someone reports that the C-terminal part (residue 104-130) of the CRD3 of DR5 is only before the transbilayer helix of supposition, shows Rigid Body in Rotation With in one direction; Shown in another report in six DR5 acceptor molecules, this region of four DR5 molecules is had to be all highly unordered.But in RANK/RANKL complex body, the extracellular regions (residue 154-199 comprises CRD4 region) of the contiguous trans-membrane region of RANK is very orderly and shows the conformation of a rigidity.
What is interesting is, do not combining and bonding state, each RANK monomer observes a metal ion combined with it in the coil region between territory, CRD3 subprovince and territory, CRD4 subprovince.According to the average bond length of binding site, the average calcium key-valency summation (CBVS of this metal ion calculated, calcium bond-valence sum) be 1.2, for sodium ion, the desired value of CBVS is 1.6, potassium ion is 0.6, calcium ion is 2.0, and mn ion is 3.2, and magnesium ion is 4.2 (Mulle etc., 2003), so infer that this metal ion is sodium ion; By at wavelength 1.698
under the abnormal diffraction disparity map (anomalous difference map) that calculates of the data collected, this deduction is confirmed further, not yet has similar report so far in other TNF receptor family member.This metal ion can with the carbonyl group chelating of the side chain of Cys-134, Ala-135, Phe-138 and Val-163 in complex body and skeleton (trunk, backbone) Ser-161.
RANKL structure in 2.2 complex bodys
RANKL one-piece construction in RANK/RANKL complex body and the structure of unconjugated RANKL closely similar.Identical with other TNF superfamily member, RANKL monomer comprises two flat β-lamellas: beta chain A ", A, H, C and F, it forms interior β-lamella and joins in the interaction between inner subunit; Beta chain B ', B, G, D and E, it forms outer β-lamella (see Fig. 3).Compare the RANKL of RANKL in complex body and free state, by with receptors bind, the ring (loop) on RANKL surface there occurs the change on some configurations.The RANKL molecule of overlapping bonding state and free state, based on residue 161-316, the RMSD obtained is 0.57.The AA that height under unbound state is bending " ring, CD ring, DE ring and EF ring be all very orderly, among these rings, DE ring is considered to the important region (see Fig. 3) of mediates receptor-ligand interaction.
2.3. the interaction of RANK and RANKL in complex body
Receptor-ligand binding in RANK/RANKL six aggressiveness reflects characteristic common in TNF superfamily complex body, and namely a receptors bind is in the gap formed by adjacent Liang Ge part subunit, and the six aggressiveness complex bodys of RANK/RANKL have 10,161
2solvent accessible surface, acceptor contribution 5,340
2, part contribution 4,821
2.Interaction between RANKL and RANK can be divided into two regions easily, this similar to TNFR1-TNF β complex body and DR5-TRAIL complex body: upper contact area and lower contact area (Fig. 4).This differentiation is mainly based on the position of two rings of acceptor: ring 70S (residue 73-86) and ring 110S (residue 114-132), they have mediated main phase mutual effect.
In the upper contact area of TNFR1-TNF β complex body and DR5-TRAIL complex body, hydrophobic interaction is main interaction force, and the tyrosine-216 in Tyr-108 and TRAIL in TNF β is considered to the key amino acid in this interaction.Residue around this region and some hydrophobic residues, the Ile-220 be included in Val-112 and TRAIL in TNF β forms hydrophobic bundle and outsourcing (outpocketing) hydrophobic residue, comprises Leu-57, Leu-61 in Leu-67, Leu-71 and DR5 of TNFR1 respectively at the first half regional interactions (Fig. 5) of CRD2.In contrast, this contact area in RANK/RANKL complex body, although there is hydrophobic residue, but the Ile-248 in RANKL (is equivalent to the Tyr-108 in TNF β, tyrosine-216 in TRAIL), in hydrophobic interaction two other important hydrophobic residues, namely be that Val-112 and Leu-67 (being Ile-220, Leu-57 respectively in TRAIL and DR5) is replaced by two charged residues respectively in TNF β and TNFR1, be the Glu-84 (not shown) in His-252 and RANK in RANKL., analyze from crystalline structure figure, remaining two hydrophobic residues in the upper contact area of RANK/RANKL complex body are also each other from far meanwhile.Therefore, hydrophobic interaction and other two composite bulk phase ratios of RANK/RANKL interaction place, seem not play a decisive role.Except hydrophobic interaction, interaction place in the upper contact area of RANKL-RANK complex body is made up of some polarity or coulombic interaction.Such as, the Lys-281 in Asp-85 and the RANKL of RANK and Arg-283 forms polar interaction, and the skeleton of the Gly-191 in Glu-76 and the RANKL in RANK forms hydrogen bond (Fig. 6 A, Fig. 6 B and table 2).
Table 2 RANK participates in the interactional residue of RANK/RANL
| Site | Amino acid | Corresponding to the residue in RANKL | Chemical bond |
| 76 | Glu | Gly-191 | Hydrogen bond |
| 85 | Asp | Arg-283；Lys-281 | Sat linkage and hydrogen bond |
| 94 | Asp | Arg-222 | Sat linkage |
| 97 | Lys | Asp-299；Asp-301 | Sat linkage and hydrogen bond |
| 124 | Asp | His-179；Lys-180 | Sat linkage and hydrogen bond |
| 126 | Glu | Lys-180 | Sat linkage |
| 129 | Arg | Glu-268 | Sat linkage |
| 130 | Arg | Glu-225；Asn-266 | Sat linkage and hydrogen bond |
Lower contact area in RANK/RANKL complex body, much larger than upper contact area, has 6,579
2solvent accessible surface, be about 64% of total area occupied.The ring 110S of RANK occupies the major part (65%) of formed area, and four residues on this ring, Asp-124, Glu-126, Arg-129 and Arg-130 play decisive role for contact.As indicated with 6, the Asp-124 in RANK and His-179 and Lys-180 in Glu-126 and RANKL forms polar interaction, Arg-129 and Arg-130 in RANK interacts with Glu-225, Asn-266 and the Asp-268 in RANKL respectively.In addition, skeleton (trunk, the backbone) carbonyl group of the Ser-123 on ring 100S and the Lys-180 of RANKL interact and form hydrogen bond.The ring 90S (residue 93-101) of RANK also participates in (about 28%) in the interaction of lower contact area.In this ring, the Asp-94 in RANK and Arg-222, Asp-299 and the Asp-301 in Lys-97 and RANKL forms polar interaction.
To the interactional residue of RANK/RANKL be identified oneself with and identify oneself with other TNF superfamily member compared with the residue in the interaction of its acceptor, only can find the combination pair that of equal value in TRAIL-DR5 complex body, i.e. Asp-67 to Arg-191 (see Fig. 7).Some residues in other complex body, Arg-77 in such as TNFR1 and the Glu-98 in DR5, Arg-101, Lys-102, it is equivalence and the position Asp-94 in RANK respectively, Glu-126, Arg-129, Arg-130, also participate in the interaction of acceptor and part, according to structural analysis, the residue that they combine is not equivalent to those residues in RANK/RANKL complex body.From the low conserved residues of ligand-receptor interaction, and lack in hydrophobic interaction, we can draw the interactional specific structural features of RANK/RANKL.
3. utilize biacore instrumental analysis to verify RANKL albumen interacts to RANK/RANKL relevant several critical sites further:
The binding constant of table 3 RANKL mutant and RANK
| Wild RANKL and mutant thereof | Binding constant (KD) |
| Wild RANKL | 4.48×10 -11 |
| 180 | 3.37×10 -10 |
| 222 | 6.90×10 -9 |
| 225 | 1.50×10 -9 |
| 266 | 8.84×10 -11 |
| 268 | 5.23×10 -10 |
| 281 | 9.23×10 -11 |
| 283 | 1.43×10 -10 |
| 299 | 1.05×10 -9 |
| 301 | 7.22×10 -10 |
Result shows, after the amino acid mutation of these critical sites becomes L-Ala or glycine, its binding constant value all increases considerably, show that the binding ability of RANKL mutant and RANK declines to a great extent, the vital role of these sites of sufficient proof in RANK/RANKL be combined with each other, also the sufficient proof accuracy of Computer simulation results.
Illustrate in conjunction with above, the present invention has disclosed the crystalline structure of RANK/RANKL complex body, from above-mentioned crystalline structure, is not difficult to find the important residue in these composite bulk phase mutual effects.According to the analytical results of Blast on the www.ncbi.nlm.nih.gov of NCBI website, the RANK extracellular regions homology of mouse and people is 83%, the RANKL extracellular regions homology of mouse and people is 90%, therefore, utilize these information, we can continue the bioactive activated pathway sought caused by RANK/RANKL, design the medicine that can regulate associated biomolecule activated pathway, thus reach the effect of required physiologically active further.According to the crystalline structure of described complex body, arbitrary polypeptide that can block described complex body and be formed can be designed by computer simulation, protein, antibody, immune conjugate, inorganics or organism, wherein said polypeptide, protein, antibody, immune conjugate, it is one or more that the site that inorganics or organism can act on is selected from the group of following action site composition: the action site of Lys-281 and Arg-283 in Asp-85 and the RANKL of RANK formation, the action site of the skeleton formation of the Gly-191 in Glu-76 and the RANKL in RANK, the action site that Asp-124 in RANK and His-179 and Lys-180 in Glu-126 and RANKL is formed, Arg-129 and Arg-130 in RANK respectively with the Arg-225 in RANKL, the action site that Asn-266 and Asp-268 is formed, the action site that Lys-180 in the backbone carbonyl group of the Ser-123 on RANK ring 100S and described RANKL is formed, Arg-222 in the ring 90S residue A sp-94 and Lys-97 and RANKL of RANK, Asp-299 and Asp-301, and the action site that the residue of ring 120S on described RANK monomer chain is formed, wherein, make the polypeptide of the bonding force decline 30% of RANK and RANKL, protein, antibody, immune conjugate, inorganics or organism are candidate compound, the application of described candidate compound in treatment bone resorption class disease or anti-bone resorption medicine is determined by biological experiment.
Above, preferred embodiment the present invention is described based on specific, but when without prejudice to the spirit and scope of the present invention defined in claims, can increase multiple change and amendment, this should be apparent for those skilled in the art.
Sequence table
<110> Chinese People's Liberation Army General Hospital
Beijing Epigen Biotechnology Co., Ltd.
The crystal of the outer complex body of <120> mouse RANK/RANKL born of the same parents, prepares its method and application thereof
<130>P21357BYSW
<160>2
<170>PatentIn version 3.2
<210>1
<211>625
<212>PRT
<213> mouse rank protein
<400>1
Met Ala Pro Arg Ala Arg Arg Arg Arg Gln Leu Pro Ala Pro Leu Leu
1 5 10 15
Ala Leu Cys Val Leu Leu Val Pro Leu Gln Val Thr Leu Gln Val Thr
20 25 30
Pro Pro Cys Thr Gln Glu Arg His Tyr Glu His Leu Gly Arg Cys Cys
35 40 45
Ser Arg Cys Glu Pro Gly Lys Tyr Leu Ser Ser Lys Cys Thr Pro Thr
50 55 60
Ser Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Thr
65 70 75 80
Trp Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Ala Gly
85 90 95
Lys Ala Leu Val Ala Val Asp Pro Gly Asn His Thr Ala Pro Arg Arg
100 105 110
Cys Ala Cys Thr Ala Gly Tyr His Trp Asn Ser Asp Cys Glu Cys Cys
115 120 125
Arg Arg Asn Thr Glu Cys Ala Pro Gly Phe Gly Ala Gln His Pro Leu
130 135 140
Gln Leu Asn Lys Asp Thr Val Cys Thr Pro Cys Leu Leu Gly Phe Phe
145 150 155 160
Ser Asp Val Phe Ser Ser Thr Asp Lys Cys Lys Pro Trp Thr Asn Cys
165 170 175
Thr Leu Leu Gly Lys Leu Glu Ala His Gln Gly Thr Thr Glu Ser Asp
180 185 190
Val Val Cys Ser Ser Ser Met Thr Leu Arg Arg Pro Pro Lys Glu Ala
195 200 205
Gln Ala Tyr Leu Pro Ser Leu Ile Val Leu Leu Leu Phe Ile Ser Val
210 215 220
Val Val Val Ala Ala Ile Ile Phe Gly Val Tyr Tyr Arg Lys Gly Gly
225 230 235 240
Lys Ala Leu Thr Ala Asn Leu Trp Asn Trp Val Asn Asp Ala Cys Ser
245 250 255
Ser Leu Ser Gly Asn Lys Glu Ser Ser Gly Asp Arg Cys Ala Gly Ser
260 265 270
His Ser Ala Thr Ser Ser Gln Gln Glu Val Cys Glu Gly Ile Leu Leu
275 280 285
Met Thr Arg Glu Glu Lys Met Val Pro Glu Asp Gly Ala Gly Val Cys
290 295 300
Gly Pro Val Cys Ala Ala Gly Gly Pro Trp Ala Glu Val Arg Asp Ser
305 310 315 320
Arg Thr Phe Thr Leu Val Ser Glu Val Glu Thr Gln Gly Asp Leu Ser
325 330 335
Arg Lys Ile Pro Thr Glu Asp Glu Tyr Thr Asp Arg Pro Ser Gln Pro
340 345 350
Ser Thr Gly Ser Leu Leu Leu Ile Gln Gln Gly Ser Lys Ser Ile Pro
355 360 365
Pro Phe Gln Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu Ser Gln
370 375 380
Cys Phe Thr Gly Thr Glu Ser Thr Val Asp Ser Glu Gly Cys Asp Phe
385 390 395 400
Thr Glu Pro Pro Ser Arg Thr Asp Ser Met Pro Val Ser Pro Glu Lys
405 410 415
His Leu Thr Lys Glu Ile Glu Gly Asp Ser Cys Leu Pro Trp Val Val
420 425 430
Ser Ser Asn Ser Thr Asp Gly Tyr Thr Gly Ser Gly Asn Thr Pro Gly
435 440 445
Glu Asp His Glu Pro Phe Pro Gly Ser Leu Lys Cys Gly Pro Leu Pro
450 455 460
Gln Cys Ala Tyr Ser Met Gly Phe Pro Ser Glu Ala Ala Ala Ser Met
465 470 475 480
Ala Glu Ala Gly Val Arg Pro Gln Asp Arg Ala Asp Glu Arg Gly Ala
485 490 495
Ser Gly Ser Gly Ser Ser Pro Ser Asp Gln Pro Pro Ala Ser Gly Asn
500 505 510
Val Thr Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val Met
515 520 525
Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser Gln
530 535 540
Glu Gly Pro Gly Ser Ala Glu Pro Glu Ser Glu Pro Val Gly Arg Pro
545 550 555 560
Val Gln Glu Glu Thr Leu Ala His Arg Asp Ser Phe Ala Gly Thr Ala
565 570 575
Pro Arg Phe Pro Asp Val Cys Ala Thr Gly Ala Gly Leu Gln Glu Gln
580 585 590
Gly Ala Pro Arg Gln Lys Asp Gly Thr Ser Arg Pro Val Gln Glu Gln
595 600 605
Gly Gly Ala Gln Thr Ser Leu His Thr Gln Gly Ser Gly Gln Cys Ala
610 615 620
Glu
625
<210>2
<211>316
<212>PRT
<213> mouse RANKL protein amino acid sequence
<400>2
Met Arg Arg Ala Ser Arg Asp Tyr Gly Lys Tyr Leu Arg Ser Ser Glu
1 5 10 15
Glu Met Gly Ser Gly Pro Gly Val Pro His Glu Gly Pro Leu His Pro
20 25 30
Ala Pro Ser Ala Pro Ala Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser
35 40 45
Met Phe Leu Ala Leu Leu Gly Leu Gly Leu Gly Gln Val Val Cys Ser
50 55 60
Ile Ala Leu Phe Leu Tyr Phe Arg Ala Gln Met Asp Pro Asn Arg Ile
65 70 75 80
Ser Glu Asp Ser Thr His Cys Phe Tyr Arg Ile Leu Arg Leu His Glu
85 90 95
Asn Ala Asp Leu Gln Asp Ser Thr Leu Glu Ser Glu Asp Thr Leu Pro
100 105 110
Asp Ser Cys Arg Arg Met Lys Gln Ala Phe Gln Gly Ala Val Gln Lys
115 120 125
Glu Leu Gln His Ile Val Gly Pro Gln Arg Phe Ser Gly Ala Pro Ala
130 135 140
Met Met Glu Gly Ser Trp Leu Asp Val Ala Gln Arg Gly Lys Pro Glu
145 150 155 160
Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser
165 170 175
Gly Ser His Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly Trp
180 185 190
Ala Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val Asn
195 200 205
Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His His
210 215 220
Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu Met Val Tyr
225 230 235 240
Val Val Lys Thr Ser Ile Lys Ile Pro Ser Ser His Asn Leu Met Lys
245 250 255
Gly Gly Ser Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe His Phe Tyr
260 265 270
Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile
275 280 285
Ser Ile Gln Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala
290 295 300
Thr Tyr Phe Gly Ala Phe Lys Val Gln Asp Ile Asp
305 310 315
Claims (11)
1. a protein complex crystal, wherein said complex body comprises the aminoacid sequence of mouse RANK/RANKL extracellular regions complex body, and the crystal of wherein said complex body has spacer P6
3hexagonal space group, unit cell dimension is a=b=121.23 and c=94.67; Wherein, in the complex body of described mouse RANK/RANKL extracellular regions, the aminoacid sequence of the extracellular regions of rank protein is: QVTLQVTPPCTQERHYEHLGRCCSRCEPGKYLSSKCTPTSDSVCLPCGPDEYLDTW NEEDKCLLHKVCDAGKALVAVDPGNHTAPRRCACTAGYHWNSDCECCRRNTECAPG FGAQHPLQLNKDTVCTPCLLGFFSDVFSSTDKCKPWTNCTLLGKLEAHQGTTESDV VCSSSMTLRRPPKEAQA; The aminoacid sequence of the extracellular regions of RANKL albumen is: AQPFAHLTINAASIPSGSHKVTLSSWYHDRGWAKISNMTLSNGKLRVNQDGFYYLY ANICFRHHETSGSVPTDYLQLMVYVVKTSIKIPSSHNLMKGGSTKNWSGNSEFHFY SINVGGFFKLRAGEEISIQVSNPSLLDPDQDATYFGAFKVQDID.
2. crystal according to claim 1, the extracellular regions of wherein said mouse RANKL refers to that described RANKL monomer chain C-end is from 161 amino acids to 316 amino acids, and the extracellular regions of described mouse RANK refers to that described RANK monomer chain N-end is from 26 amino acids to 210 amino acids.
3. crystal according to claim 1, wherein said complex body is three mouse RANKL monomer chain formation tripolymer kernels, and another three mouse RANK monomer chains are combined on described RANKL tripolymer kernel respectively, form six aggressiveness.
4. crystal according to claim 3, is characterized in that described six aggressiveness have 10,161
2solvent accessible surface, by RANK contribute 5,340
2, contribute 4,821 by RANKL
2.
5. crystal according to claim 3, wherein in described complex body crystal, the ring 120S of described RANK monomer chain, namely residue 122-127 is combined with described RANKL completely, and the residue 182-185 of described RANK monomer chain and residue 194-196 forms 2 short β-sheet conformation.
6. crystal according to claim 3, wherein in described RANK/RANKL complex body, the residue 154-199 of the contiguous trans-membrane region of described RANK is very orderly, and described residue 154-199 region comprises CRD4 structural domain.
7. crystal according to claim 3, in described complex body, there is a sodium ion in described RANK monomer chain, described sodium ion can with the carbonyl group chelating of Cys-134, Ala-135, Phe-138, the Val-163 in described complex body and the Ser-161 side chain on skeleton.
8. crystal according to claim 3, in described complex body, Lys-281 and Arg-283 in the Asp-85 of described RANK and described RANKL forms polar interaction; The skeleton of the Gly-191 in the Glu-76 in described RANK and described RANKL forms hydrogen bond; His-179 and Lys-180 in Asp-124 and Glu-126 in described RANK and described RANKL forms polar interaction; Arg-129 and Arg-130 in described RANK interacts with Glu-225, Asn-266 and the Asp-268 in described RANKL respectively; Lys-180 in the carbonyl group of the Ser-123 on described RANK ring 100S skeleton and described RANKL interacts and forms hydrogen bond; The ring 90S residue A sp-94 of described RANK and Lys-97 forms polar interaction with Arg-222, Asp-299 and the Asp-301 in RANKL in described RANK.
9. prepare a method for RANK/RANKL extracellular regions complex body crystal, comprising:
(1) Triptide thioltransferase fusion rotein mode is utilized to express solubility RANKL, and utilize Glutathione Sepharose 4B by this fusion monomer of affinity purification purifying, cut with PreScission proteolytic enzyme, cut RANKL agarose 75 chromatography, thus obtain the extracellular protein monomer of the RANKL after purifying;
(2) obtained the extracellular protein monomer of the RANKL of purifying in RANK monomer and step (1) by expression and purification RANK extracellular regions albumen step, be condensed into 10 mg/mL at the 1M TRIS of pH 7.00 in buffered soln, and mix with 1:1 ratio;
(3) in temperature be 294K condition under, utilize high-throughput nano to rise and sit and drip the crystal that vapor diffusion method forms RANK/RANKL extracellular regions albumen; And
(4) described crystal is in 0.1M SODIUM PHOSPHATE, MONOBASIC, 2M sodium-chlor, 0.1M Kdp and 0.1M pH value are grow under the MES condition of 6.5, or by described RANK/RANKL extracellular regions albumin crystal 20% PEG 3350 and 0.2M ammonium formiate in grow;
Wherein, described expression and purification RANK extracellular regions albumen step comprises the following steps:
A () builds the carrier of the cDNA with coding RANK extracellular regions, by described vector in prokaryotic cell prokaryocyte, thus obtain with the restructuring RANK of inclusion bodies expression;
B () carries out supersound process to described inclusion body, be then again dissolved in the Guanidinium hydrochloride of 6M, obtains the described restructuring RANK of dissociated state;
C () be folding described restructuring RANK again, described again folding process comprises:
Described inclusion body is diluted in comprise 20mM, pH value is the Na of 7.3
2hPO
4, 1M L-arginine, 20% glycerine, 10mM reduced glutathione and 1mM oxidized glutathione renaturing inclusion bodies solution in, obtain the first renaturation solution;
Being diluted in by described first renaturation solution containing 20mM, pH value is the Na of 7.3
2hPO
4, 0.5M L-arginine, 10% glycerine the liquid of folding buffered again 1 in 4
odialyse 12 hours under C, obtain the second renaturation solution;
Being diluted in by described second renaturation solution containing 20mM, pH value is the Na of 7.3
2hPO
4, 0.2M L-arginine, 5% glycerine the liquid of folding buffered again 2 in 4
odialyse 12 hours under C, obtain the 3rd renaturation solution;
Described 3rd renaturation solution is diluted in 20mM, pH value is the Na of 7.3
2hPO
4, in 0.2M L-arginine 4
odialyse 12 hours under C, obtain the 4th renaturation solution;
By centrifugal for described 4th renaturation solution, obtain supernatant liquor;
D described supernatant liquor utilizes agar 75 post to be separated by ();
E () is collected by SDS-PAGE and is analyzed correct folding RANK;
In the complex body of described mouse RANK/RANKL extracellular regions, the aminoacid sequence of the extracellular regions of rank protein is: QVTLQVTPPCTQERHYEHLGRCCSRCEPGKYLSSKCTPTSDSVCLPCGPDEYLDTW NEEDKCLLHKVCDAGKALVAVDPGNHTAPRRCACTAGYHWNSDCECCRRNTECAPG FGAQHPLQLNKDTVCTPCLLGFFSDVFSSTDKCKPWTNCTLLGKLEAHQGTTESDV VCSSSMTLRRPPKEAQA; The aminoacid sequence of the extracellular regions of RANKL albumen is: AQPFAHLTINAASIPSGSHKVTLSSWYHDRGWAKISNMTLSNGKLRVNQDGFYYLY ANICFRHHETSGSVPTDYLQLMVYVVKTSIKIPSSHNLMKGGSTKNWSGNSEFHFY SINVGGFFKLRAGEEISIQVSNPSLLDPDQDATYFGAFKVQDID.
10. method according to claim 9, wherein, in the albumen step of described expression and purification RANK extracellular regions, the product that the mRNA that described cDNA is mouse RAW264.7 cell obtains through reverse transcription PCR.
11. methods according to claim 9, wherein, in the albumen step of described expression and purification RANK extracellular regions, described prokaryotic cell prokaryocyte is E.coli bacterial strain BL21-Gold.
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| Title |
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| Shuichiro Ito, et al..Crystal Structure of the Extracellular Domain of Mouse RANK Ligand at 2.2-ÅResolution.《THE JOURNAL OF BIOLOGICAL CHEMISTRY》.2002,第277卷(第8期),6631–6636. * |
| Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.;HAILING HSU, et al.;《Proc. Natl. Acad. Sci. USA》;19990331;第96卷;3540-3545 * |
| 张勇,等..人可溶性白介素24 受体(sIL-4R)在大肠杆菌中的表达和纯化.《中国生物化学与分子生物学报》.2004,第20卷(第6期),778-784. * |
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