AU2017200239B2 - Designer osteogenic proteins - Google Patents
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Abstract
Abstract of the Disclosure The invention relates to novel designer osteogenic proteins having altered affinity for a cognate receptor, nucleic acids encoding the same, and methods of use therefore. More preferably, the novel designer osteogenic proteins are designer BMPs and have altered affinity for a cognate BMP receptor. The designer BMPs demonstrate altered biological characteristics and provide potential useful novel therapeutics.
Description
The invention relates to novel designer osteogenic proteins having altered affinity for a cognate receptor, nucleic acids encoding the same, and methods of use therefor. More preferably, the novel designer osteogenic proteins are designer BMPs and have altered affinity for a cognate BMP receptor. The designer BMPs demonstrate altered biological characteristics and provide potential useful novel therapeutics.
2017200239 17 Mar 2017
DESIGNER OSTEOGENIC PROTEINS
The present application is a divisional application of Australian Application No. 2015202418, which is incorporated in its entirety herein by reference.
FIELD OF THE INVENTION
This application relates to the field of osteogenic proteins, methods of making improved osteogenic proteins, and methods of treating patients with osteogenic proteins.
BACKGROUND OF THE INVENTION
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
The cystine knot cytokine superfamily is divided into subfamilies, which include, the transforming growth factor β (TGFp) proteins, the glycoprotein hormones, the platelet-derived growth factor-like (PDGF-like) proteins, nerve growth factors (NGF), and the differential screening-selected gene aberrative in neuroblastoma (DAN) family (e.g., cerberus). In turn, the TGFp superfamily comprises approximately
43 members, subdivided into three subfamilies: the TGFps, the activins and the bone morphogenetic/growth differentiation factor proteins (BMP/GDF).
The TGF-β superfamily members contain the canonical cystine knot topology. That is, cystine knots are the result of an unusual arrangement of six cysteine residues. The knot consists of bonds between cysteines 1-4, cysteines 2-5, and the intervening sequence forming a ring, through which the disulfide bond between cysteines 3-6 passes. The active forms of these proteins are homodimers or heterodimers. In each case the monomer topology is stabilized by the cysteine knot and additional cysteines contribute to additional intrachain bonds and/or mediate dimerization with another protein unit. See Kingsley, 1994, Genes Dev. 8:133-146; Lander et al, 2001, Nature 409:860-921.
BMP/GDFs are the most numerous members of the TGF-β protein superfamily. The BMP/GDF subfamily includes, but is not limited to, BMP2, BMP3 (osteogenin), BMP3b (GDF-10), BMP4 (BMP2b), BMP5, BMP6, BMP7 (osteogenic protein-1 or OP1), BMP8 (OP2), BMP8B (OP3), BMP9 (GDF2), BMP10, BMP11 (GDF11), BMP12 (GDF7), BMP13 (GDF6, CDMP2), BMP15 (GDF9), BMP16, GDF1, GDF3, GDF5 (CDMP1; MP52), and GDF8 (myostatin). BMPs are sometimes referred to as Osteogenic Protein (OPs), Growth Differentiation Factors (GDFs), or Cartilage-Derived Morphogenetic Proteins
2017200239 17 Mar 2017 (CDMPs). BMPs are also present in other animal species. Furthermore, there is some allelic variation in BMP sequences among different members of the human population.
BMPs are naturally expressed as pro-proteins comprising a long pro-domain, one or more cleavage sites, and a mature domain. This pro-protein is then processed by the cellular machinery to yield a dimeric mature BMP molecule. The pro-domain is believed to aid in the correct folding and processing of BMPs. Furthermore, in some but not all BMPs, the pro-domain may noncovalently bind the mature domain and may act as a chaperone, as well as an inhibitor (e.g., Thies et al., Growth Factors 18:251-9 (2001)).
BMP signal transduction is initiated when a BMP dimer binds two type I and two type II i0 serine/threonine kinase receptors. Type I receptors include, but are not limited to, ALK-1 (Activin receptor-Like Kinase 1), ALK-2 (also called ActRla or ActRI), ALK-3 (also called BMPRIa), and ALK-6 (also called BMPRIb). Type II receptors include, but are not limited to, ActRlla (also called ActRII), ActRllb, and BMPRII. The human genome contains 12 members of the receptor serine/threonine kinase family, including 7 type I and 5 type II receptors, all of which are involved in TGF-β signaling (Manning et i5 al., Science 298:1912-34 (2002)), the disclosures of which are hereby incorporated by reference). Thus, there are 12 receptors and 43 superfamily members, suggesting that at least some TGF-β superfamily members bind the same receptor(s). Following BMP binding, the type II receptors phosphorylate the type I receptors, the type I receptors phosphorylate members of the Smad family of transcription factors, and the Smads translocate to the nucleus and activate the expression of a number of genes.
’0 BMPs are among the most numerous members of TGF-β superfamily, and control a diverse set of cellular and developmental processes, such as embryonic pattern formation and tissue specification as well as promoting wound healing and repair processes in adult tissues. BMPs were initially isolated by their ability to induce bone and cartilage formation. BMP signaling is inducible upon bone fracture and related tissue injury, leading to bone regeneration and repair. BMP molecules which have altered affinity for their receptors would have improved biological activity relative to the native proteins. Such BMPs include proteins with increased in vivo activity and may provide potential improved therapeutics for, among other things, tissue regeneration, repair, and the like, by providing greater or altered activity at lower protein levels thereby providing improved protein therapeutics.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
SUMMARY OF THE INVENTION
According to a first aspect, the present invention provides a designer BMP protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 70 and SEQ ID NO: 12.
According to a second aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a designer BMP protein of the invention.
2017200239 17 Mar 2017
According to a third aspect, the present invention provides a method of producing the designer BMP protein of the invention comprising introducing a nucleic acid encoding the designer BMP protein into a host cell, culturing the host cell under conditions where the protein is produced, and purifying the protein.
Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.
The invention includes a designer BMP protein comprising at least one mutation in at least one type I or type II receptor binding domain, wherein the mutation confers altered binding to the type I or type
II BMP receptor compared with the binding to the type I or type II receptor by a corresponding wild type BMP.
In one aspect, the protein is selected from the group consisting of BMP2, BMP4, BMP5, BMP6, BMP7, BMP8 and BMP9.
In another aspect, the protein comprises at least one mutation within: the type II binding domain
A; the type II binding domain B; the type I binding domain; and any combination thereof.
The invention also includes a designer osteogenic protein comprising an amino acid sequence comprising at least one mutation in at least one type I or type II receptor binding domain, wherein the mutation confers altered binding to the type I or type II BMP receptor compared with the binding to the type I or type II receptor by wild type BMP2.
’0 In one aspect, the mutation is a mutation within the type II binding domain A wherein said mutation is at least one mutation selected from the group consisting of a mutation at V33, P36, H39, and F41 with respect to the sequence of SEQ ID NO:1.
In another aspect, the is a mutation within the type II binding domain A wherein said mutation is at least one mutation selected from the group consisting of V33I, P36K, P36R, H39A, and F41N with respect to SEQ ID NO:1. 2a
2017200239 13 Jan 2017 in ye? another aspi?et the mutation is a mutafcon within the type H binding domain S wherein .said, mutation is at least one mutation selected from the group consisting of a mutation at £83, S8f>, M83, 193, £94 ES6 K97, and 799 'Ait?· respect to the se-quence of SEQ ID NO: f in =f NiUf'Oi aspect the mutation is « nutot’i-n mtnn the t»pe 5 pindipy dooia-n U whcrem scud 4 mufatson Is at feast one mutation Oisiaeted from -he group consisting of E83K, S35N. M89V. L.Sf^F, t-9«D. E98S, K97N. end 793! with respect to of SOQ iD NON.
in another aspect, the mutation m a mutation within the hyps i binding domain Wberpin said mutatson is at least one mutation selected from the group consisting of a mutation at H44. P48, A52, 053, L55. 557. N88, S69, V70, an insertion of a single amino acid after N7t, §72, K7S, 174. Λ77, and VSO with 9 respect to trie sequence of SEQ tO NO: t.
in yet. another aspect, the mutation is a mutation within the type I binding domain wherr in eaid mutation is at teas? one mutation seiected from the group consisting of !7<>. P48S. A52H, 0S3A: t obM S57A. N88H. S59L. 770M. insertion of P after N?t S72E, K,?3Y 17-17. A77P and V89A. wth respecs re the sequence «· SEO 10 NO ?.
a in a further aspect, trie protein comprises a mutation at each of ammo acids H44. P48 A52. 053.,
L65. $5?. N0d. S69 V?0 insertion of a single arnino acid after N?1. 372 K?'S, :?4, A7?, and 780 With respect, io the sequence of SEQ ID NON.
In ann-hsir aspect, the protein comprises s mutation at each of amino acids ί ϊ«4. FM8 Ab? Dob LSS, S57. Nod. S>59, V70, insertion or a s-ngle amino acid after NZh S72 K73, I74. A77< ano \-30 with.
n respect to the sequence of 5E0 43 NO't wher&in the mutations are H44O, EN3S. A52N. I353A. L55M. 55 ?A, NOSH. S59L, W0M. insertion of a P after N? 1.5?2E, K?3V. l/4V, A77P, and V80A.
in yet another aspect, the protein comprises a mutation a; each of amino acids '733, £36. H39. ohb M89 L32, £3-1 £96 W9?, joe 799 with respect to the sequence of S£Q if? NO t ' ji'ctoe aw ί>ρπίη>< v,. <se^ c maN os m ρ& > of c n o< ecd- Am “33 -Bn NN
..5 ?V;89. L92. L94 £36, K3?< end V39 with respect to the sequence or SEQ ID NON. wherein the mutations are V33I. P3SK. K39A S86N. M33. L92F. £94 D, E96S. K97N and 7931 in a further aspect, the preteir; comprises a mutation at each of amino acids 733, P38.1-139, i-N4.
P43. A52. 053. ESS. S57. N68, 539, 770, insertion of a single amino acid after N?1. S?2. Kf'3. 174. A77. arid, '780. S8h. MSS.197, ES< £96. K97, and '799 with rospact to tha sequence of SEO ID NON in yet another aspect, the protein comprises a mutation at each of amino acids '733. P36, H39.
i-144, P43. A52. D53. tab, 557, N68. S09 '779. insertion of a single amino acid after N71. 572 K73. I73. A??, and VS0. S3S, M89, 192, £94. £96. K37, and '799 with respect to the sequence of SEQ ID NON wherein die mutations ary V33I, P38K, H3SA. B44O. £485, A52N, D53A. L55M, 557A, N88H. S59L. V70M. insertion ol a P after N?1. S72£. K?3Y. f/4V. A77R. and V80A, SS5N, M89, L92F. E94D, ESoS
K97N. ano V99I.
In yet another aspect. ?he protein comprises a mutation at each, of amino acids 733. P38. 1139. ΗΠ4. prig, AS?. D53, 1.55 S57 N6S. 569, 779. insertion of a Single amino acid after 'ir t, S77, K73. ΐ74.
9:
2017200239 13 Jan 2017
A77, and VSO. Sdte ·Μ89> L92, E94, B96. K97. and VSfS w4h respect to the sequence of SBCt fO NO:i wherein the mutations are V33t. P3CR. H3SA, HU3. P4SS. A52N. DS3A; L55M. S57A. N68K. S6^i... V70M. insertion ot a P after N71. S7?E K?3Y. f74V. A77P ami VSOA, S8SN, M89, L93F. £9413. E&6S. KS7N, and V99I.
in another aspect Vte protein hinds: toe At K? receptor with a K;; not greater then about2 nM. toe
ALK3 receptor with a Ko not greater then about 2 rtsM: the AlKS receptor with a Ki; not greater toon about 1 nM; to* AetRiiA receptor wah a Kr. not greater than about 2 nM; the Ac-RHB receptor with a Ko not greater than about 0 S nM. and the UMKRtlA receptor with a K.-. not greater than about 3 5 nM, in one aspect the protein further comprises t, 2, 3. 4. 5. 6. 7. 8. 3.. or 10 amino acid mutations not fotvsied wtlhsp tt $>. tyHr. f w toe type fi bicdne '(.gions
The invention includes a designer osteogenic protein comprising tbs? amino acid sequence or any one? of SBO ID' NGs.8-73.
The invention includes a designer osteogenic protein comprising: the amino acid sequence of SBC ID NO-12
The invention includes a designer osteogenic: protein composing toe amino acid sequence of
S£O ID NO,'f4.
The Invention includes a designer osteogenic protein comprising the amino acid sequence of SBC ID NC-36
The invention includes a Cosigner osteogenic protein composing too amino acid sequence of h BBQ ID NO.37..
she invention -qu-udes method of producing a designer BMP pmioitt compne-ng at feast one mutation in at iaast one type f or type if receptor binding domain, wherein the mutation confers altered bind-ng to the type I or type II BMP receptor compared with the b-nding to too type I or type II receptor by a oomeAportdirig wild rype BMP Tne metho* comprises introducing a nucleic acid encoding the protein
-nto a host oeff. suffering the cel· under condition? where the ptolem w produced and puntying the protein.
to one aspect, the nucleic acid comphseb » sequdhpe selected tosm toe nuelbte acid sequence of any one of SBO ID ΝΟ?:?4Ί39.
The Invention Includes a designer BMP» protein comprising an ammo acid sequence comprising 3tf at least one mutation In at least one type i or type If receptor binding domain, wherein the mutation confers qitorerj binding to the type i ni type if BMP receptor compared with tne binding to the type f or typo if receptor by wild type SMP6.
to otiifi aspect. the mutation ;s a mu-afco within the typa ft binding demem A wherein said mutation a? at least one < nutation selected from toe group consisting of a mutation at 157, K30, <551. A53.
AS V'u ^Ct an< Gfo , n re\p«ct iv-oc*· -, f --..7-3
2017200239 13 Jan 2017
In another aspect ttie mutation w a mutation within tbs type Is biridlng domem B wherein said mutation is at least one mutation selected from the group consisting ol K10S, Ml 10. A111. VI14, F117, 01 19 NISO $1'21, N122, V1?.3, and H24 With respect to tbs sequence o? ;3EQ ID NON.
in yet another aspect. the mutation sc a mutation within the type i binding domain wherein said 5 mutation is at tees! one mutation selected from the group consuming ot a mu-afcon at $72, N7$. A77, H78, M79. N8Q< AS1 N-33. VS?. Ϊ39. H92, L93, MSN. N95. P96, £97, ¥38, V33. end P10D with respect to the sequence of $EQ iD NON.
n aroTet n\ ><\i inc r ta for ώ e motion at e» 0 <4 c mu; u< t’\> it c 13- td> Ob'
N8ti, V6G, end DCS wish respect Io the sequence of SEQ ID NO 4.
η a miner asm ci du' t Uii<s a notai’on «t eau ot arm's oci<. fc^chc* νλ33 N'% A'I
VI H, Ell ?. 01 -9. Nt 20, $121, N't 22. V123, and B24 with respect to the amino acid sequence of SEO ID NON in yet another aspect, the mutation is a mutation at each of amino acid residues $72. N7i>. A??,. H78. M7P HS0. ASi. N83. W: 789, BS7. 1.93. M94, N9h. P96, £9?. Y98. V99. or PW0 with respect to is the amino ac:d sequence of SE.Q ID NO:4, in another aspect, the designer BMP8 pattern composing an amino amd sequence comprising at least one mutation in at least one type I or type II receptor binding domam. wherein the mutation confers altered binding to Ute type I or typo li BMP receptor compared w4h the bind-ng to the type ΐ or type ϋ receptor Py wild type DMEfl further comprises 1, 2. 3, 4, 5, 0. ?, 8. ft, or 10 ammo acid mutations not n located within the type I or tho type II binding domains.
The irtverthon includes an Isolated nucleic acid moiscule comprising a nucleotide sequence encoding an ammo acid sequence selec-ed from the group consisting of -he sequence of $EO ID NOs8 to Z3.
in c-ne aspect. ids nucleic acid encodes a protein comprising an amino acid sequence selected ..5 ftotn the group consisting of tbs sequence of SECi ID NO:12. $ED ID NO.14, SEO ID NO:38 ano SEQ ID
NO,3?,$S ?'Ite inviSnfiori includes an :soiaied nucleic acid molecule composing a nucleotide sequence selected from the croup consisting of SEO ID NOs 74 to 139.
In one aspect, thy nueie<c aetd composes a nucleotide sequence selected from the group 39 consisting of the sequence of SEQ ID NO;?8. $EQ ID NO.$0, SEN ID NONOZ. and SEO ID NO;103.
I liis invention includes a method of producing the designer BMP8 pfolesn comprising an amino acid sequence comprising at least one mutation in at least one type I or type ii receptor binding domain, wherein the mutation confers altered binding Io this type I or type II BMP receptor compared with the omd ng t>> tb typ< of type epio; by rN tvo< cnVi i' I h« o< in-. < - u- 3 on Nuc nc a t u<. or.
acid encoding said protein into a host cell. cuftunng said cel: under conditions where said protein ss produced, and punting said protein.
2017200239 13 Jan 2017
The invention inetadaa a method of treating a hone disease aesociaied with hone loss in a patient in need thereof, I he method comprises administering a therapeutically effective amount at a designer BMP protein comprising at least ane mutation in at feast one type I or type ii receptor binding domain, whetem the mutation centers altered binding to the type I or type li BMP receptor compared w-th the 5 binding to the tvpe i or type il receptor by a corresponding void type BMP* profem to the patient, thereby treaiittg bone disease in the patient.
Tire invention Inoludes a msfhoO of Beating fibrosis <n a patient in need thereof Trie mathoO comprises οοηνηιαίοηηρ a tnerapeaficaiiy effective amount of a designer BMP protein composing a- kmsi one mutation m at least one type s or type ii receptor binding domain, wherein the mutation confers h aiferod omdmy fe the hpo = or type d BMP receptor compared voth the omd:ng to the type I or type ϋ receptor hy a corresponding wild type BMP to the patient, thereby treating fibrosis.
The inygntian includes a method at Inducing bone formation iff a tissue. The method comprises contacting She tissue with a designer BMP protein comprising at least one mutation in at least one type I or type ii receptor boding dosnam wherein the mufef:on confer··:- aitensd binding ta the. type i tv iyne ϋ S BMP receptor compared with the binding to the type ! or type If receptor by a corresponding wild type BMP, thereby inducing baneTqm-iaison m sate tissue.
SeDESGRiPTOlOL^
For the purpose of illustrating the invention. there are depicted In the drawings certain P embodiments of Ihe Invention However, the invention :s not limited to the precise anangements and iosh vmenfefliios of the embodiments depicted so the drawings.
Figure t. comprising panels; A~C. is a diagram showing the alignments? various wild type ssno designer BMP amino acid sequences and indicating iby being within a box) the regions el these proteins potentially involved ip type I and type ii receptor interactions. Figure 1A show·:, the amino acid sequence
..5 alignment of wild type BMF2, 8MP4, BMPS. BMPS, BMP?, BMPS and tsMPB, Figure IB shows the amino acid sequence alignment of various designer BMPs where the corresponding wild type BMP Is BMP2. Figure 1C strews tfia ammo acid sequence alignment of various designer BMP8 molecules where the corresponding wild type BMP is BMP6
Figure 2 is an illustration of a structure! model showing a wid type BMP2 homodimar binding to
TO two type I and two type fi BMP receptors
FiCiite T, composing panel' Λ and B w a>- ifiagtac· ot a itieeiotal model ehov>mg (he petif.cn >it the histidine doorstop fhlbdl in human BMP2 produced in Chinese Hamster Ovary (CHO) (Figure SA) and E. call calls {Figure 3B;
Figure 4; composing panels A and B. is a diagram illuvtreling the iocation of the glycan tether and 'x potsntiai histidine (His; deorstep Figure <fA shows the glycan tether fN-linked giycan si H56;· and is ttosi ' it .1 '« u d<»< ^opofcnreton -« II ο<» K nkUo'u) ,1 - yi c it v «» λ fn >- k> af in
GHO-produced BMP/ Figure 4B shows the giycan tethet (N-imked giycan at NBO) and the histidine in
2017200239 13 Jan 2017 the γκϊΓε-doorstop configuration at K78 in I3MPS. as weF as the R39 corresponding to R16 in BMPS.: Th© sequence allgomeot of BMPS { VKSSCKRHP) end B?vtP6 (35-KTACRKHEl showing me corresponding amino acids between BMPS and BMP6 is shown atoog the fop of the figure.
hi jf o w Ίίρη^'Ό pat ' v? A E stiow^ '^neu^ stops s > the pu?' for or iVd m t of BMP'· an ’ 5 designer ΒΜΓ-’a. Figure £>A shows a chtornatogram showing gradient aiufton of BMPs using a ceSiufina sulfate culutnn. Figure SB fe an image of a Coomass-e stainedSDS-PAGE inon-red'uced on file fefi and reduced on ihe doh- side; get containing sampias of frao-ions. from the celkrflne sulfate column step Figure SC shews a chromatogram showing the protde kern preparative reversed phase purification step Figure SD is an image of a Cooma&sie stained BOB-PAGF (non-t'educed on the left and reduced op the 0 right) gel of BMP containing samples of the fractions obiained oy Ute prepat aft ve reversed phase purification step.
Figure 6, composing panels A-D show images of Coomasste-stained BDB -PAGB protein gels showing perilled BMP2 wild type and various mutants as Indicated along the fop of each gel image. The gels wise run under either non-reducing (Figures nA and 68) and recusing (Figures 6C end 60) conditions.
Figure 7 shows aikalme phosphatase assay results in G2CI2 pre-myoblasts comparing trie osteogenic activity o? wild hype BMP2 and BMP2/-3 heterodimer Io the various cosigner BMPs as indicated in the graph legend
Figure & shews tire results el a C2C't2 BMR-Respcnse Element luciferase teRfEluciferase) (f asirsy indica-ive of Smad actlBty showing stronger signaling hy BMPB compared to BMP? end equivalent signaling to 8MP2/S,
Figure 9, comprising paneis A and B. shows -he ectopic imns formation mediated by various BMPs f igure 9.A is a graph showing lire amount of ectopic bone (calculated as milligrams of hydroxyapatite; mg HA) as determined bv eCT analysis for each limb which was implanted with the „3 indicated BMP (EMP2, E-MPE. and BMFBm) at the cose Indicated {0.1 or 0,5 pg). Figure SB is a graph shewing -he amount of ectopic done (calculated as milligrams of hydroxyapatite? as determined by pCT analysis, lor each limb which wets implanted with the indicated BMP (BMP2. BMPG SMPA. and BMPFat the dose indicated {0B cr 0 6 rigs The data presented arc from 2 separate experiments.
Figure 10, comprising pausis AO, shows images cf radiographs shewing the results of a nonbt human primate (MKB) fibuia osteotomy mode! nt 4 and S weeks.. Radiographs. are shown of Ihe tlbuias; of 7 r-?prvsentot:vc Ml IPs that meowed BMPE and UMBO, respectively, at 6 5 mg-'ml (250 ftg total BMP dalsvereddlmb) Each MHP renewed WT BMP2 ai l.ho same doss in the contralateral limb. Figures, KlA poo IBB shew the todouiapns for ihv \f IPs ncfoutec at the ten of e«-sh digram show.ug *ho effects of BMPE compared with BMP? wild type at 4 weeks and B weeks, rsspsotiveiy Figures tOC arid WD show the radiographs lor the NHPs indicated at the top of each diagram showing the eliects of BMPG compared with BMP? wrld type ,at 4 weeks and 3 weeks respectively.
2017200239 13 Jan 2017
Figure 11 fs si grant? showing tfse bone volume of tha iTues treated w?th BMP-E vernes contraialefat limbs treated with SMP-2,
Fifitii'fe 12 ss, a graph showing rosuits of an alkalir-s phosphatase assay m C2C? pm-myoblasts comparing She osteogenic activity of wild type BMP2 and EMF-GER, BMR-GBP and BMP2/B 3 hetarodimer.
Figure 13 Is a graph showing the amount of ectopic done (calculated as milligrams of hydroxyapatite* as determined by gCT analysis for each «mb wbsch was; implanted with the indicated BMP {BMP-2, BMP-2te, ΒΜΡΈ, BMP-GER and BMP-6; at the dose indicated {0.05 or O.25pg},
Figure 14 is a graph showing -ho arnoun· of ectopic bone (cafouiated as mitiiO-'ams of 0 hydroxyapatite} as determined by pCl analysis tor each limb which was implanted with the indicated BMP {BMP-2, BMF-2A>, RMP-E, BMP-GER. arte BMP-6; at the dose indicated {0,06 or 0.25pgj. These are tee resufishom an experiment separate from that shown In figure 13.
Figure 15, comprising panels A and S, shows images ot radiographs and pCT images showing the respite <4' a non-humors primate {NHP; fibula wedge osieolomy mode! at 5 and to weeks Figure ISA 5 shows linages of 5-week radiographs obtained In a NHP fibula wedge osteotomy model- Figure 15A shows images of the iibui.es of 4 representative NHPs which asceived BMP-GER In one limb artd WT BMP-2 In the confrafatsraf limb at 0,5 mg/mi (260 pG total BMP delivered/limb} at 6 weeks, Figaro 15B shows uCT images s? the same smbs a; 10 weeks shewing tf*e Serge calluses of the BMP-GER treatisd mb-. co' 'outeu ws,l tea ote-Y tecUm' ' onfmtateral mt -< ‘a each an mat d Figure :6, comprising panels A-C shows graphs Illustrating the strength {Figure ISA}, stiffness {Figure 16B), and callus hone volume ('Figure 16C) el the BMP-GER treated Iseths versus the BMP-2 treated contralateral limbs.
Figure 1; cotftpnsing panels A·(...-. snows radiographic images ci the healing over time of 3 nonhuman anmaies {NHP; fibulas treated wdh BMP-GFR at O.b mg/mi and BMP-2 in -he contra lateral limb
S at : Smg/trti using a culcium phosphate Based cement as a earner following lire wedge defect model. Figure 1 ?A. upper panel. shows results tor NFIP number 1 left arm treated with 0.5 mg/ml GBR as follows, panels i r-nd 2 show LAf fiateral) mu AR {antonor-posterior* images respestiveiy, at the msfel time pomt, panels 6 and -I show LAT and AP linages, respectivesy at. 2 weeks, panels 6 and 6 show I.AT and AP images, respectively, at 4 weeks, panels: ? and 8 show LAT and AP images, respectively, at 6 weeks.
TO panels 0 and 10 show {.AT and AP images, respective:'/ at 7 weeks; panels 11 and 12 show EAT and AP images. (eepecftveiy at δ weeks: figure i’7A fewer pane: shows results tor NHP numbei I doh! arm boated with 1 5 mg/mi BMP-2 as follows panels and .? show FAT {lateral: and AP {anterior-posterior; images respectively, of tee initiof time point, panels 3 and 4 show t.AT ano AP Images, reapeativety, at 2 weeks panels 5 arte 6 show LAI and AP images. respootixcfy, at 4 weeks', panels 7 and B show LAI o-rd
AP Images, respectively, at 6 weeks; panels 9 and 10 show t AT and AP Images. respectively. at 7 λ„ο\^ | ones m an< 2 Anu y ^ui' 'F mages w'V i!£A« < te> sge 5teotWit\'
2017200239 13 Jan 2017 radiographic tbit r;ts;ult.y lor NHP number 2 as described for NHp #1 in Figure 17A, end Figure i ?O sets out the results for NHP nutnber 3 as described for NHP #1 in Figure 1 ?A
Figure 13 ts >s diagram of a structural raodsi showing representations end comparison of the crystal structures BMP-E and 8MP-6 VVf fhe differences in the length oi the giycen resolved Is 3 highlighted showing that fhs gtycao for BMPE tnat :s resolved is much longer than that for BMP6 This indicates that the BMPE glycen ts more coniormationoify constrained than that oi BMPb such that more oi the glycan can be rendered in this model. The histidine doorstop residues for both BMPE and BMPb me shewn in simile? non-doorstop conifgura&ons Also, the ergs?;tnn glycan ''tollter stabilizing the EfoPE glycan is shown by dotted Smet, representing the interactions of the arginine with the glycan.
Figure 19 ts a closer view oi She histidine doorstop end urgfhfne tether o’ the BMPE and BMP6 comparison shown In Figure IB. This image shows the similar coniormalion of the Ή54 histidine residue o' BMPE and :fee equivalent hlafldlre of BMPS both in the ooh-doorstop positron The image also shows the R;6 tethering I via interactions o' the BMPE. giycan such that the giyoan is more rigid and therefore mom ?s rertoe?ed by the model compared to ihe more floppy” and less constrained giyoon of BMP6 such S that less of the BMPb glycan is visualized In this model. The diagram of this model also shows the similar positioning oi o¶gine Mfefe oi BMPE showing N-iinked rsffachii;c;?'if of the giyson and the equivalent and similarly positioned asparagine of BMPS. The diagram also iiteslrates the potential additional glycan tethennn interaction of BMPE P110 shown by dotted ilr-es between the ammo ecid residue and Ute distal end of the givc-un, The differences m foe length of foe glycan resolved is f-ghligbfed showing Ihel less of n fh« darker BMPG giyoan can be resoived compared with the lighter shaded longer glycan rendered for BMPE indicating that fhe BMPE gfyean is mote conformatonaig’ constrained and f??us snore ss rendered upon sirnctural analysis.
Figure 20 is a graph shewing the rasults of an Asline phosbatese assay using C2C12 premvobiasis comparing the osteogenic activity of BMP-2, BMPE and BMP-6 with their F.ndo-M treated deglycosyiated fOeuiy.} counterparts.
Figure 2t Is a diagram Illustrating the structural mode! of BMPE showing Ihe location oi Ihe glycan tether at PI6 and itfnstraiiog foe stabilising interactions between the arginine (PI island glutamic acid sE 1 10 corresponding to E109 of BMP2) residues. The diagram shows that Bib and ΕΉ0 both form rfoilbgie hydiogen bonds wlih foe third {d-mannose) and fourth iu-mnnnoea) giycert moieties. fhe
3ft diagram also shows the position of 1-:54 polsnbal doorstop' and asparagine 56 (Nbb) which provides the N-linkod aitechmoni Site of the giyoan.
Figure 22 is a graph showing ihe resuits of an alkaline phoshatase assay using C2C12 premveblasts qompnnng fhe osteccenio activity ot BMP E with BMF E MB, BMP-GEB and BMP GEB-NP »; fhe presence of increasing doses of Noggin - a natural inhibitor oi BMP-2. The data demonstrate Inal
BMP-GER-MP comprising sequences derived from actrvin was not inhibited fey Noggin even a! high Goncenirallons bid that BMP-GER was sensitive Io Noggin inhibition, Thus, addition of sequences derived tram acte/in caused BMPOER to become Noggin rasistenS (NR) These results demonstrate that
2017200239 13 Jan 2017 at least lb this m Vitro assay. 8MR-GFR «snd 8MPE- which are Noggin sensliiye, become Noggin resistant ,NR' upon replacement of the 0-terminal regie·· ! of the protein with sequences derived from aciivin.
Figures 73 is a graph showing the bone score as determined by ironmnohistochemisiry (IHG for rat, ectopic -mpianis treated with the indicated BMP at the specd-ed does, Ihe data show that the bone 5 forming activity of BMP-GBR was greatly decreased when ;he Olerrnirtsi sequence of the rnolecuie was replaced with a seyuanco derived bem aotivin ‘NR), f bus. ike data demonstrate that BMP-GER-RR was •nc'i le < ac'b ~ fh tc B\ P*t F*. no.
Figures 24 la a graph showing the bene score as determined by immuncriistociiemistry f|HG) for rat ectopic implants treated with the indicated BMP at the specified doss. The data show that the bone If forming activity of BMP-E was greatly decreased, indeed, ft was completely abrogated, when the C· terminal sequence of the molecule was replaced with a sequence: derived from aoiivm (NR),
This invention reisies a designer' bone morphogenetic protein, referred to herein as designer S BMP.' 'designer osteogenic protein and ‘designer protesn ' The designer BMPs of the invention may correspond f»theam>no acid sequences of wild type unmodified BMP, such as, but not limited to, BMP7. ΒΜΡΊ. BMPS, BMPS. BMP?. BMPS, and BMPSj. in particular embodiments, the designer BMPs show altered binding to a type I and'or typo ti 3MP receptor when compared Io its corresponding wild iype BMP. in further embodiments, the designer BMP may be modified to have altered hail-life, b immenogenicify, or any pharmacokinetic/pharmacodynamio -'RK/PO) parameter when compared to its eofrespohd ing BMP.
igfioiy&s®
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have -he meanings that are commonly understood by those of ordinary s.-Pifi in the
S an '<!,i 'f ui I -w other λ ^e »ec< ir --e I \ jc^'cm s nqulqr s< r' s ah' i kd okruii 's ar i oLr i! u »r'» shall include the singular. Generally. nomenclatures used in connection with, arid techniques of. ceil and tissue culture, molecular biology, immunology, microbiology, genetics arid protein arid nuciek acid chemistry and hybridization described herein are those weil known and commonly used in Ihe art.
The methods .md techniques· of the present invention are generally performed according to ${f methods well known in ihe ah and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise Indicated Such references include, e.g,, Sambrook arid Russell. Morecir/ar Cte/m/g, A Laboratory Approach. Cold Spang tiarbor Press Cold Spring Harbor. NY {?Pf) t}, Ausuhei at at.. Cwfcv! Pio/oco/s L? Afofecuiar Biology. John Wiley & Sons, NY 12007), and Hgfiow and Lons Antlbod'es. A Laboratory Afanuai. Cold Spring Haibor 'x Laboratory Press. Cold Spring Harbor, NY GSSOi, which are incorporated herein by reference. Enzymatic rsacFons .grid purification techniques are performed according tc manufacturer s speed leaflet is as commonfy acoompiisned in the art or as, desicnbed herein. The nomenclatures used In connection with.
2017200239 13 Jan 2017 and toe laboratory procedures cod techniques of. anal yheai chermsffy, syrilheiic organic chemistry. arid medicinal and pnarmaceudcai chemistry described herein ere tense well known end commonly used in the art Standard techniques ere used fot chemical syntheses, chemical analyses, pharmaceutical preparation, formulation. end delivery, and treatment oi patients;
As used herein, each of the following terms has the meaning associated with it in this section.
The articles “a” and ‘an are used lierain to refer to one or to more than one t?.e.« to at least ortoj of the grammatical obieot of the article 8y way of example, an element’ means one element or more ifisn one element.
in this application. the use of tor means ’'and/or unless stater’ otherwise, h Conventional notation is used herein to portray poiypept-de sequences: the fefMtand end of a polypeptide sequence is the amino-terminus: tbs right-hand end of a polypeptide sequence is the carboxyt-terminus.·. As used herein, the twenty conven-lorias amino acids and toeir abbreviations hi oa conventional usage. See toirnyno/ooy-A S/nf/tesr’s {2nd edition. E. 8. Golub and D. R. Gren, Eds,, Sinaaer Associates. Sunderland,. Mass (·;ίΑΤΐ)Τ which is incorporsleo tienen by reference. As used
S herein, ammo acids aie represented by the mil name thereol by the three letter code corresponding Ihareto, or by too one-letter cede corresponding thereto, as indicated as follows-
Fu|l Name | Three-Letter Code | One-Letter Code | |
Aspartic Acid | Asp | 0 | |
Glutamic Acid | Glu | : HE | |
bK | Lysine | tys | K |
Arginines | Are | P | |
Histidine | His | H | |
Ί yrosine | Tyr | V | |
Cysteine | Cys | c | |
5 | Asparagine | Asn | H |
Glutamine | Gin | G | |
Serine | 8er | 8 | |
Threonine | Thr | T | |
Glycine | Qty | Q | |
to | Alanine | Aia | A |
Valine | Vat | V | |
Leucine | Leu | ,L | |
Isoleye-ne | fie | hi | |
Methionine | Met | M | |
o | Proline | Pro | P |
Phenytotonino | Pho | F | |
Trypiophan | Trp | :¥ W <: |
1::
2017200239 13 Jan 2017
A 'conservative amino acid substitution is: one in Miicn an amino acid residue is s-ibsiitn-ed by another amino acid residue having a side chain R group with similar chemical properties (e.o,, charge or hydropbobioify). ;r; general, a conservative amino acid substitution Will not substantially change the functional properties of a protein. in eases where two or mote aimno aosd sequences ciifiet irom each 5 other by conservative substitutions, the percent sequence identity or degree of similarity may be adius.-fed upwards io correct for the conservative nature of the substitution, Means for making Inis adjustment are wefi-known to those c-f skill in the art. See, e,g . Pearson, Methods Mo/ S.o/ 2)-1-3:307-31 (1934-.
Examples of groups of am-tio acids that have side chains with similar chernieai properties include ) iii that! io* 'no >-x gly ce nntste /s.r-. lakii <snd vek ire 2; a! ph*tc-t \ lro<p ioe-nnn9 tonne and ihroontne 3} umidewootoiosog side >.hams asparagine <and ginfannnc 4i aromatic tide chains· phenylalanine. tyrosine, and tryptophan; 5j basic side ebams lysine, arginine, and histidine; δ) acidic Side chains aspartic acid and glutamic a-vd; and 71 suitor· containing Side chains, ovsteme and methionine. Preferred conservative amino acids substitution groups are. vaiinsneucine-ssoienelne, phenylaianineiyrosiniti, fysine-arginine, alanine-valine. glutamate-aspa: rate arid iesparagiiie-giiifaruiui; s Alternatively a conservative replacement is any change having a positive value m the ΡΆΜ259 ing-likelihnod malax disclosed m Gonne? et at.. Science 756Ί 445--1445 (1997). herein incorporated by reference. A “moderately conservative'’ replacement is any change Having a nonnegative value in the RAM/EO log likelihood maf»x.
Pretence amino acid substitutions are those which. ft) reduce susceptibility to proteolysis. (2) n ‘e<'u> a sccoes-bP! ty to oxs< Uron ,3) alter h ndi tg aftirhto h-m tg r.-ot-te romp‘e<*s ape (4 s rorter or modify other physicochemical or functional properties el such analogs. Analogs comprising substitutions, deiefions. and/or insertions ran include various muiems of a sequence other than the specified peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made m the specified sequence /preferably so the portion c-f Ihe polypeptide outside ,,:5 the domainis) forming intermnteeuiar contx-cts e g , oulsihe of the CORs. or the type I w Iype II recaptor binding sites). A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g,. a rapat onu-ni amino acid should not tend to break a hehx that occurs m the parent sequence or disrupt other tyaes of secondary structure that characterizes the parent sequence·. Examples ot ad-recognised polypeptide secondary end tertiary structures are described in Pmie»rj3. Stmcteros arte? Mo/ecu.'ar P;;ne',o/ev (Creighton. Eo,. W, I I. Freeman and Company. New York ·19&1}). Mtrodncfio:·? ?» Pro/em Structure <C Sraixlsn and .1. Tooze. eds , Garland Publishing. New York N.Y. (1991)-, and Thornton of a! Nature 394.195 (19315. which ate each incorporated herein by reference.
flic terms “polynucleotide'’, nucleotide sequence”. '’ndeielc Acid, Khucieic acid melecuiqA nucleic acid sequence. and 'oligonucleotide’ refer to a series of oucieotide bases tai&o called '’iiuoleohdes: m DMA and RNA and mean ant chain ot two or more nucleotides, The polynucleotides can he chimeric mixtures or derivatives or modifier! versions, thereof, single-stranded or double-stranded. The
2017200239 13 Jan 2017 oligonucleotide can be modified at the base moiefy. soger moiety, or phoophafe backbone, for example, to tmprove stability of the molecule, its hybndizebun parameters. etc A nucleollde sequence typically carries genetic infonnation iociuding the Information used by cellular machinery to make proteins and enzymes. These tenmy include double' or singie-ytranded genomic and cDNA, teNA, any synthetic and 5 genet erh, marn 'fofed ,>oterun eot.de md note sem-n. ^nd antisas'xe po.yj -,ole. t-dex Ήιη as., w Ldcs -r foil uudx orfo s x, t0v< sued ray; fo* < xarnalo fo e Ravi Meg c » >e ;1< ite. c u a< I or containing carbohydrate or fipxis.
in the context o; a nucleotide sequence, the term 'substantially -deni-esi” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minim orc number of nuoteotides that are b -dontKai to aligned nucleol-des in a second nude-c ac;d sequence such that the iirst and second nucleotide sequences encode a polypeptide having common font lionet activity, or encode a common abuefurat polypeptide domain or <3 common functional polypeptide activity. For example, nucieoltde sequences having at least about 85%. 90%, 9i%, 92%, 93%, 34%.. 35%. 33%. S?%. 38%. or 99% identity to a reference sequence.
it By 'designer BMP nucleic acids. and grammatical equivalents here-n is meant nucleic ac ds that encode designer -BMP&
The terms ‘protein'’ and 'polypeptide'' are used interchangeably herein, These terms refer to a sequential chair; of ammo acids linked together via peptide bonds The terms include one or more proteins that function as a d-screfe unit If a single polypeptide is tbs discrete functioning unit and docs b not raqulrs permanent or temporary physical association with olhet polypeptides In order to terns': the discrete functioning omt. foe terms polypeptide’· end protein may bo used interchangeably, if the discrete tunctlanal unit is composed of multiple polypeptides that physically associate with one another the term “protein” its used herein refers to foe multiple- polypeptides that aie physically coupled and function together as the discrete unit Z* protein to bo expressed according io the present invention can he a protein therapeutic. A protein therapeutic is a protein that has a biological effect on a region -n the- body on which II eels or on a region ot the body on which If. remotely acts via intermediates examples of protein theiapeutioa are discussed in more detail below
Designer BMP,' as the term Is used here»·, relates to a BMP protein comprising at least one am no ad Xifaf. i mm ό-<· I fo s < «η u?< «Ming wi I %{ n b\'t w ‘t.gvt 'he mt teho-i w tew i 1 ie designer BMP has detecfahly altered binding for at least a typo I receptor and/or al least one type is receptor compared with foe binding of the corresponding wild type BMP for the type I and/or type it receptor.
By “corresponding wild type prole»! if is meant toe wild {ype version of the designer I3MP prior to the introduction ot any mutations. Fo? example, if foe designer BMP is a designer BMP2, Iris corresponding wild-fyps BMP Is wild-type BMP2. Thus, in one embodiment, design oi a designer BMP iian; but need net, .begin with a void type BMP sequence whefe-n mutations (e.g„ amino acid substitutions, deletions andte:·»· insertion? are introduced ink! the wild type sequence Therefore the
2017200239 13 Jan 2017 desxsner BMP can coneapond with a wifd typo BMP, and the locations of the mutations can be said, for instance. to correspond with, be relative to ano-'or ha respective with the ammo ao-d sequence of the wiki type corresponding or 'mterenoe'’ BMP sequence
Fite proteins of the present invumioi: include iragmunts, derivatives, analogs, or variants of ihe v polypeptides described herein, and any combmatlan thereof The femis. 'fragment.' “variant' 'derivative’ atfo ’analog” when referring fo proteins ul the present invention inciuda any proteins which retain at feast come <4 the tuhcnonai pm-gertiei· of the protein from which it was derived
By the term 'Iragmenr as used heroin refers io a polypeptide and -s dei-ned as any discreteportsoo of a given poiypeptide -ha- is unique to or characteristic of that polypeptide The term as used b herein ateo rotors to any discrete portion oi a g-vun poiypuptidu that retains at toast a traction of the activity of the fuli-tengih polypeptide in certain embodiments the fraction of activity retained n. at least 10% <?f foe activity of foe iaii-tenyth potypeptide. in certain embndsmente. the fnaetion of activity refined u> at least 20%. 30%, 40%. 50%. 00% 70%. SQ% or BOB- of the activity of the lull-length polypeptide, in κ 'item enmods I'ente fo, *t\ ie> <4 aetvky >ttei »«» i - -I s >asi ’fe 96' >< 5 . a ufo'·' p,c
S activity oi the full-length polypeptide, in certain embodiments, the fraction of activity retained is “00% or more of the activity of the ftiit-Seriqlft paiypepiide Alternatively or shciinc-nally, the tenv os used hraein also refers io any portion of a given polypeptide that includes at toast an established sequence element found in the foil length polypeptide in some embodiments. foe sequence eiement spans at least about 45; 10. 15, 20, 25, 30, 35. 40. 45. So or more amino acids of the teii-lenyth polypeptide. Fragments of b. proteins- of foe present invention thefude pioteoiy-ic fragments, as; welt as; deletion fragments
Vananh; of the proteins oi the preserti Invention include fragments as described above, and also polypeptides with altered ammo aox; sequences due to ammo acid substitutions, deietlons or insertions Variants may occur naturally or be oom naturally ecpunrng. Non-naiuraily occurring variants may bo produced using an-known mutagenesis techniques Variant proteins may comprise conservative os non..5 conservative amino acid, substitutions. deletions or additions.
The proteins of the invention include proteins having one or more residues chemically dehvafized by reaction of a fuocbormi side croup Also induced as; proteins; of the ihvention are polypeptides that contain one ot more naturally occurring amino acid derivatives of the twenty standard amino acids For example 4 t\di νκ«η«- n \ <u « r <i\tec fo pi<4fo<· t ·,<’ ocvtysiue ' i/ e xub- fete c’m Iv^nc % 3-me?hyihistidine may fee substituted for histidine hontosenne may be substituted for serine; and omithme may txr substituted for Iyulnc.
'’Recombinamly expressed polypeptide'' and ’recombinant polypeptide as; used herein refer to a pelypepifoe impressed from a host coil that has; beer- manipulated Io express that poiypeptide In certain embod-meuts. the host coll Is a mammalian ceil, fo certain embodiments, flits manipulation may compose 'x one or more genetic modifications Far example the hos; cells may Psi genetically modified by foe 'teoduehot' of me or more n< tenMopo< gc'tes 'ncortug fo< j.. vpte-id. to %> eq'msscc the heterologous, reeambinantiy expressed polypeptide con he identical or similar to polypeptides that sire
2017200239 13 Jan 2017 normally expressed in the host ceil The heterologous recombinant!'/ expressed polypeptide can also be foreign to the host ceil. e.g. heterologous Io polypeptides nomcaliy expressed in the host ceil, in certain embodiments. the heterologous recombinantfv expressed polypeptide is chimeric. For example portions of <5 polypeptide may contain amino ac J sequences that are identical or similar io polypeptides normally 5 expressed in the host ceil. while other portions contain ammo sox* sequences that ere foreign io the host celt. Additionally of alternatively. a polypeptide may contain annno acid sequences from two ei more different polypeptides that ere both normally expressed in the host ceil Furthermore a polypeptide may contain amino acid sequences from two or more polypeptides that are both foreign to the hosf cell. In some embodiments the host cell is genetically modified by the acitvatlon or upregulation of one or more 0 endogenous genes.
Calculations of homology or sequence identify helwesn sequences (the terms are used Iniurehangeably herein) are performed as follows Io determine the percent identify of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned tor optimal comparison purposes te § , peps can be Introduced m one or Path nl a first and a second amino acid or nuclei::. amd s sequence for optima: alignment and non-homefogous sequences can he disregarded for comparison purposes) in a typical embodiment, the length ol a reference sequence aligned for compati^rs purposes is at least 30%. at least 40%. at least 50% or 6Q%. or a? least 70%. 80%.. 90%. or 100% of the length of the reference sequence The ammo acid residues csr nucleotides at corresponding amino acid positions or nucleotide positions are then compared When a position in the first sequence is occupied by the b same amino amp; residue or nucleotide as the corresponding position in Hie second sequence, then the -'oc Jocose dentin th.. t j λ t m a., iw Vi >an n)t»s. i mciovOc S ιοί,’^ν ·-< u \a u t to amino acid or nucleic acid homology ).
To determine the cement identity of two amino acid sequences or ol two nucleic acids, trie sequences are aligned for optimal comparison purposes -e.g., gaps can he introduced m the sequence of a ilrsf amine acid er nucleic acid sequence for optimal alignment with a second amino acid >,t nucleic acid sequence). The percent identity between the two sequences Is a Inaction of the number of identical positions, shared by the sequences |i.e . % homoiogy-# ol identical positisns/tota! ?“ of positions x l0Q>. The determination ol percent homology between two sequences can be accomoii’Ped using a mathematical algorithm. ,A preferred. noniimibng example of a mathematical algontnm uhtised for the
III usn e iwis m iw> \cqae> ..w- s tie a'goifhm <<f Wf ” a ‘'.st ,V/> Ata-> f c> U b '* 8r 2..ΌΊ 8 , modified as in Karlin el at., Pmc .h/a/; Acad Set I.J S A 90 587;%? f l993). Such an algorithm Is incorporated Into the NBLAST and XBLAST programs of Attschul et ah. J Vo/ S?o; 215.403-10 (1990), BLAST nucleotide searches can be performed with file NBLAST program, score- IOt). w»nJSength~12·.
BLAST protein searches can be performed wTh the XBLAST program. score«58. word!ength=3.
To obtain gapped alignments. for comparison purposes. Gapped BLAST can be utifised as described In Aiucl u at a A'uC'r ’r. Asms Gee 2o VA9-402 J99?' Wncn uh. zing BLAB I and Capp'd Bf Ab programs, the default parameters of the respective programs (e.g.. XBLAST and NBI..AS71 can be used
IS
2017200239 13 Jan 2017
The percent identity between the two sequences is a function of the number qfideniieai positions shared bv the sequences, taking info account the number of gaps, and the length pleach gap wheh need to be introduced tor optimal alignment of the two sequences
The comparison of sequences and determination of percent identity between two sequences can 5 he ar-eomplished using a mathematical algorithm In one embodiment. the percent identity between two ammo acid sequences is determined using the Needfeman-VyUfisch afgorithm (Needleman et ai, J Moi Biol 48:443-53 {1970)1 which has {seen incorporated into the GAP program In the GGG software package available on a? gcg.com), using either a Bfcssum 62 mutex or a PAM2S0 matrix ano a gap weigh? of 16 >4, 12 10. 8. 6 or 4 and a length weight of 1 2 3 4. 5, or 8 in yet another embodiment the percent h Identity between two nucleotide sequences is determined using die GAP program tn the GCG sohwate package (avallabie on the internet at gcg.com! using a MWSgapdna.CMP matnx and a gap we-gh? oi 40 50 60. 70' or 80 and ε; length weight oi I 2,3 4 ;j, or 6 One typical set of parameters {arid the one that should be used unless otherwise specified) are a Blossom 62. scoring matrix with a gap penalty of' 12« a gap extend penalty of 4, and e -rameshfi- gap penally of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of n Myers and V/. /visitor -Myers et ai.. Compu' Αρρί Stcsci 4:11-7 (:988)5 w-hich has beerincorporated Into the ALIGN program {version 2.0). using a PAM 120 weight residue fable, a gap length penalty of 12 one a gap permit y of 4.
instrueticoei matenai, us that term w used ivm, includes a publication a recording, a ft diagram tx any other medium of expression which can be used to communicate the usefulness of the omooot d so >’ΊΟ i -ti d < > (Ό-ινογ < i toe iw.V >i to? v* tor wle< t η>, u h vm ng <>> fmati w the various diseases or disorders recited herein. Optionally, or aiternetofy. the instructional material can ib< of\ » Ό' v> I; vs x' c i< viau g the dn* eo c dijorde * in a -v I a '.i·-. r >. a mat’ mat
Including ne disclosed elsewhere herein .5 the instructional material of the kit may, ior example, be ah-xed to a container that contains the compound and/or composition of the invention or be shipped together with a container which contains the compound and/of compositiori. Alternatively, the instmctiorsai maieriaf may ha shipped separately horn fhs container with the inlontion that the recipient uses the ir-sirosiiooai material and the compound cooperatively,
Except when noted, the terms “patient* or ‘'subject1' are used interchangeably and refer to mammals snob as human patients and non-human primates, as; well as veterinary subjects such as rabbits, rats, and mice, and other animals Preferably. patient refers to a human.
f if\'Wi am <t to c tnos}( x t > ally 5 '* vf v urn ' m to to * t x ns >< e u^v r< η.I u xn il to neo i 1 s on -= m<uni. 3 „d v-ne t«. Jm us fe< 1 >0 ., vc e r « r'ir’nnai 0 .fefeby a Kmar ’udiatvs detsotabie -herapeuiic response compare;! to the response detected in the absence of the compound, A therapeutic response, such as. but not iim-tsd to, inhibition of and.-or decreased fibrosis, increased bone
2017200239 13 Jan 2017 mass or bone density, end the site can be readily assessed by a pletficra of art -oseogrifeed mefh-ods. including. ax?., such methods as disclosed heroin
The '•biisati mfisan wouiit under-- tand thal tet -^eciv*· amaun' or the os .mpr.·· sto r->· cranp-saitton administered hefeirt vanes and can he readily determined based on a number of lectors such as the S disease or condition being treated. tee stage o? the disease, the age and health and physical condition oi the mammal being treated, tee severity of the disease, the particular compound be-ng administered, and the like
As used herem. to “treat- means πχΐυριηρ the Ireguenoy with which symptoms of a disease (eg.. decreased hone density, fracture. fibrosis. and the like) rare experienced by a patient The term includes P the «dmii-ist» ahon ct tee cv«-icurds c- age its ο* I te p’esent -uv-^t-t-cn to p'ewrt or ;tete-, the ans<l of the symptoms. complloaftons. or biochemical indicia of e disease, alleviating the symptoms or arresting or Inhibiting ieriher development of thia disease, condition, or disorder Treotmeot may be prophyiaebe (to prevent or delay the onset of the disease, or to prevent the manifestation of clihicai or subclinlcal symptoms fhereoi; or therapeutic suppression or alleviation of symptoms artier the mnnifesiahon 0; tot? s disease.
By the phrase “specifically binds as used fierem. is meant 0 compound. n o. a protein, a nucleic acid, an antibody, and toe like. which recognises and hinds a specific molecule, but does not substantially nttoognise or bind other molecules in a sample For instance art BMP protein, an antibody or a peptide inhibitor y.hich icucgs-izev auc tones ·. cognate mxxptor -,ee 0 afdP tope | or hoc ii receptor an b antibody that binds With its cognate antigen. and the like;· in a sample, but does no- substantially recognise or bind other mtoecutos it* tee sample Thus, under designated assay conditions, the specified binding moio-y (e g., a BMP or a receptor binding fragment thereof) binds preferentially to a particular target mciecuie and does net bind in a significant amount to olhet components present in a test sample A variety of assay formats may be used to select an antibody that specifically binds a molecule of interest.
S f or example, soifd-ohuue LUBA immunoassay vumunopm-e-pkatioie olAcore, I ACB. C-ctot. and yVeito-m blot, analysis are among many assays; that may he used to identify a BMP that specifically reacts with a BMP receptor Typically, a apitoific or selective reaction will be 0· least twice background signal or noise mure preferably. at least five-fold greater than bacitgrcund signal or noise, and more typically, more than 'tO times background, even more specifically a BMP is said Io 'specifically bind'' a BMP receptor whan
TO the equilibrium dissociation constant (A,,) is c 101) pM: more preferably e 10 pM. even mere preferably < f uM, yet mote preferably s WO nfyi and most preferably s' 10 nM
The term 'Thd refers to the egu8ibnum dissociation constant of a particular ligand-receptor tefe; cction, bind ! q Ml-ntv u-ieuA .wj t>- tee shennte c ;h - .cm tote ef uoKewteu'' n teraeborv
-x between a binding site of a molecule fe.g.. a BMP ligand) anti its binding partrior <a.g. a BMP type I or tvp< ϋ fvcepfcrt Unless indicated otnonvae os used I cfcitt cinding effete’ tutors io inlnnsic oindfng affinity which raflecis a I interaction between members, of a binding pair fe.g., BMP and its cognate
1?
2017200239 13 Jan 2017 receptor), The affinity of a molecule X for iis partner Y oar·, generally be represented by the dissociation constant (Kd),
Affinity < ao bo mesaurod by common methods known in the art. including those ilescdb&d herein Low-affinity BMPs generally b-ne a receptor slowly and lend to dissociate readily. whereas hlgh^iffinlty 5 BMPs generally bind a receptor fester and fend Io remain donnd longer A variety of methods of measuring binding affinity are known m -he art, any oi which can he used for purposes oi the present invention. Specific illustrative embodiments ere described eisewnere herein
The term %?. as used herein is intended Io rater io the association or on mte constant. or specific reaction rate of-hs forward, or comptex-form log. reaction measured -rs units: M sec ’.
It The toon ''k, ’’ as used herein, is intended to refer io ihe dissociation or oft note constant, or specific reaction rate, for dissociation of an antibody from the antibody/antigen complex, measured in units sec
The term “K2 as used herein, is intended tc refer t.c Ihe dissociation constant oi a particular ariiibody-sm-iaen interaction I- la ease-mated by the formula
The term altered boding as used heroin means Ihe designer BMP comprises a dilferen· specificity for at least a type I receptor and/or a type II receptor when compared with the binding of a eoirespartding wild typo BMP to the same type I and-'or type li receptor. The deetgnor BMP mey bind wm >. cate m amyitv vd ifv χ oeote' ' o^ , a w to tv fdme o' tec ,- d t-pe CM'-' o het b receptor Fot instance, if the wild type BMP bound a certain type i r&oepfor with a certain binding affinity, tee cones poixhng designer BMP binds that receptor with greater or lesser affinity compared with the odd type BMP. h may oven be that the designer BMP will specifscaliy bind a receptor that the Wild type BMP did not dotec-tabiy hind end v:ce-a-versa where the designer BMP will no longer delectably hind a receptor ihal rite wild type BMP binds. Thus, altered binding encompasses any detectable chance In bn d ig I - o d 'Si<ti ' BVt~ to a 'w1' <' iyv if r w lor r -' oar-. 2 -vet ,1 ¢. n? J sg ?i J o, r coMo- b, tee corresponding wild type BMP. II may be Inal the designer BMP has a greater or lessor 2..., value compared With the k... value for a corresponding wild type BMP printer the designer BMP has e greater or lesser value compared with the K-e value oi ihe corresponding wild type BMP such that the Kd of the designer BMP is greater or lesser then the Kn of a corresponding wild type BMP1 for- the same BMP
3tf receptor Thus, any difference in a binding characteristic and/or affinity value between a designer BMP and a corresponding wild type BMP are encompassed by thri term aitered binding’ as used herein.
The term “surface ptasmen resonance”, as used herein, refers to an optical phenomenon that allows tor the analysis of real- time biospectho interactions by detection «» alterations in protein concertifKhons 'within a biosensor matrix, tor exanipto using the BIAccre system (Pharmacia Biosensor
AB. Uppsala. Sweden and Piscataway N.d.>. For farther descriptions see, e.o Johnsson. et at.. Ann. ?h;c/. Cte; 51. 13-26 (1933·; .johr-sson, et at,, f&jtectorgttes 11'620-62? (1931 ). Johnssott, el at.. «?. Arfcf, Pecyg.'-iB 8' 36-131 (1699). and Johnnsori. et ah, An»/. S/ccben? '596: 268-27? (1991)
2017200239 13 Jan 2017 ?b
As used hereto. substantially pure'* means an al>jecl species Is the predominant species present g.e.. on a molar basis it is more abundant than any other Individual species in the composition·, and preterabiy a substantially purified fraction is a composition wherein the object species leg., a designer EfMF) comprises at least about 50 percent (on a molar basis) ot all masromotecular species present Generally. a substantially pure icmpositiori will immense more than about 30 percent ot ail ; naoromotecuiar species present in the composition, more preferably more than about 35%. 90%. 95%. and SS%. Most preferably. the object species te purified to essential homogeneity (contaminant species cannot tv detected m tne compeuhen by conventional detection methods) wherein the composition -'onusis essentia:^ et a single marromoieutiar species.
As staled previously elsewhere herein. BMPs are members el Iho TCF-3 o'cteln supedamily all of which anti cheroKtenxed by six-so: iservad cysteine residues (La” 1> M a, ,, AiON Nature, 409 860-92'ΐ The BMP/GDF subfamily Includes, but is nol limited to, BMP2.8MP3 (osteogenin) - sea, e.g.: US Patent No 6,177 406·, BMP36 (GBF-10) (see. eg , US Patent No 6 294,04?;, BMP4 tSMP2b) (sea, e.g US Patent No. 6.245.839). SMP5 (see. eg., US Patent No. 5543,304). 8MP6 (see. e.g.. US Patent No. 6.813,744). BMP? (os.teogenie proisln i or ΟΡΊ ? (see. e.g.. US Patent No, 5,141,906), BMPS (OP2) (see. e.g„ US Paten! No. 3.663,673). BMP8B (OP3) (see. e,g.: <JS Patent No. 5 354,071), BMP'3 (GDP2; tone, e.g. US Pa-anl No 6.287.815) BMP10 (see. e.g. US Patent No 6,703,643). BMP 11 (GOPi1iis.ae.eg., US Patent No. 6.437.Till. BMP12 (GUT?) (see. e.g , US Patent No 6027,0195 I5MP18 tGOI‘6. ( PM'A (se« ec 1% °ntenf No nt >7 Ρ?ι Ρ5.?τ·< 5 ; ;Γ>Γ<0 ; ic smef No -> L M >''3$
BMP16 (see, e.g.. US Patent No. 3,331.312), 6DF 1 (see, e.g,. US Application No. 2004/0039162) GUF3 (sea. e.g. US Patent No 8026.475). GOF5 (COMPi: MP62) (see. e.g, US Patent No. 5.994,094); and GUF8 (myostatic) (see, e.g.. US Patent No, 5,62?.?33).
BMPs specifically bind their cognate receptors, which include Type I receptors: At.K-l, ALK-2 (also called ActRla nr ActRI). ALK-3 (aisn called BMPRtei. and ALK-6 (also called BMpRibi. anrt Type d receptors, ActRlla (also called ActRII). ActRllb. and BMPRsl. The BMP-recepter binding interactions have been studied extensively, and (he binding spssiitcities of each wild type BMP for each type I and/or type H erepter o gene-a vlown nJ 'Ui.orx on u ow < r > 'abl 1 eg Nkv ' =. >c 2,
Fac/ofP/ev 20:357-7? (2009·; Heinockg cf al.. SA-fG Β.ό/ ?:50 -:2009).
TABL5 1
ALK 1 | ALK 2 | ALK 3 | ALK 6 | ACTHA | l· ACT) IB ) | BMPRII | |
BMP-2 | No Binding | No Binding | + ·ϊ -t -t | !: 4-1% <' '' | 1 A. 4. A......... | ||
BMP-4 | No Binding | No Binding | ... ; φ< .<:. ... | Hl· <: .< | |||
BMP-6 | No Binding | No Binding | 4-4.· | :>· *<S**:#‘‘K ' | 1 |
T9
2017200239 13 Jan 2017
ALK 1 | ALK 2 | ALK 3 | ALK>3 | ACTliA | AGTIIB | BMPRIi | |
BMP-? | No Binding | No Binding | <M4., | 4- -A- | ; ΉΉ , | ’Τ^'Τ-Τ ., .· | jLIjL^s.^.1, tttt |
BMP-9 | No Binding | No Binding | No Binding | 44 4 ' | .. 4 | •·!!·.-,-3-. |
This application is based tn part on the utiderMandihg that each BMP dimer binds to four BMP receptors two type i receptors and two type it receptors. The specificities of each BMP for each receptor a arc known in the art as shown above tn tabic 1. Aiso, the receptor binding regions of venous BMPs that mediate binding of the BMP’ for each receptor have been mapped and ato shown tn Table 2 her instance. it is wet; established that wiki tvpe »MP2 and PMpa ρ»;d type ; BMP receptors Atr-,> and ALK-6 veto high ^ίήπίίν .tnd h-si-d type II BMP :ereptor«< weh tower affinity On toe -sthe: hand. Wild type BMP6 and BMP? arc known to have bind type II receptors Ask HA. Acts! IB, and BMPKi! with high affinity but bind type I receptors: with tower affinity than they do to type H II is believed that the differing cellular responses from fhe epproxtrnetety Sortythnss TGHT suosirtamily mssmbem signaling through inisrachon with approximately twelve receptors Is believed to he due to each ligand chiming a specific repertoire of receptors wTh which ;f binds with diflering affinities. The type i and H binding domains are described ir· Table ?.
TABLE 2
BMP..... | Type II domain A ammo acids | Type! domain ammo acids | Type II domain B amino acids |
BMP? {SBQ ID NO 4 | 31-44 | 48-76 | 83-100 |
BMP4 fSEQ ID NO:2T | 3444 1 | 5244 | 65-102 |
BMPS (SBO ID NG B) | 54-67 1 | 71-100 | 107-130 |
BMPS 442 ID NO 4} | t 65-69 | 73-102 | 104126 |
BMP? (SPQ ID NO'S} | ; 55-69 | 73-102: | 108-126 |
BMPS (SBQ ID NO St | 5546 | 73-102 | ios-i’is |
BMP9 tSPO tb NO4I | 25-88 I | 42-71 ........ | 78-06 |
in one ombodimont, the invention comprises introducing an ammo soid mutation m al least one ?t! receptor binding site thereby providing altered binding to type I and type is BMP receptors by designer BMPs compared to the binding ot Tip corresponding wild type BMP to those receptees That is. if is well shown in the art that wild type BMP? shows a relative?'/ high affinity for type I receptors, white wild type BMPS shows a high affinity for type si receptors It is further Known m toe art that liaferodimors oi w;td type BMP2 and BMPS bind to both typo i and type fl receptors with relatively high affinity each BMP apparently providing the higher afhndy binding site for each meeploc Ban fable 3, below The ΒΜΡ2Ό
2017200239 13 Jan 2017 hetar'obi.msrx are known to be more active that PMP2 or BMPS alone or as homodimers. in both At wiro and vivo ix-tis formation assays able 3 shows on example of BMP2 end BMPS binding afFnlties to type 1 pod f 1 receptors<
TABt-E 3
Type I : | Type II | |||
Ligand | AI..K3 KuthM) | ALKo K:UnM) | ActRil A Κ-,ίηΜ} | AciPdB K , - nMi |
&MR2 | 0.69 | o.w | 141 | 42 |
BMPS | 150 | W2 | &73 | 2.0 |
BMP2/6 | 1.57 | 0.43 | 2.S5 | 1.15 |
Accords; igiy. it is an object of the ww:t;oi; to provide bessguor BMPs With improved binding to type i arxhof type I; receptors As; shown in Figure 1.A and Table 2. each BMP comprises three binding ados ibid oon'-nbsjie to receptor binding t neni N- to C-fermmus coot! BMP composes a type It rcccpior binding Site A. a type I receptor biridlng site and a senond type II receptor binding cite Ft. Although an exemplary alignment of wild type BMP?. BMP«. BMFb, BMPS, BMP7, BMPS, and BMPS? is ilinstmtod in Figure 1. the skilled artisan wilt appreciate that there are well-known alignments providing the relative gos-bootnq of venous ammo acidSs among the members of tlx? ΤΟΒβ soperfornily. Snob alignments me provided among others, in international Btiblscailon Nos. WO 2009/088131 (e g.. Figures 15-17.. Figure 31A-, WO 20(?SA?b1526 {Figures 9-12), WO 2Q8M18835 {Figure St, WO 7303 ; in.'Wb, WO 2005/113585 (I igurc 3b WO 2001 '92208 {I ;gure SA-5C). Kir soli et ai.. cMSO J. 19 ) B -h )324 {2000) Figure I). NB Patent Application Publication No.. 20Q7/G293425 iFigure 6s. Ooppe ef a; . iVad/ro 420 .835-542 {2002?. Nickui et al. J. Sone Jo.e.4 bmg. Am 83 /-1 -I <20015, and Weber et ab, SMC S'meinra: .0/o< 7 G (2007), Thus using protein sequence alignment algorithms and fools; well-known in the art, including the alignments of the amino aod sequences oi trie venous TO) β superfemliy members: as well as the disctosure provided herein, the corresponding ammo acid m one BMP.-GOF protein relative to the eim-io acid at any posrtson in another RMPKJDs protein can be determined in one embodiment, the corresponding ammo ackl residues in BMP-2, BMFM. BMP-5. BMF'-G BMP-7. BMP-5 arid BMP-9 are shown {see. e.g.. Figure 1 Ah in some embodiments of the inven-ior·. -he designer BMP comprises mutations In a type I binding domain or a type |i binding domain. wherein the mutations confer altered binding to a tyee I or type is BMP receptor, in some embodimenls. the designer BMP comprises one or more mutations In both a type I bindmq domain arid a hrst t binding domain At or second toindsno domain B) type ϋ binding domain Irs
3b other embodiments, the designer BMP comprises one or more mutations in both type II Sir-ding domains. Ir; other embodiments, the designer BMP composes one or more mntMions In the hr si type 3 binding
2017200239 13 Jan 2017 domain, in the second type i| binding domain, and m toe lyps I binding domain in some embodiments, the designer BMP comprise;: one or more mutations in the type I binding domain.
in some- embodiments. the mutations improve binding to a type I receptor. tn other embodiments, tbs mutations improve binding to a type II receptor In other embodiments, the mutations dsorsase 5 hind ng to a type i <; Ape -i mcemom n mm a rn onnin'tmLs to- m-,: vioi's cteafe <·-> de-hmm a qUran tether as more fu»y set forth fcetow. in some- embodiments, too mutations create or destroy a His doorstop as; more tolly set forth below.
Because BMPs are so welt characterised and understood in the art. 4 would be understood, onceprovided with the disclosure providers herein. She location of possible mutations ihaf can be made that do b not further affect the activity ol the designer BMPs would be understood. Accordingly, the designer BMPs of the invention encompass vahani BMP's which difi-ar from a corresponding wild type or designer BMP in that it contains additional i-nsortions. deletions, or substitutions winch do riot effect (be receptor binding affinity of the variant BMPs. in some non-limiting embodiments, those ol skill in the art would understand thaf the cysteines involved m cysteine knot forrnafion ano amir·» acids involved in receptor interactions s should not he mutated or should he changed with conservative substitutions, white other amino acids may fie more freely substituted, inserted or deleted wilhouf adversely effecting biological activity of the designer BMP.
II should be noted that unless otherwise slated. all positional numbering of designed or modified BMPs is based on the sequences of the mature native BMPs. Designer BMPs are characterized by the b prerieisrmined nature of the variatlan. a feature mat sets them apart from naturally occurring allelic or iuterspecles variation o; the BMP sequence. Variants of designer BMPs must retain at least 50% of the activity of the corresponding wild lype or designer BMP activity in one or more celt types, as determined using an appropriate assay described below. Variants that retain at least /5%, 80%, 00%, 90% or ?:>' & of wild type activity are more preferred, and variants that are more active ihan wild type are especially
..5 i of< u 5 A d< wgr v.t BM^ < w i.c m iny fn to- ’< t tie- a id or ^u c-u < to x ot * ο N tor η xo C terminus, or Infernaiiy In a preferred embodiment, designed or meddled BMPs have at. to-ssi 1 residue that differs from toe most similar human BMP sequence, wlto at least 2, 3, 4., §, -3. 7. 3, V, '10 or more different residues being more preferred
Be->g iu OVP- etti -wo-'ten i to-'to c ' ( tot 50' „t Met > I ι< i v 8-' at teas-l\. .
at least? I . at feast at least 33 o rsf to-est c/o at ea%h?/i a* -est<?9% at -ea% 9? > m ;est 01%, at least $32%, m least 93% at least 94%, at least 93% at least 90%. at least 97%. at least 93% or ai leas- 90¾ toeniity with the corresponding wild-type BMP protein sequence.
Designer BMPs ol the invenfion may maintain at least 00%, el least 81%. at least 82%. at least 83%. at leasl -35%. at least 85%, at least 88%. at least 87%, at least 88%., at least 39%, at least 80%, at least Stoto. at least 93%, at leas:· g,j% at least 34% ,-sf least 93%, ai least 90%,. at ieesi 97%, at least 9-3%, or at least 09%. identity with the conserved cysteine domain Of the C-termmai region of the corresponding wiid-typs BMP pro-esr- sequence.
2017200239 13 Jan 2017
Designer BMPs may contain further modifications for instance mutekcns the! oiler addiSbhai protein properties such as stabtiliv or iromonogenicity or which enable or prevent postfransialionai modifications such as PFGviation or giyeosyiahon Designer BMPs may be subjected io co- or postiro!V.ud o-u! nOdH< at'vtis, ,rx ac'mg hot net »r >tes i<’ aptii dons shannon c' one o. ret vdo chairs 5 or ienminl. givcasylation, PPCiyiarion, circular permutation. cyclization, fusion Ιο proteins or protein domains, and addition al peptide Ians or labels.
Due in the degeneracy of tbs genetic code, on extremely large number of nucleic acids may be made, all oi wiiich encode the designer BMPs of the present invention, by simply modifying the sequence of one or more codons io a way that does not change the amino acid sequence of the designer BMP. The d designet BMPs oi fee invention do not comprise these sequences eel forth in yVG20d8'd5152d or W02d09'0«B12f,
As desenbed above, BMP’s .ere naturally expressed as pro-proteins comprising a long prodomain, one or more cleavage sites, ano a mature domain. This pro-protein is then processed hy the cellular machinery to yield a dimenc mafere BMP maiacuie, ir- a preferred embodiment, the designer
T BMPs ol fee invention are produced in a Similar manner. The pro-domain Is believed to aid in the correct tokiing erst processing a? BMPs Furthatmore, in soma hui not all BMPs, the pre-domain may noncovuienily hind ins mature domain and may act as a chaperone, as well as an inhibitor (e.g,. Thies ei al -2001} Growth f-'actors, I8?biTbife; Preferably. fee modified BMPs of Ibe invention are produced andor administered thatapeuticaiiy in this term. Alternatively', BMPs may be produced in other terms, n inckidlng, but not limited to. where the mature domain is produced directly or refolded from inclusion bodies, or comprises fuil-ienoih miaci pro protein, l ife designer BMPs oi the invention will be useful «η these and other forms.
in particular embodiments, the designer BMP ot fee invention comprises a backbone BMP. <.e. the Wild type BMP. to which ibe designer BMP corresponds. in particular embodiments, this backbone
..5 beawieiy tet’kfe? BMW- 2<MS ' oMk > BNP’ 2<M~o or oMP-J badree «<
In some embodiments of the Invention, the designer BMP comprises at least one muiafion in a type s binding domain and/or a type II binding domain, wherein the mutation centers altered binding to a tvpe i or type it BMP receptor compared with the binding of a corresponding wild type BMP not composing the mulshon in soma ernixxiiroenfs, the designer BMP comprises at least one mutation =0
TP both a type I binding domain and at least one mutation in a type H binding domain, in other amfeixiimente fix? designer BMP composes a- fees? one mutation within the type ii binding domain A arid the type II binding domain B. in other embodiments, the designer I3MP comprises at least, one mutation Vi type II binding domain A, typo II binding domain B. and a type I binding domain
In certain embodiments, trie mutation may comprise an amino or nucleic acid substitution.
e®*i >n ana ortest-iffo! 11 o m ten -d m1 oK >i»-p' in-mutet 'i c* 5 ns-a ay a- i m ocx i»shuton
2017200239 13 Jan 2017
V in some embodiments. the backbone BMP <s e wild type BMP and fba mtitatiens are one or mor® of the mutations listed io Tables 4 to 8. The designer BMP may oorsiasn any combination and any number of mutations listed in those tabiss in some embodiments, the backbone BMP is a wsid type BMP and the mutations are one or more of the mutations listed In Tables 4 to 8 The designer BMP may' contain a pet mutation and any and ail ci the mutations listed in these tobies or disclosed elsewhere herein fABLE 4
Type i Bis BMP2 | •ding Dorn. BMP4 | do Ms.daf.io BMPS | ns BMPS | : BM P? | BMPS | »M*.X\xKxXxxx\k>»w»kk«» BMP9 | .....KKVK.XKK.KKVKKV<.VK..KKVKXKKKV.........K........K......K.......... Possible mofatiahs |
P43 | P5Q | 371 | 372 | A72 | 37.2 | P42 | F 3. N, A, P ................. |
F49 | f 72 | 673 | F73 | 773 | F-43 | y | |
A52 | A54 | hi?5 | hl?6 | ws | Π76 | A48 | hi, A. |
D53 | 055 | A7S | AT? | :w; 7 ? | 37? | 04 7 | A, L. D |
K54 | H58 | H77 | hi 78 | V78 | ¢78 | 048 | 0. c |
L»6 | L5? | M78 | M79 ...... | )«7?'7 | ?M7p77 | 749 ...... | M. 7 L |
hi 86 | hi 88 | N?9 | N89 | N80 | NSO | T50 | T, hl |
So? | S59 | A86 | Adi ..... | '7 | A82 | P51 | A P |
N.6S | N61 | N82 | N83 | N88 | N83 | K53 | K. M |
763 | 766 | 766 | 787 | ?787? H | 1.87 | 707 | 1.7.L |
T65 | 78? | T88 | T89 | T83 | $89 | TS9 | A?.7,<3 ............................................... |
N88 | 5470 | HP1 | HP? ....... | 'TO 7 77 | H92 | H62 | Id. N......... |
369 | 371 | 1.92 | L93 | 693 | L.93 | LS3 | I... $. P |
770 | 772 | MPS | MP4 ....... | 794...........' | 04? : | $64 | Μ, $ I. V ...... |
hi?1 | N?3 | F34 | h!9S | N95 | rM95 | f65 | b.hl.M........ |
P95 | P98 | P98 | P96 | P66 | INSERT $. P; DELETE P | ||
372 | 374 | DBS | ES? | 39? | D97 | re? | 0 . 7 . p/d |
K?3 | $75 | i-|9? | Y98 | 798 | A96 | $66 | y. i-i. r, a, $ |
!74 | f?8 | 798 | 793 | 799 | 7799 | 769 | A. V, i |
P75 | P77 | P99 | PI 03 | Pt oc> | P100 | G70 | $, G |
n« TABLE f,
T ype II Binding Domain A Mutations
BMP? | BMP4 | BMPS | BMP6 | BMP7 | BMPS | BMPS | Possible mutations |
733 | 735 | I56 | I57 | ?l$7'7 | IS? | I2? | i 7 |
2017200239 13 Jan 2017
P38 | P3B | B59 | K60 | εβο | 080 | K3Q | P R, P. B, Q |
G3? | G39 | G6Q | G<61 | G5f | G61 | ESI | G. E |
H39 | Q41 | A52 | A63 | A63.......... | S83 | £33 | A. B. S. Q |
MM | 143 | T94 | NSS | Ϋ55 | Y£O | T35 | R. Y.i |
V42 | Y'tel | V65 | Y68 | V68 | Y66 | B38 | Y, £ |
H44 | P-46 | Du? | D88 | BBS | IB | IB | Η, B. K R. £ |
TABLE 6
Type II Binding Domain B Mutations | |||||||
BMP2 | BMP4 | BMPS | BMPS | BMP? | BMPS | BMP9 | Pes&ibte mutations |
BBS | £85 | K1G7 | RIGS | OIOS | h i OS | K7S | Q. K. B |
BBS | S3? | NWS | DM0 | DM0 | SMO | B30 | N: S |
ASS | ASS | AMO | AMI | AMI | AMI | PSf | P, A |
MSS | M9I | Vi 13 | VM4 | V114 | VM4 | V54 | Μ. V ' |
1..02 | I..S4 | PMS | PM? | PM? | Y11? | KS7 | £, K, L , V |
£94 | ESS | DI 13 | Di IS | DMS | DM >3 | D89 | OgE ......................... ' |
NS5 | YOr | SMB | Di 2.0 | St 20 | SMB | MS0 | Μ, H, S |
£96 | Bis | SI 20 | SI 21 | Si’2i | ST20 | G'9'l | S,G,D |
K9? | K99 | NV21 | DI 22 | D122 | R12T DI 22 | V02 | N. V. K |
V9& | VI00 | VI22 | V123 | Vi 23 | V12S | BOS | R. V |
iviST..... | P23 | 112-1 | Β·24Γα | 3®....... | TS4 | 'T<£V < |
In some embodiments, tee mutations miptove binding to a type I receptor. m other smbod'-mante 5 improve binding to a type II receptor, in other embodiments the mutations decrease binding to a type I or tvpe it rsieeotora.
Tabies 4-5 above provide a non-limiting compilation of example mutations of the present invention where (he posttion of (he mutation is provided relative to tee corresponding wiid type BMP amsno acid sequence, thus, in some- embodiments, Ute designer BMP composes the following preferred * if -combinations of mutations..
in cerfam embodiments. the corresponding wild type BMP to the designer BMP is BMPS. Further tee at least one mutation within the type II recaptor binding domain A Is, a mutation selected from the group consisting o: V33 PBS, GV, tUS. i 41 Y42 AM? I i44 in ether embodiments, tea designer BMP comprises at least one mutation within te« type |t ίό receptor binding domain A and further comprises at feast one additfonaLmatalion within a type i receptor binding domain. The mutation within the type f receptor binding domain is at least ona mhtatlon at PiB,
2017200239 13 Jan 2017 '40 ''-2 Pod I 1¾ Nbw Bo? N-* v§s Sv’> V/5 K'! S?' w/3 1/4 ordE/'-wnb respect io the sequence of SEQ HO NOT, in yet f-rnbei· em Pediments. the designer BMP comprises at least on© mutation within a type is receptor binding domain A. at least one mutation wilh-n the type I receptor binding domain, and further 5 comprises at ieast one additional mutation within a typ» IIB receptor binding.demaih. Thp mufafipn within the type If receptor binding domain S is a* feast one mutation at EB3, S83. ASS, MSB, LS2, EBf, MBS, E9S KS?. V9&. and V89 with iespscl to the sequence. nt BBQ IG NG:1
In some embodiments, the designer BMP composes mutations at each of ammo acids H44. P48. AE2. B53,1.55 Sb? 1468 BBS V70. insertion of P after N?1 S72 K73 174, -V7, and V® with respect Io b the sequence el BUM ID NO.'I.
in one embodiment: the designer BMP cone’s the (bltbwthg mutations. H44D, PASS, AS2N, D53A L85M S5?A N8SR S69L. V70M, insertion of a i’after N71 S72E, K73Y 174V, A??P and VSOA with respect to the sequence of SEG IB NQ:1.
In some emtxidiments the designer BMP comprises mutations at each of amino acids V33< P38, 5 H39. SS5. M89.1..52, E34, B36. K97. and V95 with respect to the sequence of SBQ IB 140:1.
in someembodiments, the designer BMP composes mutations at each of amino acids V33i, P3SK. H35A; S85N. M89: 1.92E, E34G, BBSS. K97N. and VWt with respect to the sequence of SEG IQ NOB.
In other embodiments, tbs designer BMP comprises the following mutations: V33I. P36N, H39A, b B44D. P48S. A52N. l£< S66M. I466H V7QM. S72E, K73E. insertion oi s Y after K73, 174V. 77AP.
S85I4. M8DV. L92H. E94B. EB6B. KVZN and VuVi willi respect to the seguencs oi BEG IB 140:1.
in yet ether embodiments. the designer BMP comprises the following mu la-ions: V33L P38R.
' 3W-OE P43B ABEN LMM Bb?M NhBI ι V ?M A, 2L K<<L rm>t on of a b am - \/3 n’iv 77AP. S85I4. MS9V. L92P. E94G, E96B. K97N. and V99i with respect io the sequence of SEE fD ΝΟΊ
..5 in certain embodiments. the corresponding wild hype BMP io the designer BMP is SMP4. In certain embodiments, the at least cos mutation within the type II receptor binding domain A is at V35. P35. ¢339. 041. f 43. Y4a, and HAS of SEQ Hb NO 2
l.n other embodiments, the designer BMP4 comprises at least one mutation within the type II receptor binding domain A ano further composes at least one additional muta-ion within a type I receptor
5b binding domain. The mutation within the type I receptor binding domain is at least one mutation at P50. AM, Bbb. H66 Lb?. N58, Bb9 NbE Vbb i‘6Z, f470. SZt. V72, N?3. S?4. 373 176. and PZ7 of BEG IB NO;?.
In yet further embodiments, the designer BMP4 composes at least one mutation within a type L receptor binding domain A. at least one mutation with-n the type I receptor binding domain and further comprises at least one addition©! mutation wirhlo a type I IB receptor binding domain I'he mutation within the type ϋ rocentot binding domain 3 is at Ic-syi one mutation at Edo, 35/, AS8 M91, L34. ES6, KSA Vgg and V99 of BEG IB NO:2.
2017200239 13 Jan 2017 in certain embodiments. the corresponding wild typo BMP to the designer BMP is BMPb. in certain embodiments, foe mota’icn within the type II receptot binding domain A is at toast one mutation at I5F PF9 !>>() ,,6? EG4 'ΤΑ O: ΓΤ7 of BAQ lf> kiu ?<
in other embodiments, the designer BMP oompnses at toast one mutetion within the type if receptor dinning dom{ain A and further comprises at least one additional mutation within a type I receptor binding domain, The mutofion within the type i receptor binding domain is at least one muiafion ai S?1. F72. Ν?δ. A?G. NT?, M78. N79. AGO. N82. 78G T8S. H9?, I 9?. M93, F94. PS5. D96, H97, 798, or P99 of SEQ ID NO:3.
in yet further embodiments. the designer BMP comprises at toast one mutation within a type it receptor binding domain A, at least one mutation within the type I receptor binding domain, and iurihot comprises at leas? one additional mutation within a type HB receptor binding domain The mutation within the type It receptor binding domain B Is oi feast one mutation of Kt07. NI99 At 10 Vi 13. F116. 0118. S119. S129, N121; 7122, or 1123 of SEQ ID NO:3,
In cerfaui embodiments, the corresponding wild type BMP to it-·® designer BMP ίο BMP9 in s certain embodiments, the mutation within the type i! receptor binding domain A ts at least one mutation et
I57. K80: Goto AGS. Nob, YGG, or DBA of SEQ ID NG.4 in ether embodiments, the designer 8MPG comprises at least one mutation within the type II receptor binding domain A and further composes at toast one additional mutation within a type I receptor binding domain. The mutation w-intn the type I receptor binding domain w at isos? one mutation ai S72. η N7G, A77, H78, M79 N80, A81 N83, V87 T83. H92, I.93, M94, N95. P3G, t~97. Y9S, VS9. or P100 of
SEO IB NO:4.
in ye? further embodiments, the designer BMP6 comprises at least one mutation within a type ϋ receptor binding domain A at toast one mutation wVhm the type- I recc-ptoi binding domain and further comprises at least one additional moiarion within a type IIS receptor binding domain The mutation within
..5 the typo i| receptor binding domain 8 ts at least one mutation at KIDS. N1'!0. AHI, 7114, T 117. 0119. N120, StzI, N122. 7123. or H24 oi SEO ID NO:4.
in certain embodiments. the corresponding wild type BMP to the designer BMP is BMP7. In certain embodiments, the muiation within the type II receptor binding domain A Is a? toes? one mutation at io?,E60. GS1. A63, Y86. Y8G. or BBS of SEQ IO NG 6 in other embodiments, the designer BMP? comprises a? leas? one mutation Within the type It receptor binding domain A end further comprises ot feast one additional mutation within a iype i receptor binding domain. The mutation within the type i receptor bidding domain ;s nr least one mutation at A72, F?3, N7S. SAto Y7G, M79. N60. ABto N63, 767, TB9 f-192, B?>3. 194 Ν9ό. P9o B97 Ted. 799. or PICO of BEG ID NOT.
in vet further embodlmsHs. rhe designer BMP7 comprises at -east one mutation within a type ϋ receptor binding domum A at toast u-tie mutation within the type i revepfor binding domain, and torthsr composes at leas? one additional mutation within a type Itn receptor boding domain The mufation within
2?
2017200239 13 Jan 2017 the type It receptor binding domain 8 is at least on«: mutebon-ai. OIOS, N110, Alft,. VI 14, F117, 0119. SI 20, S121. N122, V123, or 1124 of SEQ ID NO:S, in certain embodiments. too connsgoriding wild rype BMP to the designer BMP is BMPS in certain ernhouttnetito, the mutation within the type 1 receptor binding doma-n A is at least one mutation at
157, Q69 GSt. 863, Y85 Y88. or EOS of SFQ IP NG’8.
norm r tOd mart» the d< signet JM^'4 o’j fo-s a, te-tP, o t< ' to tot >„ n wires tot wpe receptor binding domain A ano t'urrh&r romprises ol least one addificna: mutation άιΙΙίΙο a type I receptor omdiqy domain the mutotsns· v'ctoin toe f>pc t receptor binomg domain w at toast one mutation at 8/2 P73. D7S, S77, C78. M7S. N80 A82 N83. 1.87 S89. H93. IBS. MB«, MBS. PBS. D97, A9S. V99. or P1G0 of SEQ tD NQ;G.
In yet further embodiments, the designer BMPS comprises nt least one mutation within a type II receptor binding domain ,4 a; least one mutation wtthm tire type | receptor binding domain and further comprises at least one additional mutation within a type 113 receptor binding domain. The mutation within toe type It receptor binding domem B is at toast one mutation at KW8, SHO, Alii, Vila, Vi 17, D118.
S119 8120, N :21. N122. V123, or 1124 or SEG IO NQ'5
In certain embodiments toe mutation within ihw type it receptor binding domain A is at ieaet one mutation at 127. K3Q. E31, E33. V3S. or E36 of SEQ IQ NO:?, in other embodiments, toe designer 8MP9 comprises a; feast one mutation within toe type It ereptor hciciou domain A ut d 'urtoc ou^pnae^ at i< u*t cm- addthora: tn tt dor w fust o type t ( oopto· ft binding domain. 'i'hs.- mutation Within the type I receptor binding domain is si toast eno mutation at F42. f-4,\ A4S. D47, 048. V49. 150, P51, K53, V57, 199, Hb2, 183, K84, roS. PSS, i'S/. KS8, V9&, or G70 of
BEG ID NG:7.
in yet further embodiments. the designer BMPS comprises at least one mutation within a type It receptor binding domain A. at toast one mutation within toe type I receptor binding domain, anti further comprises si least one additionai mutation With:?! a type HB receptor binding domain. The mutation within the type It recegtot binding domain B is at. teas’ one mutation at K78, SSO, P81 V-34. K87. DS9, M90, <591. V92. P93, or TB4 ot BEG ID NO ?.
Exemplary amino acid sequences of designer BMPs are set forth In Table 7. below. Table 7 shows the name and sequence of toe designed molecules.
TABLE 7
NAME | SEQUENCE | SEQ: ID NO |
BMP-A | x Μ \ > Μ. Λ V V \ > icr , £ -. to r ,. - Oto 1 a street: rr-’tacr'Cii'rsiais.iSi-swt^itototo-e.Ki'ivpritwViitccoc | 8 |
BMP-B | CAism-ysri^.xmie’u'torwviwscvi.reaoireeR’niGVSAesci-isscston^’aus-’sresisivqs tcre re - ' < ' c c -, | 9 |
8MP-C | 0-1:1-:--1 ocx? uxsscxr·? re.wecntorewinre:.: ’ Asitoysr-cYifeoirrrefwii.cci-icns’iireA.rrey:”.,:??··· ...... --......' .....·.....-....................'......i...............'·................................................................... | 10 |
BMP-D | QAiH,.·. tCWi'to.,-> > ;i. go-iec., «re^retA’- c ΡϊΑ,ο r i- seremtWi :Rto-1 -tow. s * \ t , v re- ,. v * - | 11 |
2017200239 13 Jan 2017
-ΒΒ'Ί | SEQUENCE | SEQID NO |
BMP E | t x ' , '5„, 0 ,,7..,.--te'.'A A-·-' | ta |
BMP-F | Q?au5K0?£KBcs<:crim&'iteV'>:tei:i'.'«umA';iiH?KGXAAi'XCs-iGiscs-i: PUAiXiuteTNiSArcgrAvo ,. Γ.-A··.··.··' '' : ,.v.....,-,-- ;·'·,· ; ., ·'?................................................................. | Ό |
BMP-G | 0ΑΚ5ίΕ^Κ^ΙίΚδ3ΟΚ?ΗΡ^Λη;ψ30ν®«\’θνΓΙΤΑΡΧ^Λ?'ΕΥσΗ<ΪΚΟ?ί'ί·υ>,·>5ί8Ν5ΐί»ϊ<\ϊν<>τυ\!5'? :Y W«x;ΡλΑΟΟΥΡο 055-7: : ,?rDKii!Vΐ · .Z5-VOb}iVVSG''C,C?; | 14 |
BMP-K | 0ΑΑί:70ϊ'Α?ί,ϊΙ.Ϊ?0ΐ;57?Ι.770Γ7·75Ο77ί:?;’ΐΑ:·;τθ7:-5Α3:7 '-HORS'?? 7'.Ο':ΐ?Η:,βτ375·,'3':Α.ΐ 0-^^3..75, ' - 1 - Λ ' '' ο | 15 |
BMP-S | ί,,Όο?;: ?;::.···Si:-. :-.5::-:::::,77:,::<”-<;,α7-ο:α: : Λ οοο,αανχοκ;ses· ?ί.Λ.::>;=.:.ν·7ϊν·<-, ::7C?:: ί,νκ 57070. rXitecvp-Titei-ix r 775,700:5:-5:70-,.-; 5?.χυ??βλα:'ϊια0Ρα | 16 |
SMP-J | , . -, V 1- - 1 ' · . A 1 χ' - ' , 55:5577577 ?K?CCr\PTULMAt 50i0iY?OK5x?7Jx7V5^K7<5i55VV55OCC5 | 1? |
BMP-K | W.\573A,7 7-,5.i'A'’, οΙΟ,ΪΟΡΟΑ ΌΧ'ί- C 7:057' / :\χ,ιΟ,Λϊ' 0 ,3k--,' 5 s'UVix-OxCXS.' t x V 575 '» - ’ f < ' - ' , ' 7 Kx , 5 -, - | W |
BMPM | 0' KSi.S C-AP P.O.KS SC:<'®Hf<:,77CPS0\f«VS'5S5<?7APSGY!iAFYCYiGnCSr?iiAS>iiiS'i8TTKAiVgYiAS'i ,- ,,.- x’ , - | w |
BMP At'* | ' , O' 0' ' 1 ' ' ' ’ ' ' - ' ' - ' v ' ' * - ' -- | 20 |
:44' AP | gtraWrStSSSC’SiiHPuyv»e.Sb-.teWbO<.’UAe:<riAAi.S?CHG3PS-i· P0Aiif;I5i8T58teSV0tei,vy ASI'T ?i<ACCV?T£5SAi355Yi,O5ii7i:777S5Y6OA77£7.0CC? | 21 |
Bite’ .¼ | 0 'Ktii:75?75ASSO-\?S357?75?75Vte0N57IiAF-A!?7.A:!i57OS557SC75?.i505}5AiS05HAI7575^7i'i ,- ,,, 1 ·, , , 1 ’ | 22 |
BMP-A:' | ,>Xi:HKOr.Ot^SSi7XRh?i.W»FSDVSMO?tIAFKGVA?OJVCHG£C»F?I^nH5t5S:TTt«A.tVOTr..Vrj N'SS : ϊΎΑΟΟνΟΟΡίΥΛ.ϊcm:.,0 5.:05:70,κηw^noVSCCYOP | 23 |
BMP-DP | QAAiU;-/A:: foomso·' 50-ίΐ :,χ·7:05-7::θ77Οχι:-Υ00/Α;γι0;θί;Α!:'ΟΟ!·5Α:70ΐ·::·?:,5Α!·5:5?··Οχ;ϊΑϊΥΐρΐ·Ο7:: - ,, , , ,- | 24 |
8MP-E9 | QAKHKQKK? ICG 5X0x37} ίΊ.ΥΥΪ!?'3Ο\:·8*ϋΥίίί ΥΑΡΡΟΥίίίίΡΥΟΑΑΟοΡΓΑίΤοΰΟΧ/'ΤΑ·: 5777 Yg'VJAYi XX X -- > X | 25 |
BMP- Eli) | .,5 -sac?u.- ,-,οοί-,;λ,α, οο , ' όροο,ό, - ο-χ,iο,',;- ο τα ο t ο ο.....,?.,, ?;Χϊ-,α;. χ-Αο , ο u; NSC5A3 55 A CCV 0 V £ 7 SAI. SKL s 0 5SJ, OSCWO,· KfiYg^/VKCCGCP | 26 |
BMP-BK | OiTi’.KKOSKRt.^SSCRfxHPOAVOFSibVGXiJrAitVAFPoOYHAFYCOGFi.'SPST.tiAHt-SSATtSKA.tVOTT.VH , x x , XX 1 ' ' X „ ' 0 | 37 |
BMP-E7 | 5055:0:^:7:50:.0^5:.:.5::-:0:,,-7:0:70070-.,::,5::7:: vA-’iCsYf-AsoiCc-OiBCS·: PS-NXi-KNA 0-:-:5- ::77::5,7:: > --- x' x 1 | 28 |
BMP-IP | 7070-::'COOT;- 77007.70- 53 PLnmFSOYSo^Oii’.'tVA&EiOYSiSf'YOSteKC70-7UA^tetSifS-YYiO’QYUyN ' ,'Vx- ' - i , , , , ! ,1 0' ,1-x- ' | 29 |
BMP-535 | Q?,Yi3i\OAi;A5,?SSC0;5H?:.77O?SexXOp^5pS:::;.yipp.G7fi^s7ys:Gj.,;055.p£,;iOH5B?7f.iS5A7.35JT5RS:: S5-:-i?5:sr?P'O077O55:.SA5:5N3,7i:.5iR3xS:K-;V5..57f7QO:-r.JV5O0GOS | 30 |
BMP-ER | OA5iS<SO'5«5:i:.KS50-<-;’Oi75705iFSiOxfGv;N>:7;?7APi:cOYi5AFY055G55.0SrO-IO\:>t5:0-i50-50iAJ:VOO-3AO: X - XX - X X | 31 |
BMP-GP | /--,5-:5- x-,'. ' : ,;>A.',·','O' ' i '·)'.·' ' >1 O'-.'x-,!· :: S; ·»·.?: ....A- STOSS.:;· ARAOOViO'ROOxAOSVi-APOOOS-OOT OSOTVOAOWSOCOiOP | 32 |
BMP- GP | , ' x - - XX, - x , 1 ' X ATOSi' Ϊ SKAxWi'TSWtAi707., 000035.-350, L-Kt'-YgOSiVOteACOCA | 33 |
BMP-GK | QAtOtAOEKA!,55550i:KfiP0YOO7SCi70-7:?OPIO 0A7RO7AA5'70550000Sf7£,AO5O,OS5KOA5OAJTO.OO: 575-585: TA?ACOVST53,50;AXS7O«7?5iOOTS:77I 5.505-: Ϊ0 0500? £0-000? | 34 |
BMP-GT | t, X x x> ' X - H f- xl -V I· x- x -χ 7-1 ' H ? ,' ι - 5 οχ ο, ;·· :: 5:A;0OO'/:-o'i:?:.5-A: :ί»ί.·7::ϊ>:3Ν5ον 5 :,:::00-00:-:7755: ::000:0 | SS |
BMP-GB | .- X : :--,- ' , 5,705? KYVPi^OOASOOt.UATSVtA-YOOOSi-iOOO.OiAteSOYVSOOiOCA | 38 |
BMP-GE P | 1 X- - X - : 7 χ -ίΑΟ-χΑτΟΫ'χΟΤ-ΐΟΟιΟΟΑΟ’Ο-ϊϊΟ-ΟΑΙΪΙΥΟΟΟΥϊϊΥχΟΝ'χΤΤΟιχίίΫΟιΟί-ί'Λί&ΟΟ:-:::-:.!: | 37 |
BMP-JP | ΟΑ355ϊί\Οΐ:ΚΑ!,ΐ:550θ;Α5-ΟΡΙ,Υν·ΪΟ?5?70ϊ'0:5200 .00707 707-07? vCJOOOOOiteiOsAiOPSsATiteA.S 777570 3AS'3?5:7V?A75CA7755:.:'3ATS7?7FO?3x5iiV7I.?5te0T>:i'.?7?O05OA | 38 |
BMP-JR | O' xt t - 0' r XX χ -x 1 « x'xf , 5'- x-' 5i70-o:;Vi-:; C-o 5 ? ;-:-:;,:.!A:i 57::.7 οοοοίογο-ϊ,ϊο-ίοοϊοχοχγ,ίΟΟοο? | 39 |
BMP-JK | i χ- ' : x ' ' - -' x - ' v :α\'-·-5χ'7- 0' 00:x' 350 \7,tt\ 5 ,7'>?t:t;'',7 '·? '·7-χ :- C-OP,,-?' | 40 |
BMP-2T | QAKiiKQ? 55555:5Si'-::i<5:?3,77:5:teTOA;7Y:?J0,: ocoooo a? oc;ooocoo: ?u5Siuspi«rr5iA s vqoavu 57-te?S7V?S?OOAPT&:::OAlSV5?7S5x55-?A05ix?705m57iAOOO:?i: | 41 |
BMP-A9 | 0θΟχ5-!.00:0000.Οί:0θ:θθ:ί:7;,07Τ0Χ00:70?0?·000:0.ϊΑ?>:7:θϊΑ-:'0:0-:&0ϊ;.005-'0.:,;000ί.0ΐΟΐ0;ί000,0·\?00ί007;': 77050:7 0 PKAOOVPT S5SA-7:OI,7550S7:?AV7550xYOOS-i773xOOOOR | 42 |
2017200239 13 Jan 2017
ΝΛΜν | MUBNCE | «id NO |
8MP BV | ^XEM?OfiX?t,KSaCKJiBPf.Y?toFSnVP5'.’t3r;!'!’νΑ?·?<5ΥΧΑ?ΥΒΟΚ?ίΓί·ΙΑΟΗ·\Ο.ζΤί·ϋ’:Αϊν·0·?Ι,νΝ | 43 |
BMP- PPP. | ................................................................. QAiYPAC-Pitab?SAP”'iiWus ; Vi- PKiliiSA? 3f ChOKCSf ΡνΝΑί;ϊ<Ν';'3'3χ:Ά4. 3 vgYWV-3 i.iu\!PS?V?'P PCCAPTSAito ISYAPK-AAA3VP ΓΰΑίίΡΑ3ΑΑ VitoCCCF . | 44·'··...... |
BMP-GP | 4-?iiK0KKet,SSSCKRHPLVVerSC-.-Q>e!C>it 5.A.PA&? SACS Oto SC?!· PUtoHk.NsTNtJAiVyTi-'xto ' .. ( ' I” < t - ' ’ λ p A ' | 45 |
BMPB22 | <' 1 1 ' , A Ϊ O'' X “''V ' v I \ M \ „ I ' 1 - \ - , - | 46 |
BMPSS9 | C> rum·' CKV?t,x$scq5< st.?ws,5>cr-S'fosi'!x r Apr eyaa vAyttoACSPAPWAHEPiATfiYA s vqtcvk A.\3x': Ύ ' P A P H A'.i ''V . P'A >s V'S | 4? |
BMPQA K no SAC4 | QAi'isAY?ΡΑ3Α35ΑΡΟ“Γ5 Stomp AiiAOACSAi ΪΑ PASS VAX PCJtovCAP PVAiJPV!. pyaaa3 VYYAVP a U χ' > p - \-u x u s — „'>i'' - | 46 |
BMPOAKS A GAG | QA!mAQs34Si4ASA3ASS;40X:33i5:£S3BMaP2aMSp52AiB;iS3SABd4Ae3Pi?&SA&iAiPS'5A8A 3 £XraSiiA?S34®25W®!-;AefiW’5’KiiSSBABl,AAS3S'3SV A:ii4y.i4B3SiSVA55Si53®X..... : | X |
BM'P- GPP | ' ' X. ' ' 3v3. V- A P VHI POCA PYSS35AXSV5W PO3A3SNVXί,-ΑΝΧgXSKV'ASueOCP | ® |
BMP6- BA | VxSSASOTOS^BPKTAC^HKb’AASFOPiZ^Q^'ira-FKQYAAS-iYCPGSdSFS-UnAAi^^ieUklvriTb vmT.1tm.:-s'.A.V:i';<pvc\\PTK'VA>..5svf.A'raiNSS'A:t,KX'.!'Pixto''AWxCOVH | SI |
BMP5- SL | VAAAYPi3xSRPA3'iAxPPAS-.iVY!to£3:.<;Y2PA:T ·ΛΡ;;ΡΑ\..ΛΝΥΡί;ΑΕνΑνρ·;>.!ΛΐΑ.ί··Λ?Ν··{Λϊν2·Α. Η ' ' ' 'P \ ' ' ' 1 i i ,' --. Ά Ά V ' | 52 |
SMP6A | SASS?PA3QSAteSr03QC'iAAVS$A2OVNSSSAP'XAppiiAi,.XVSPQaX.pAges<X’APAPAAANXCD V ’ χ i. to χ ' 3 ' \ ' ' ' ' , > A Ax ' ' ' A. .BABB.................................................................................................................................................................................... | 53 |
BMB6B | .... .',.< 3SxtotAY AATPAsAYYto to\xP\PAi to PAxVA\\tox33, OSC?'jP--P,AA'UP^S'K.AV\XVS'TPA’PiSViXSPXPto'A3V;5--pAAmA,AA,AA''rSi3’ASi?V.ii,.P.AYFNAVV.S.A;'A·; CH.. . . | 54 |
BMP6C | SASS?rti?o<3Srv>.5’«S'iQSS>n’?Ar<s?SSA$f.;yr?ssb-K?ACPxksiJYVSFOPjpfjA’Onw;'-/AP?-GVKAP’ynD G?.Cd? Pi.-NAHAPA'PNAAXVQ? ϊ..ν”<ΐΑΪ·ίΝ?ΚΥν?Μ:ί?ΟΑΡΤΑί,ΝΑϊ'3ντ.,??ί''?:,·ΓϊΟ$?ί'ντχ1ΚΑ¥ΑΝ«νν?Α<2 ‘to;--............................................................................................................................ | 56 |
BMP6B | PASSAAPQgilPiXASiQSQWAPPiiXiiSAYtiSASPAVACAAASUYVSi'geAAtoovji iAi’POYAAAYCP !’ΑΧ<?:ν;:ΡΧΑΡν’ΑΑ.ΡΤΝΡΑ5'ν^·ΤΧ.ν·ΑΑ·ΑίΡΡ';·''ΡίΑ·;'2Αϊ:&;Ρ3ΑΧΡΑ;χΧΡ·Α5Ρ!νΑν;ϊλ,Αΐ··ΧΪΑΡΓΛ.'3Αννν CBv v v v v ...... : V | 56 |
BMP5 ABN I | νΑΑΑ5»?ιϋ5ΧΚΑ·<ΑΑΑΪ·ηΑΡ.?νΧ?'ηίΑ,ηνςί·Α·:ΧιΑΡΧΡΐνΡΑ··<ΥΡΌ'ΪΑΡ2Α?ΧΑΑΑ;Ρ·ϊΑϊΑΑΑΧνθΤΰ HAT χΡ χΡ'Ό t ' V '· ' ' to' - | 57 |
BMP6- BK-KB | νΰΰΑ3ΡίΑ3ΰΡ!:ΑΪΑχ:··!'Ι;ΑΡ;νΑ?·Ο£;ΐ..ΑνγΡΑ;ΡΐΑΡΛΡ3Α?:ΑΑΥΡΑΑΛ<:ΑΡΡΐ.ΑΑ;Ρ··?·ΐΑΤΑΚΛ3·νί>ϊΡ YHtoStoAYVAP PCCAPVV -Αχ'Αϊ SVr-.YPhC-riSNVX ixAAYt-i-ePAtoACCiipA | 58 |
8MPS RK-KN ABBL Song | X Ax' , ' ' ., ' X ' X 'totoiNitoPVi-P PYC is P Γ P VNAA'SVSAi PAtoiSNVSAAP XANAVVAXAVXCP | 59 |
BMP6A BK-KB | x ’ „ χ' x \ ' ’ ' ’ χ -- X - xx \ <3CASPf‘'-'“ONi.xX3.STm’A t ν’ς-'·''.ΡΡχ£.;'χΑΑ·Α·Ρ«Ρ3<?Ο'-;'ΑΡ'Τ·Α£Α;Χ rgvLYFnONSh'.n PPAYPia-PtoPAC >:>;'i;. | 60 |
BMPS ADBL long | SA$A?PP2C'SSN?$rP$QirVAeVA$ASP>yAS38i.3V:-A<:Aim5V WAAOQXAXxgeiPT ΓΑΡΡΥΥμΑΝΥΟΏ C-X:P$rX--XUV!AXAA'Ptt AAxP.’A'X ν'.Χ·-·;ΧΧ·ΑΆ·£ΧνΑ:Α!·χ'·'ΆΑΡ'ΧΑΧ.ϊ3ΑΧ OCNSiA'X UAAi PNN:/”iiAC GOto : | si |
BMPS BK-KB ABBl | χχ' X X ' Y X X _ > - ' ' ' t, X „ A X * x ’ A'x XX X I f· X AOtox < : | 52 |
BMPS HK*KH onq | a' > x ' ' Xx' 'Χ-Χ'.’''$χν-νίΧΑ5-Α-:ΑΑΧ«ΑΑΧ'·'Α'Άχ'·Χ·-;ΧΧ·ΑΆ·£ΧνΑ:ΑΡχ'·'ΆΑΡ'ΪΑΧ.ίΧΑΧ X'AA'P OANuitoi SPAY PAtotoxAP* GCA: : x : | 53 |
BMPS8- BK-KB | .X-ΡϊΧ Ρί.γΧΡΡΑΡΡί’ΟΧςΑΑ'Ρ' νχΑϊϊΤΡ-'ΆΑΡΡί !V:-J..;\pPSA '·.'.- ·:0Ο·:;',λ<··λ'.···· · APA'A AANYC- P'Pito'toi A,x 3,- T A A~A A- ; to'l' to xtoi- A t .xA-' to 3; A P„N«3 A' .... s 3- toY toP'P ’ ;. P AY 113 '!'· A PAto's | 543: ; |
2017200239 13 Jan 2017
NAME | SEQUENCE | SEQIO NO |
<;Ή· | ||
0Mp9C2 | - X X ' t ' , .. --,, « X ,. .. ' X | 85 |
BMP9E8 | A - - \ ' , λ ' , , 1 --------J....... r\' e w: κ.,-π,' .-.v,. rt„o a ..-, .............. | 68 |
BMP6- Short | ---- i q e χ s>„ ,.- 'κ'’<χ’ΐνχ' x VKtzereervaeueeAurr.t.NSTSvbi.seopsu vrf.n-iYAmiYvaAoccq | 67 |
BMP6- SA | i x- «' x x ~ -. ' ' νΐί12·^·.Γ?ίΥν?ί1ί<ΟΑί,';Κί.?·ί»3·9ν!,ϊΤΕ>ΟΝ.ΐ,ίΤ“?£.?·ΚΥΧ^·!'Λί?Αί%χΡ?ί | 68 |
BMP5- SL | VS»isSOy»£SSdX'f SCKXftelt.T.rSFgOPGfygP'XS ΣΑ&ΚΟΥΑκΑΥΟΟΟΚΚ3Υνί«1-!ΑΐίΙΑ-ΐΑνΚΑΑΫ52;ΫΚ vni<t-k«Y£YvncxecoAeiPi,uAtsvi.Y?'ooNSP',xtiw;KYPiiN\-viuc;-;qei-.· | 63 |
BMP-L· NR | QAi’HXOrKRtKSSCSRt-mUVVOFSOU'nWtOJt^fAFK'A'fMFVnOGSdSFeU'SteWfATm&lVg^W ϊ^χΦΖΥνΓχ5\;-χ7ΡΑΡτίΐί.:χ?,ΐΥΚί'..?ε.η?ΡΐΥκ:Λί?..·<;?'?0ΡίΓ.η,ί'ί:ρίΛργ» | 70 |
BMP- GE.R-NP, | qA.Ki-ii'Of:K?us.->aex?m ιυ.υι^ρουαυοου:; rAs;;'0YAA;;-Yr-'ixciY-5io-;A;AAi.s.;A-:'m;AYVq-rEw iAe!?eYV?ct<eA?At:i.?n-A;SA:i..';'Yni>yoi:i'rx;m:O»A!'YeSxPCS | 71 |
GMP-E·· NR-6 | 0AieiSQKK?t«AS305xAimcy\m?'30VfAoiOYxVAAedYPi,eYCO03seSAC’u%w%YiatAA%ds%w ' ueei?PYv?AiCd«'x&'C Kerris s s vuyfconsov t cr ΚΥΑΟίΊνν-νΑ'ΧΟϊ | 72 |
BMP- GER- NR-6 | v ’SKIP' Yl- ΓΑΧ,-Υχ’ x? UV'„ A,'--·- SOVPPl-lPy?I · A»KOYAAYYeqOSqSKe-UPAi<iiPA'itetAXyQYtA?At ΐΑ'-ί-'Κ','' -.'ye. ΆΓ’!,' -y- ; -ζ yp ppi’SJZ.’l LKKy31?ervmiACGCK | 73 |
Althougl s the above listed designer BMPs cornpr;se embodiments o? the if :vent<on, the invention is not limited in any way to any specific molecules. Msteaid. rha Invention enoonipa&ses. any designer EMI-'·’ comprising altered receptor binding whore toe designer BMP comprises at least one mutation within a typs Is receptor tending domain A, even more preferably, iho designer BMP comprises iat least one furihsr mutation within a type I receptor binding domam mosf prefer,ably tne designer BMP comprises yet «another at least one further mutation within a type II receptor binding domain B,
In ofher embodiments·, the designer BMP ot tint: present invention comprises an amino acid •if sequence at least about ?Q%. 75%. 805(,. 8552, 87%. 95%, 92%. 35%. 38%. 97%, 99%, 99% or identical io one of the sequences descnfxtd above in another embodiment the designer BMP comprises an «amino acid sequence at toast about 70% 75% 85%. 85%. 87%, 90% 929.., 959,35% . 97%. 985s. 99% or identical to the sequence of SEC ID NOs,8-7J in yet another embodiment, tbs designer BMP comprises an amine acid sequence as set forth in i.s «any one of SEO ;D NOs'8-73. in «another fembsadim&nt, the amino acid sequence of the designer BMP consists cl one of lire sequences of SBQ ID NO&:8-?3,
Further, in one emdodiment. the designer BMP comprises an amino «acid sequence at least «about
70%, 75%. 80%. 9555. 87%, 90%, 92%.-. 95%, 08%.. 3/55. 93% 99%, er identical to the sequence ot ShQ ID NO' 12 Isa another embodiment. the amino acid sequence is the sequence of SEO IO NO· 2. In yet nnotfisr embodiment, the designer BMP is SMPE in an additional embodiment, the designer BMP comph&ss an ammo acid sequenos at feast about 70%. 75%, 805% gb%, §7%. 90%, 92%. 95% W%, 97%, 99%. 99% or Identtcalfo the sequence of
M
2017200239 13 Jan 2017
SGQ iO ΝΟΊ4 in another embodiment. the amino acid sequence is te soquenoo of BEG ID ,NO;14, in yet another embodiment the designer BMP is BMPG, in another embodiment, the designer BMP comprises an amino acid sequence at least about 70%. 76%, 80%. 85%. 87%: 99%. 92%. 05%., 06%. S7%. 98% 99% or identical to the sequence of SBQ 5 !G NG30 in smother embodiment, the amino acid sequence is the segnshoe of SPG sD ΝΟ:36 in yet another embodiment the designer BMP Is BMPOE.
in .-another embodiment, the desigoe*· BMP compnse.s an amino acid sequence at least about 70%, 76%, 80%. 85%, 87%, 90%, 92%, 05%, 06%. 37%, 98%, 99% or identical to the sequence of S&Q % NO3-? In another embodiment the amino acid sequence is the sequence of SPG :D NO3? in yet 0 another embodiment, the designer BMP Is BMPGER;
A designer Bs'dP of the invention may compass a fragment of any One of the sequencesdescrtbed above in an embodiment. a designer BMP fragment may compose a fragment of at least an uninterrupted 20, 22. 24, 25. 26. 27, 28. 30. 32, 33, 34 35, 38, 37. 38, 40. 41 43. 44. 45. 47. 50. 63 54. 58, 58, 50. 62 65, 66 79. 71 74. 77 89. 83 65. 88. 00, 9% 03. 95, 0? 99.100. %S2, 105, 106. ηδ, 112. 3 118, 117. 119, 130, 12% 123. or 135 amino acid sequence from the sequence of any one of the sequences of SPG ID NOs.:8-73, it is welt known in the ad that BMPs are often heterogeneous with respect to the amine and/or carboxy? termini at the profem That is, the presard invention comprises a designer BMP comprising an amino acid deistionrtranoaflon at the amino and/or carboxyl terminus comprising a deletion oi at leas? iQ d amino acid residues, preferably. 0 amino acid rasukiss, even more preferably. 8 amino acid residues, yet mom preferably, 7 amino acid residues, preferably 8 amine acid residues, even mere preferably, 5 annuo acid residues preferably 4 amino acid residues, mare preferably 3 armno acid residues. even more preferably 2 ammo octet residues. and stoat preferably 1 ammo acio rondo from the s,' uou or N terminus ef the designer BMP'.
In another embodiment, the invention comprises a designer BMP protein comprising an ammo acid sequence of ?iny one of ihe sequences of SPG do NO:8-7S .arid further oompnsing a deietion/fruncation from die ammo and/or carboxy! termini of the protein in another embodiment. the invention comprises a designer BMP protein derived from a BMP protein comprising an amino acid sequence of arty of sf?e sequences of SEG © NGs'6-7'3 wherein the protein comprises an ammo acid deletion/truncation at the amino and/or cerhoxyi terminus comprising a deletion of at ions; 10 amino acid residues, preferably 9 ammo acid residues, even more preferably, 6 ammo ncic residues, yet more preferably, 7 amino acid residues, preferably 6 amino a&d residues, even more preferably. 5 amino acid residues, preferably 4 amsno acid residues, more preferably 3 amino acid residues, even more preferably 2 amino acid residues, and most preferably I amino acid reside from too C and or N terminus of trie is designer BMP protein amine acid sequence.
S:B:!3ff8ALdeoign.gfBMPs.with altered roc^L^^y.m^y=3ted by.siyop^teti^
2017200239 13 Jan 2017
The data disclosed herein demonstrate that 3MP2 homodifriers pregused in E coii (referred te herein as Έ. ceil BMP2'}, which ere net glycosylated, ere less active than giycosytated BMP2 produced in :Γ>;δ?'(ίΠΊ3ϊί3η cetis, such as CKO cells; treferted fa herein as; OHO BMP?.). In addition, data disclosed herein further demonstrate that t cok produced BfvIPb hoinodaners are essentially non'functional 5 compered with BMP6 hornedtmers peadtjced in mammalian ceil cMture ' he eeto >{ setosed ae < !t d< mo tot ate <b<.j met ? are ·.-> jr toon wr mfor m h!i pf Έ?. coti BMP? compared With CKO BMP? In -he tvpe I receptor binding region.
in one embodiment tire designer BMP composes an altered conformation mediated by gtycosylafic-n thereby affecting a binding motif that in tom. mediates altered blading to a type I receptor:, b This Is based on the present discovery that ,ri mammalian (e.y.. CHOt ceil produced wild type BMP2, D53 points towards the receptor interface while the KS4 pointe away from the r’cepfor. Phis is In contrast to E coii-prcdoced BMP2 when- the D53 res-due points away from the receptor interface and the H54 residue lines op toward the receptor, stacking against -s proline reside as illustrated m Figure 3. apparently acting as n doorstop.' in addition, toe dare disclosed herein damnnshsto for the first »ims best s CKO-produced BMPd, which is hilly glycosylated ano active, also comprise?; a histidine residue pointing toward she incoming receptor. I.e , a fvsfidine doorstop
Without wishing to be bound by any pariiotjiar theory, the data disclosed herein suggest, for the first fii-rse, that moving a doorstep residue away from -he receptor interface. can mediate increased binding bohv.-,n the shfh Imane ano ds «vseotcr ' he cate tudnor demonstrate that die doorstop rowdus b may be either mutated itself to remove the doors,lop or ether residues may -o mutated to shift the position of toe doorstop residue E orths?, the data dssciosed hete-n further demonsttute that other sesidues maybe mutated fa provide a 'givean tether” which then, in rum can orient a gfyean se;h that the tethering of the gtycetn will motioni. toe doorstop residue.
Therefore, in t-otno embodiments,, a designer BMP can be produced by inogrporsting at teas<t one .5 amsno acid mute-ion iftef affects the gfyean tether and/ot removes a histidine doorstop structure thereby providing a designer BMP with altered receptor binding.
in summary, in wrie embodiments the designer BMPs of -he invention may comprise ai teas!
one mutation In the type I and/or type II binding domains of BMPs that confer altered type f and/or type II receptor binding Ir! one embodiment, toe BMP sequence is engtneerec to alter tpe receptor affinity of
BMPs in order to alter and Improve the receptor binding and/or osteogenic activity of toe engineered or •designer BMP In one embodiment, this engineering involves identifying toe residues involved in type I and type II receptor binding and repfactng them to Croats designer BMP molecules that show, among other things, higher affinity to both type I and type toracepters toan toe parahtel BMP horn which the designer is derived in other embodiments. the designer BMPs of toe Invention comprise mutations that create a new atgsmne 'givcon tell·<' o> destroy on -.owt-cg one to m scape toe type 1 -'e; ?pter Lmdfcg de-mam ' not w tiie mutation fa an arginine in toe position two residues C-termlnai from the first cysteine, equivaienf to
2017200239 13 Jan 2017
BIB of BMP?, appears to cause -tie olyoan chain. to be tethered' cute -ha BMP surface end consequently alter the conformation of the pre-hebcal loop region compared with the wild type BMP that lacks the mutation, to ather embodiments. the designer BMP of the invention may comprise at least one mutation that alters, creates or destroys (abolishes) the “doorstop'’ residue that blocks typo I receptor Irom 5 furlher engagement with BMP That is;, the mutation of H5<f ;n the designer BMB, or a corresponding equivalent residue thereof. that is oriented in such a way that ?t impedes or increases interaction of the designer BMP with a type s receptor.
la some embodiments, the amino acid mutation affects the conformation of the designer BMP such that the mutation mediates the creation anti or abolishment of an arginine “qlycan tether otherwise h present m t-v, \ori spot osng wA; hco BMP ir soi m embodiments 'he mutation i-edv-de* an alter* d conformation which creates or removestobolishes a histidine doorstop conformation it? the designer BMP where such doorstop conformation ;s either not present or active, respectively. in the corresponding wild type BMP.
Therefore. the skilled artisan, once armed with the teachings provided herein, would appreciate s that the presence or absence of an arginine giycan tether” and-’or a histidine doorstop’ in a TGFf> euperlamily mamher may be assessed using any method known in the art for -fie structure; analysis oi proteins, including, but not limited to, the methods exemplified herein. Once tec presence of a 'doorstop'· residue has been .identified, then at least one mutation can be introduced into the molecule to reorient the histidine away -tom the receptor binding interface. Alternatively, a nictation can ns introduced that will (I create or enhance a glycan tether such that the inhibitory effect of the histidine 'doorstop, it present, is; decreased or, more preferably, eliminated.
in one embodiment. where the T'Gf-β superfamiiy member is BMP'?, the mutation that removes the hrskdfoc ooetviot,· is substitution M «'-oil ammo aciu Ms 1-54 In some embodiments the H54 w replaced with alanine, glycine, senna. or threonine.
S Although the present invention discloses such doorstopT-removing mutations for SMP2. tee skilled artisan would understand, based on the Imcwtedge in the art. how to identify corresponding mutations for other ΤΌΡ'β superfamify members and readily produce mutants lacking a doorstop.-' i.e . removing or reorienting a residue Inal would otherwise interfere with receptor binding by facing or projecting irtto the binding interface. The effects of the mutation on ptotem confomiation con be
TO determined using any art-recognized method for tee structural analysis of proteins such as, but not limited to, those disclosed horem Alternatively. mutations that can remove tbs doorstop end increase iigand binding to the type i receptor can be identified to s/ifoo using computer modeling method-, available in the art Therefore, the present invention encompasses ihe design of TGFI) super-family uwnix is having Improved binding with the type I receptor ;n that they ieert a histidine ’doorstop' usidue that would jT otherwise be present in the receptor interface.
Ihe present invention further provides ihe skilled artisan wite the Under standing Of how: te identify mutations for ether ΤΒί-β family members that wouid generate or destroy the arginine giyenn tether.
2017200239 13 Jan 2017
Mutations foot add this arginine glycan tether to a protein tacking foe tether art? centen-plaieq by Ihe instant invention. Therefore, foe present Invention encompasses, the design of TGE'n superfamly members having improved binding with the type i receptor In font they contain an arginine giyoan tether that alters foe eoniormabon ot the type I receptor binding domain, in some embodiments, foe removal of the histidine doorstop thereby removing the requirement of s _ wu i t\l 1 as nice'- e ce^ gno' EVE rut < an b< t c 1 -~'v t w u < --val c > while mainfof % biological activity. For example, designer BMPs may be produced In celts, with gtyoosyiahon activity that oi V'o t o namr a a i -. < -. «' * ·{ jrow n„ » e<s b t fo s- ' a s ->' < % < fo n ract - el --. or -J w mold cells in particular emt»odtm&nte. foe designer BMPs may be produced in E coil .arid maintain biological acbvify.
Thus, in some embodiments, foe invention provides methods for designing and producing BMPs ’h i rii be pn»f n t s md tl ιρι oi' v- y! Cwt - < om -η-,,ι i ihv< f giveo- yi fu»' '«< '« 4 tt tt altered giyoan is produced which dilfers from that produced by a mammeiien ceil. That ss, the present invention encompasses methods tor introducing a mutation that removes a doonstop residue that would 3 otherwise impair or inhibit receptor binding. The skilled artisan would understand once provided with Ihe teachings of foe invention that a doorstop residue that impinges upon the receptor-ligand interface may be mutated to entirely remove the residue or other mutations can be introduced such that foe residue is oriented away troth foe Interlace Surd? other mutations include, but are riot limited to. providing a glycan tether that wilt alter Inc? conformation of a glycan and foerotp alter the conformation of ihe ligand such ft that ihe doorstop residue is orientated .away from the binding interface.
fhe invenflen also includes nucleic acids encoding designer ihe BMPs described herein. Nucleic acids encoding the designer BMPs described herein can be prepared according to a wide plethora of
..3 methods known in the art.in one. nucleic acids encoding designer BMPs are prepared by total gene synthesis or by sitedirected mutagersests of a nucleic acid encoding wild type or modified 8MPs. Methods including templatedirected ligation, recursive PGR. cassette mutagenesis, site-directed mutagenesis or other techniques that are well known in foe art may be utilised {see for example Stnahov ot el., P'oc Nah Road Scj. tcS'A ‘33,15012-1501{1990). Prodromou end Peri, Prof, Eng. 5: 927-829 (13592): Jayaramat: and Puccini.
Sib/eobotgoes 12: 392-338 (:932,1: and Chalmers si ah, B/ofechnigues 30: 249-252 12001 !
Thus, embodiments of the present Invention esc comprise nucleic acid molecules that encode the designer BMPs of fhe present invention. In certain embodiments, the invention provides a nucleic acid molecule foot encodes lor one ol the amine- acid sequences oi BEGS ID NOsB io 55.
in other embodiments, the nucleic acid molecule encodes a designer BMP protein foot comprises ah dfoiuo acid sequence at leasi 70%. ?5%. 99%. 85%. §?%. 3952. 92%, 99%, 9433, 35%,. 99%, :3T%, 9519-., 33% Identical to the amino acid sequence of SEC IP MG I? In some embodiments. lbs nucleic
2017200239 13 Jan 2017 acid molecule encodes a dossgrier BMP protein that compdess the amino ao«f sequence oS SEO ID NO: 2 in another embodiment, the nudeic acid moiacuie encodes the amino acid sequence of 8 MPE as set forth in T ania 8 in other embodiments, the nucleic acid mofeouie encodes a designer BMP ercte-n Inst comprises 8 an amino .acid sequence at least 70% ?5%„ SO% 85%, 87%. 98%. 92%. 93%. 94%, 35%. 98%. 97%,. 98%, 99%. identical to the amino acid sequence ot EDO id NO.'M. in some embodiments, tbs nucleic acid moiscule encodes; a designer BMP protein that comprises the amino acid sequence of SEO ID NO.14. in another embodiment, the nuelesc eod molecule encodes the amino ac;d sequence of 8MPG as s% forth in Table 8.
In other embodiments. the nucleic acid moiecuio encodes a designer BMP protein that composes an ammo acid sequence ri \a 70%. 75%, 80%. 8559, 87'%, 90%, 82'%,. 93%, 94'%. 95% 36%, 97%. 98%:, 99% identical to the ': i acx; sequence ol SEC1 ID NO'39. la some embodiment, the nucleic acid molecule encodes a designer 8MP protein that comprises the amino acid sequence of SEO ID NO36 In another embodiment. the nucleic oad molecule encodes the amino aoxf sequence of 8MPGE $ as set η T able 8 m ofner embodiments, the npofeic acid moieci.:ie encodes a designer BMP protein fha! composes on am nc ac% scsqocrcc at raw AT 7h% dm 8s% 8/ . 3? t % , 99 . et% 9% 9*9 S'* 98%, 99% Identical to the amino acid sequence of 5EQ ID MO.37 In sense embodiments. the nucleic acid molecule encodes a designer BMP protein bed comprises the smino acid sequence ot SEO ID b NO:37 In another embodiment the nucleic acid moieouie encodes the amino acid sequence of DMPGEfd as set forth in Table 8,
Exemplary nucleotide sequences encoding designer BMPs ere set form i> i Table 8, below. Table 8 shows the name of tlx; protein encoded and the uucfeohde sequence encoding that protein. In general, the mature protein coding sequence begins at nucleotide 84? of the sequences listed below
TABLE 8
NAME | BEGDENCL | SEQ 1D NO |
SMPfA | .,'m:.<j'mi.-,i.,<.i<.<w?.ec:i-..i.-.ec,WiTCA'rOCie:C,i,.-.c t i-.-i..'>aa., ·λcc,ci.-.eoi.?χόοαoo>-<ci-..ec,*ouci<ai<csoc- CWaty.’iAdOTi.'C^AirCGAGTTCOAdTTOdOOCTCO-rq^r.r^OTtCOfrOCTPSAACASACb’.Cr.'C.i'.CC cccAc.cAr,jooACbccf»'tqctociXc<'t?i».cAiqcT.wAce?«bT^'rqr»cArijri>cACTCArjmrqAG<x6O'3 0rCS<y;r%,cm'.CAi%cccA0%'sst'n.'aS%:.%'%.'3C.r%qTaqcc<ov%7CAAi'.'SCT0%;r%%.%0'T-.'':'%Ar.Y. < A - 4 ' r. ' ' \ a t.} v-e? \ * i ' k -''.a.’· .^•tsc:rA7CcccAr;GqACQAi7j?rA't'OAc-qTdAqcA;%v%?uiCukOGTutT%cOAq?^CAqA'rrxeAb& C‘ ' v.% i, 9 \ ' C > 'av'' sv%'tC <m ΛΧ k '>' ΟΟΑν.':ί04λ^ννΓΤσθΟΕθτΤΡ\ΓΟΑΡ;4ρ^.ιττ^ΑΟ»<70ΛαΐΤΤΡΟΤί·^ννΤ<^ΟΛΛΚ':ΟΛΤ·.ΟΟΛΟΟΤΡΟ ο.,ν^οτϊ'Λ'τοΑ-Λ orc a;>?r;coqd70T<^TCaacyrcoAr;TCOAC.^OAceeor;ci\a/;ci1TbOATii'tx?r G&K;mv.r.;TOGCdOAi.’TKY:;a,GA«AA,^^^^ TCX.>^CCrOtCATGAAC?si\AfX:T«570ACAr.;ATAAfX1CCATTCf.!7e«5AAC:rirrTOGC7sTCS'rqfii:A?’.AA r^CK’fOCTen7CACA^!^%%GA.AAAACOTr;AA.'3iXAAACAgA&AgAOC3Q4AACqgCl'rAAO'?%qM C'n'5'rAA:'$“OAf.Agqc'rTTi3-3'Ar.O'?.:x'!*CTTCA.ytoAqo’Tqr5bi3i'rsGAA'rGAq4oqATeA'rTqgAccg& Ab<%c'iWrGC'%AAo.v<:nrACTqr;CAc..%AqAftTqcocsr;?i7<'r?rcim;oci.aArqar<'-;CvSAC-iO.'';AC'V .%%iO,%iC?C\'A'ir.Tt'iCA9isnqrr%'/iOAACrd‘tOri'A«.?rC'rAAGA'r-iC.'C'rAKqoc«r'ii’;rqt'GrcC'C ..., „Λ;. .. v--,.,-ξ v-C ;-CAOCAOAAI;OAAiiAOSsTbvATTAftAdAACSATC λ .ο ,ο „ ' ,,. , , η ' <' ,c Λ ” | '7 4 ; '* |
2017200239 13 Jan 2017
NAME | SEQUENCE | SfeQ io NO |
SMP-B | a' A ’Ί < -, < -, x ' „ , -··, X x , , fo , „ Xx -. -,.. x , , x i CC te?G ΐAGAKGAAGTAAGGAGG ΛAGAAA A A CG CGAAGGAA XCATG GAGG CAGC GA A CATC C A AG x'AGT CTiAACCJAGGTACTTAxGCAxAGT'rCGA.G'T'ifoAGGCTOC-rAAGGxATATTCCACCTGiiAAA/AAAGACACA.CA cccAGqAr.;sGz'iCfiCCG’ti;oTOC'.y.'qcTACAT<;cT«GAf.'rrGTS'?c«CAr.’:GCACTC.VA';'?t;?te3cc«SG GAAG'fo'GGxCGAAAGGAAGxG:T'TAAAAGAGGGx;'x<;i:AAGGxGPfo-ACAAAAGTG7GAKGAAG'?fo'GA.Gi;’ AtOAAO^tC’m^MOAACTACCAf)U<SJ».CG>^1W7A.«AACSAiXC«%sAOATTC,rrC?TTSA-m-A A'feT-TC-rA¥CfofoACG<jAfteAQT?'rA’SCAC;?Tf':AOCA;'5AOC'’iCAaAT-r'r-TCrXfoC,Ai,CAi'iA'5TjCSAVA 't'QCT'i TAGGAAAG AA't AAASOT'fo GAA i CACCAAAA ΤΑΑΐΑΤΪ TATGAfoAX CAX AAAACA AGCAAAAA CAAAATCGxAAATTCCCAGiAACCxASACTXiTGfeifoACCxAAG'i teXGT<SA,AT-AAGAAi.GCAAGCAGGTGG x.x'-Ax::·', A:' CA. Α\-ΑΆ’<: ΙΑ,ΑΧχ.Χ,,,--,. χ,Χχχ.-ΑΑ,χΐfo'APxxCGA λ <Α:Αΐ, £w’„Αχ,Αΐ·:·-χΑ·χΑΑ·.<χχ.Ai,.fxvAAl,-’k,-AGk?G^T-A'G-X::T: ^TCA^A-mGAACAGT'WAAGAAGZkVxCxWAX'iATCTCArxAAACAAA'xtePrTAATXATAxACCxAiXC'fo.'TT TGx ACCAAG.xVxteAA.foAAGC?A;G'?-CAC'.te;A'7AAAxfoAA'foAfo7AAGAAAT'TGTGGAAtefoA'?AAAxAAA fAtex'ATCCTC'Ti'G/^CAAAAG'AGAAAAA.CtjTA'x'xtA'GAx'zi.AAAACAAACACsCxiAAAAAGCGTTAxAGTCCAx: c A'foA A;') A AAA .5. A ifo TT TO'T AAGTAG ACTA GAG ’foAifo AfojCfofojOA ATGA Ί GOAT TATGAC'rGC AX' ' ' foC t > Cfo -, ' \ '>χχχ ; G'fo ' λ 1 AATCATSCCAtT-ST'iCA'jAO'jT'rWiCAACTCTS'yrAACTfeTAAi'jSTTC.'C'TAiiOqCAreirTC-'rfji'CCC· GACAAAAAAAAAAAAiXATAX GAGA ΐ<?=·ΐ ‘AAA T i'GAT A AGAAAGCA AATGAGA’teTVA&AS XAXAfoAA - -. ' x x -, | 7S |
BMP-C | '.x < x < X x X 1 ' ΛΧ txfo'lfo XXX > Αχ-, fo X CCTCGTTCTOAAGATGGACCACAtWAxixGTTGGCfxGxTifxCGTCGTAGGGCCCGACCTCATf.'CCAACGCT CTGA.i.'GziAiGTCCTGAACGAGTTCG'AGTTGCAGGTGK'jCCAGCATG'TTCGCjxACC-AAACAGAGxACCCi'xCC GAAAGAAGGAACGAGOTGGTi'j.'foGACGTAxGAWjCTACjACCTGTATGGCAOGCACTAAGOTGAfeAGGqG r.-tCAf.ccij'.ccc’AOAccAcr.'O'STTOfeAos.C'.sOCAqccAqreqAoccAACA'fox-Tocqi'AixTTCcAcc A'rG.M.tXA.ATAWiGGAAGAAAjAGAJ'xGAAAGAAGTiJGGAAAAGAACCAGGAG&TTf’foTG'iTTAATTTA x t ' '< xx « - x'- x ' x -χ,χ, χ'χ- TGCAT ' AGAAAAAAAT AGGAGG ? TAAte ϊ AAGAGAAT 'i AATAG TTATAAAATA fo AAAAAGTAAAifo AG GGAGxGX'AGAAAXTxAGGATGAAGAGxAA'iTAGGAG.GAAAAGAii'i-GiATGAA'iGAGiteTGCAfoGGA.AGGAG AiAAAATGTTGATG'X-CAACCAAGCTGxAA'TAC'teTteCACTGCAGAGGAACAAGCCAACCA'fo-AAGTAGT GGTGiiAxAGTGGCCCAGTT&GAG'GAAxAcW.VxAGATGTATCCAxAGAAACArGTTrxGiGATAAfJOvGAGCTT TGGAACAAAi\'TAA.AG?,AA-GATGxn'GA-GA.GAfoxA-GACGAT-TGGT?xG'?xTiGTT7foGGG?,Ti>rATGiGAA;'xA AAifoATAA'r-rxCCA,A;'.AAA-AAA.AAAAA<^ATCAA0CCAi,ACAi;AAACAAAG<3Ai',ACAi;C'r-TAA0'rC;;AG AteteAfo;.teteCACGAT'GfofofoforG'GACTTA,fotefoCAteGGAte'-teAAA'?GACT;fo.AT?A'AteteAAAGA V'<!>' A. Α’Ρ,'ΊΑιΆ'tefoAfo;·-AAAtefoA 'fo. ί fofo\ fo,, te'te,CA,G”fo-Afofofoi fol - , fo “ \ fo xte fo , V fo, te fo’ ' ,\,„x x'A X- .fofo , , X AAC.foAixGGTAAATtX'CAGCTCGGTTCTrTACTTxAAGC-AArG'.GTCGxAl^TCATTTTAxAAGAixCTATC AGAAGATGGTTGTAGAGAGG TAGGGAGGTAGGTGA | |
BMP-D | ATAGG GA CCGAGAx ACGCAGTATG-CTAGCGGAAAGAATCCCA GAAGGAAG CCTGGAAGGAGCGAAGAG AA7AGT'AGAGG?,GG'fofoGGAGCAAGAAiA7TAGG::AAG;Gx;GGGAG-CGAGCAiGGGxATCATCGAPxGifoGT GWACAxAGGTCCGTAxiSCAAGTTGGAG'TTifo.Gxy.'TGCTAisi'JxGGCATGCGACCTGAAACAfJAGACf.O’.CC· iXCAccAiXxOAcoc'%rtoctGC:Xc<'t'?t».cAio?r.wAccT«3T^'T<r»CAsr»cAc^cAqsTCAG<xGo«3 CrCACCCOCAf.CAfiACCACO'A'^tejAAAAOC-GCAQ-CA'A'XCOAiXCAACACTSfoXOCAi'te'TTCCAfX· \ \ Afofo t ' ' ' x'x ,-fo fo fo A--fo fo ' Χ-te fo < x- x-· .GGGTAfoATAACAAAQAAGGAGG'te'AG'CAAGteteAGGAGxAGAG'foteGGTGGTAAGAAAAGAGATACAAGA X Gx foiTAKxAfoAACAA'fAAC AGTG'G GA AT x AGGAPxATTAAGATG TAG'GAAAG CAGAAAACATGCAAC AA GAAAATCAAAGVS'TGGGAG'foA'xCGxAGAxrGTTGGAGACGAGGGTGG’AAPGVAGi'xGAfoxGGGiG'iGAAGXri'GG x ,- - > -,- , ( , χ XX X AATAAXxAArGGAAGAAX'XAfo?AXAA«AAA.GAAA<;fo;XCTAi:ixfo:;;'xGAAATx:-';'XAAGATAXxGG?xGt/?Ar.iT , x X XX X . X ' ' X < ' ’ ' ' x · ' x’ ' CxGOsGA'fCCTC'rCCACAAAAg&OAAAAACO'rfXAA-SXAAACACA&ACAOCSrtAAACSCCWAAOTiXAC· C'lXX'X.AAG-A'GAiXAGACTX'X'G'TAf.G'AAGcGX'XCA.foGACG'XxlAGA-AAGx'.A'rGAA-XAAA'i T-A'i'GGAX'GXtAi' CGAAGTAG'GAAGGCTTGTACWXAAGGAGAATAAGA'Ai'Xfo'AACG CAAAGCAA ACAXGAAGGCAAGA - ' 'x i kx‘ - ' i. ' X ' < X -, , < X GAx'AG.foAAGA??,GGAAGATAGGAAGAAG'GTAGGTi'GAAGAGAixG<GGAiGAAGGTGGATTfoAGGfoGAGA'-fo AG-GAAAGAAGGATGGAAGGTGGTAGGTGCCAACGA | 77 |
BMP-E | ATGATGGGAGGGAGGAGCTATGTTGG'AGGGTGGfoTGGTTACGGAGATGGTACGAGGGAGGGAGGGTGG CCTC'GTTC'CAGAxfoTGGAACAAAGAAAxteTCGAGGCAGCAAteGteAGGxfoCGCACCTAAte'CCAGCCCT ATGAAGAAAGG>ATGAAAGAAG'G'-teAG?ACCAGC,'n:te'''.teAGCAfoGTAGGCAGGAAACAG.foAACCCACC CAAAAAAGGGAC-GAGGiG'AG'hGAAfo'AAGTAG.A'A'foTAAA'AAATGTA'i'TOAAGtGAAxCTAixOGTCAA-ACGAG ffoGACGCG'.GCAAGACCACAGGT'JGGAGA-GrjGGA’.'foCAqCCPAGGCAACACTGTGCAAAGC'rGCCAGC ' ' „ · fo'' ' χχ^χ’χχχΐ· i ixGT':,'ATA,TCGAA'ACGA'AAA?'GTTG ATAisCAG'GAxGA'A-GAAxGT'i'AAAiGGT'ifoACAAGAAAAAAGGCAAGA, p,-;eT'rx;7,f:.--:.VxAAAJ;V3'XxGGAG'XX'TAGAX'GAAAxGfoX''XiGV.r.iVX';'X.i?A'G'GXA'i'GA'Xxi'xi;AfoG-AAi‘.'foAAXxG CCAGiCTAGfote,x,te?AAG?ATGAAAfotAAforTxGAAA'AAAAGATGAATGteATCAGteVGC&xGCAi>GGx:G AAAAGT'T-TrGATGGCAACCACCfoTGGAAGAxGtTteteACTGCAx.AGGAACteteCAAACCAGAAA'TTAGT GGTGG.AAG'7AG<y.teACTTGCAAGAAAA.AAAAGifo'G?AT-CCAAGAAAC.ATGT?AGxterA.te:teAAiAG:CT’i' TOCAf.CAAf^.'TrAAACACA-X-XOi'iTCACAOA'rAA-XsCCATTGCTSgTAACTTT’rqOCCAl'OArOOAAAA GA’'foA'-'i:x'''foXCAAGAAAAOAG.AAAAAAATGAAGC'XA,i,.AAAi'.-AAACAAACxt3A,ixlx<Y3i'foT-t?,xfoTCCAG * ' - x A ! 1 - fo -- .fo ' ,, t. V ' fofo - | ?B |
2017200239 13 Jan 2017
NAME | SEQUENCE | SEG iO NO |
CAAGGTAGAACGAC3'3'TTAATGGGATGGAGAA'i'GC3AA3'rAAGAC3CAAGGAACAAAAGAAACAAz:CA A,<\ccic7GC:':;?<TTGTCC?AtA<;cATSd''iTAAccmA-R>Abv;:ccow\aT??:'GTccc?o'0iCi?G':A'j;?’iO'rcc GCCGACAOAAfATCAGTgCTATfATa.;AT-?:CTGTAC;CTTGAiX:kkGAA:?OAA<WX'?T'ST?sT'r<V’.Af:iSACT A A ?A: AzA'CAC· C ·3'·':.'·Α.'·.; ·<Α,·'·';':.G C ·. C-< i'CAAGGA | ||
BMP-F | AToyroggf. OQ-y acc.ao c?g ?cm cta©eg 're censer cr cc Asecrcc vee tc3<xcgaaa· At;ctcc CCTCO^CCns.->?eOt>XCi3C^<3t'jA»OT10XOO'XGCOTi:'3TCG<5'XCQCCC<:-rCATCCCAS:CC<?T G'eCACCAGCrC3eCAGCCAGTTCG.V3mCC&GCiW?'r<.'ACCA3CT-rCC<3i'XCi3A«xACN3AGACCCACC i3AGA3r373‘3CG3.C;;GCCrGQTGAAC;';G<rrAAArGA'rAGi!CC-K!rA'AC3GAGCG3t''iC3G-3i3'<AAGGC3rA5 CT^CCiXICvCeACiACCACCGG^rimxCAaVGOiCCOAGCCKSCCC/xV^VCTGKfCGCAGrf'rCC/Ay: AKACAG;AATf;eT'rc3Aa\GiccAA<'C?r3?ocr;A7icrc7GGAAiCv;3Cc;r:GrAAGA'rrc-?T':':ATTmsT'C.rA A-?:TeCTA-TC.?CCACGtC.CGArrr-~'TATCACr'T;AAGCAC<AGCTe'CAf::C'T';''reCCGGC;iA;ACAGA'?CCAAr:C TGCTTeGvrcAAACAATiCiCAA'rTrmAAecAcmsAAeeAC-TrrrTe.ACAAr'.ATCATArc.AiArr'ecAAA'i'c eCAACTiAViAAT'TCCCCCTG.Ai'C:AC;ACTTe''rCi.«>CACCAGG,rrCife»nAAeCAG?!ATSCAAG<:Af;G<S-f',A-i &.ΑΑΑΑ?·Τ'Γ·ΤΟΑΤΑ'Κ\Αζ0·:?;'Λ'Ο^7ΤΟΑ·Κ^:70^Α'Α3Α\7Ο0ΑΓ,'?ν3Οΰ&ί7Α'νΑ:ΓΑ'^ς3'Α';'ΓΑ3Α';'·?<'ΟΤ Ο.ΑΤΟ·'3Α3^Α:3ί)η,:'ΓΑΟΤΤΟ·3Αη*Α3λΑΑΑΓΑΑΑ·'Α'?ΐ<3Τ<;''.;·0<'ΑΑΟΑί'ί?ίί2Α'ΓΟ'5'ΤΑΟ<3Α';:ΑΑί$Ο.ΑΓΑΑ··.Α3'·'ϊ' 3‘ \ AWx - ' * A<> „ t m ~ V χχχ ' ' -.. » <>^CAiCCTiCtCCACAANV3AGWCUi^CAAQC?A4Mi<sAC^4U<CA0CG<3MAC3CCrtAAibtiCC^ C'm;.TZKAfA>GA<?ACCCxT:?IACG7<ZA>CTrmACmACGTCQi'A;TiA3^TCACroC:AT:iAT:mCA<;eCA ArA7CA':rATGC'K;AG'rT'rcc;TGi.>'3rsCQGAriAC<Kr;?c<7TT,r3'COGCc;:-c?rc:3ArcAri'C'X*AcfrecAr.'T AATCACJCCA'rc?:TTCC.;Acrrr-”i;G'rcAAfA'£CTfr?'rA<ACTce'AAGrr?Tc;:'?AAGGCATGf.'ef?,'?>3i.rccc GACAfAVrCTCzyiTGCTsATCTrGATOCTfATACCTrGACrkAKA.ATGAAiVxO'.iCJCTATTAAt’.C.AACTArC caca.v; Cy7-iCi»C'A-.>iG-r-Ge.>:r,7.vr:\3’;-i-;'jA | 79 |
BMP-G | - A ' χ ' χ ' ' A ' A ' X X ~ A ' X' CC‘T«'p37'ri?<'.OCAOCr<'sO<X:r.OCAGOAAGTTi70Cr3>.'»CO’3CO?<CfS?<>W3CCPCCCC,3'CAT<XCS’>S~CT ' ‘ C .. . · , ' C · , . ., Λ - v < A ,. 1 x< X < > - > < r;c\'AGCAGGQ?rOC'?Ge<2;'iOO'XCCfm,A;?Ae<5'7TAGAGC7CiA'S-roCAGGCAWCAGG'r';-AOC';-GO*3 CeCACCCG<Y:CCr\GACCAC<'GC-T'mGAGAGCGCA<Z’CAGi:GG?^eCA<\CACK:-TGCGCAGCTiCCACC ATGAACAATCrA 'ACGAACACAT AAAAxGAAAGAAGACCGAAAAAAAGCACGACAA KC ATTA AACATA AGTAi.'AAA':?GCxAC3GA£3CAAxA'A'rACCA.AAT~z'z3x3ACACGA'rx'.ACx3TAA'rCAC?xGA?xirAiAA'rGAAAGA TCCTTTA«GAAACAx>'TACC’ACrTT7CATf.\ACL«3JiTTAATAT3'TA:XCAAAT'\A?AAiV>eC:Jf;C;VJCAS iACisAA-eAri'AAAA'reCAzrrACAAxArA'AAnAAeT'r'rACACACCAG'jCr-ACrraSATAAfiAATiSiAiC'.CAAtAAtAAA Α'ΑΑΑΑνη'ϊ'Α:'ΑΑΑ·'ΑΓΑ.Α<?.ΑΑΑ·Α;;Α'ΐΎϊ'5ΑΑΤΟΑ·Αϊ·Α3··;;ί\ί'τ·?Αί','ΑΑΑί'ϊΑΑΐΑ':';ΑΟ:'Α'.·ΑΑΑί'.·ΑΑ'ί'ΟΟΑΤ··ΑΑΐ3Τ ' '· AA > ' i x. > A- ' ·, > i < C « >, *’ xx ·- . ’ 1 Λ ' \v-' > χ χ , ι Λ ' < χ A \ Λ' ! ,ilu> C»'' GCCGATCCA<'ArrAACA.CGAG)'.CAA\AA<tC'rGA.Ar33G^3'xCAC3xAAGAGCGCAAACCCGA'rAAGACGxAC CrGAAxAxx;AGAA,AGA'A'3''rA<rrAA'3’rCAAGAACAAriCAA3;CGGA-x3:'CCAxAxA<3AxAA-''3G37xA'AAAC:3;'.3SA'-3f.3'. aoa'iCTATGCTCCCr'nTrxCTGCCACGGAGAATCCCCTiTTi'CTCTCCCTCA’rC'ATi’iGArVATf'XAC'J' AA'KAATGCCA’TTGTTCAtSAi'GTTGGTCAACTC'KtTTzs.ACTCTAAnAT'rCCTAA^GCATfAtTfien'rCCr ' ' -, (. t ' ia<xv'x i r i n x · ' , ' x 'x ' 3 '1 xx Ί ' | ;W x |
BMP-ri | a t cm cccr. OAPAAACAcrGecre ATAoccmccrccr'rcccpAC'i'i'ccrcci'AsCC^ACCCQCOccAG!;· ' ί A X A ' XX X' X- » xA ' X * X- .,'·.< χχχ x \ , , X x x' < V x X- Vxx ' t \\ G» XX ' 'xx'l \ I xx x xx'A IxxC Ax,' ' X x λ-Α Αχχ\ xA' --. ^';-e;'AgcAQe«xkC’'A3ACt&rrr;cc<rccvAC.AtnATA;’$&-c<-ti3TMCccAcc;;-Aci-g«cctc.Arx'cci’sCx χ,-ΆχχχΧΧ. Χχ3Α.χ<. Χχ·ΆΧχΧ·Α :.·ΑΧ·''χ.χΑ;: .-·Χ.Α< :··.χ '?AA;x;XX:Ax.xX?xA,A< AA.AUx. XxXX-Ax:X. x-. ΑχΑΆζχΙ., '-ί Λχχν'ΧίΧχ'Ι-.χ χ. Αΐ-Σ. Χ.·'?. Άχ.χ-.ZXxA· ΛΤΑχνχί·ί.ί^·ΓΑΤ··7ΑΑΑ^'χΑ!Α'ΑΑΑΑ<'ΑΪΑΑΑ-ΑΑΑ·.Α7Αχ?Α7ΑΑλΑχΑΑόνΑΑόϊΟχ;ΑΑΰΛ'ΑΑ'Γ.Ϊ'Α'Α,Γ':';·Α·?ΤΑό:···''Γ.ΓΑ'. /v?!TTCTATCCCCACfA?A<Zb3CTT?ATCACx7-TCAfjCA.C<A«CTTCA&3?TTeex:GACAxACAG;A'TCAA':A TSC?Tez^GGAzAAC/e5.T,«5CA«TTTCCATCACC<5AAT7AATAT?:JA'rfA'>AA-rCA?ASAAirCTf;C'AACAi3 ΑΑΑ3<ΑΤλ”ΑΑ.ΑΑΤΤί.ΑΑΑ·Α:'ϊΐ'3Α'ΑΑ.ΑΑ;ΑΑΤΤ··ΑΑ·'Αΐ.'ΆΑϊΆ3Αί3Α'-;,ΤΆΑΐΑΐΑΑ'ϊ-ΑΑχ3ΑΑ·ΓΑχΑΑΑΑχΑΑί'{Ο'Τΰ<χ Α·ΑΑΑΑχ?'3''Γ3ηΑ'ΓΑ·Α':ΑΑ·ΑΑ·';?:Α\ί··ΑΓη'ΑΑΑ·ϊ··ΑΓΑ3Α'ΤΑ33ΑΑΤ0ΑΑΑ?50Μ'ΑΑΑΑ;0ΑΑΑΑςΑΑχ'Τ3:·ί'ίί'χ·Τ··'':ΑΑχΤ CGAC3AxA.G'rGGCAGACVAC;3AGGAG'AVxrA\A''3GACrrrrr:C3CV3AGACAA<rr3 3C3G'xAAiAGAAGG-'C3'-Ai trGAAGCAxAGA'sA..CA;?AC,ArA;?'''AGeCACCrC.IGCAr;A<'CA'Ai'G<'7xA'3-rA«iArerCGGx<'AC3AlGi'.;^xyx.A '.ΖΧ.'ΧΓΧ,χΑχ Αχ. ,ν X.-GAx...<Xx.-ACx<XiC.-<xXXxA;Xx'xixAx...G;x'X,AAx;XxX-.zGCxx-..,ixGAx<xiVXxx':xX.xX.-Xx-':.-.ixAAi-..xjGx.'.l χΑχ'χχ.χΑ'Α'Α.χΧ.Α· CTGiTA^<\xfA’?Ax:CCTTTO7AiX«<AC?xCTTCACTGACG‘KyArA?>?r?G^.TGiiCTGG.AT'iATTOCTCCCA AGC.;Ge;:rrcrAA3GA''rTT'TAC“AAAC'.k'r;x.A,AxATAAGCTTe'eAAeA'mGATG:rrAAAeA':'GAA'Ar.iACAAT AATG.rA'GGCACrGrrTAACAxCArTGxrrCAAGTATGTCVxCTATAxAGA'TCAATAxxAGGA.AxAGr.'eGCAeAAi·;· GACAG;V.ATGAxAACATAA'ArGA;CrAATGTAxTAC3AACAGAr'.TeACAxAAx:TACTAC-r?xA?'.xCxA?'.'rAxAA !x z'5;'x''eG -ex?-' >-χΑ'>’χχ''3·Α,-’ ζ,χΑ; ?'t Αχ?-'ΪΑ'Χ' | 81 |
PMP-i _________________ | ArGG'rGGACGGCAGACAGACA'CATCAACGG'r'rGAe'GGAACGCGAGGACGACGA'CCGGCCGGCACCmC Ax'r G; CGG 3A3C;Gx\AV'3AAx'3,AA3'i': -'.OxCGx' 33 AGx63 ''<< GACGAA.A: CAaCa «'XV A V GAAACC33;CAAGrG3A3GAC.G:A'AGCAGAeGACGCTCGA'GAGGAAGA’:'GGGG<r:i.'GA,xAAGAi'G33AGAG,AC3 AAx'AAr3AxKGGAC3r?CGACGAx;<3f;AGGGAAGAACGAA'-3xAGG,rGAA''rCGGAGGxGAGA'GxAA;G'rr.3iGx:iA3GG χ:ΐΑΑΑΑΑχΑ'ΑΑχΑΑΑχΑζ\>3Α«ΑΤΤηί?ΑΑ3·3':3ΑΑΑ':3ΑΑΑ;3ϊ:·ΑΑΑχ3Αί3ΑχΑχΑΑ'·3ΐ·Α;';'ΑΑ333χ3£ΐΑΐ'3Αι3ΑχΑ37 ζΑΑ·:.ΑχΑχλΑΑ'Τ·Ατ'Τ''Ύ.:3.ΑΑ<;Αζ'·.·ΑΤΑΑ··3Α«ΑΑΑΑ·3ΑΑ'3<;ί3ΟΑΑχΑχΑΑΑΑΑΑ;3ϊ333'.'·33χ3Τχ3’.ΐ'ΤΑΤΤ'?·ΑΑΓ.ί·Τ3'χΑ ΑΑΤ·Γ·33ΑΓΓ.ΑΑχΑΑχ^:3Αί;··3ΑΑΤ3''ΓΑΤ·3ΑΑ37Τ:??'ζΑ:χ.3χΑΑ':3ί·.ΑΓ··χ'ΑΏι3'ί'ΐ3'Τ'·?Α3':?χΑΑΑχ33ΑΑ'ί'3ΑΑχΑΑΑ 'ΐ''3ΓΑ7'3''·'Ας3Ο.ΑΑΑΑΑΑ3'Α.3·Α.Α3:33'ΐ'ΑΑ.’Α·3'ί'>ΑΑΑΑΑ''?'χΑΑΤΑ'ΓΤ·'3'Α'ϊ<ΪΑ.ΑΑ3'·:ΑΑχ·Τ'ΑΑχ3ΑΑΓΑΑΑΑ3ΑΑΑΑΑχ Ar'AAie-3AAAA3A'reAArA3<3'ACx^<3AAnA;A7C:''rGCAr;ACAAGG3'T-AC3XG'xA-:'':'3AGAAAArA'xA.!3r7?x<3Q'TCxf3 Cxx....:.' '< VEQxX< | 83 ' |
2017200239 13 Jan 2017
NAME | SEQUENCE | SEQ NO |
GGTGGAAGTGA:GAAAGTTAGAAGAGAAAGAGGGTGTGTCAAAGAGAGATGGTAGGATGAGAAGGTATT pkyGAki.-kGiAkxA'tXxATwiGii.AiAiA, x x:-A( T- ',..Ax,Ak?x‘G) kA, 7G\kG,AAk.-T G’i :’Tx ?-kxkGGi Ά-'χ,ΐ UATAGiAk «ΑΑΧΤΑΤΑΆ7ΑΤ©ΑΑΑΑΑΑ„Α©.λίΑν.ΑΑΛΑΑΤΑΑΑί>σΑΛ.ν>ΑΛΑ7>ΑΑΑΑχ·:ΑΆβΑΑΪ>ΑΑ;Α·ΑΤΓίν'.';ΤΑΤΑΑ AA'GTAAAC.G:AAG.AAGAACAA:TA'?GTGAA;T'7TATGG.G«AGATGGA:ATGA,ATxGA;ACTVAAG;TATTAC'ACG'CA : AGCGGTATGATGA'AGATTgGTAGGAeGGASAA?GGSATGTTAGTATGGATSATGATCTA;GSGTiAAAT A?,'A;\-vc';yyf.ArrGTrcAGAf\'>T'rGGw:.Aiv'7Tes:T':'AAGTCTAiU3Ar-TfG:-rAAGGCAT<A)'G'iW3-rGCC GAAAGAAG'TAAATAGGATGGAAGGTATTTAGTTTGA TGAGAACTA'CAATGTA^TTA'TGAASAAATAGA TV·* ' A- ·χΤ,Τ '....,...........7 ,, Γ GA..................................................................................................... | ||
BMP-J | ATGGTGAAeGAGAAACGCTGTCT'TATAGAATTGGTGCTTAAAAAiTGTACTAATGxiGAGGAGAGiTAAGA CG'K:GT'i'G<AAAAGG';VGG;>?G<AAAGiteG'I7CGG<A'.ZA';GGG'7A>777GGGGiX>:7GCG':':iCA\i'GC<;AGCGrAi' CTCACGAGG?C\TTT7AGCC;AG?TC:GAA'TTT7C'GGCTGATAAGA<VrGTTCGAGC?G?G.!V.'AGAGAAf.'CACG' AACAGGAGGGxVITjCC’GTGGTGC-ACCCCT&CATGCTAGAAATOTSTASAAGACAATCAGG'YAAGCAAGG GTCAGCCGGGCGAGAGGACCCiGGAGGA7AGGGCAGr>:7AGGi7G?y7:CCAACi'sGTG'7Gr;GGAi::G'?T'-7GACf: ATGAA.GAATGGTGGGAAGAA.GGA:'.GAA:AAAAGA'7TGGAAxA,AAAS'G.AAGGGAGATTC'TTvTTTGATTTA v.GTTGTAAGAAGAAGGAAGAG'iTAA'iGAA'GTAAGGAGAGGATTCAAG'TTTTCA'GAGAACAASTGJAAAGi'x Λ ,χ>' 'x' (.„ (, A \ A A< A ' xG ·. ,· * A ? xx x ^'A*.'?rCG?PATi77;>?CG'P7ACCAGACr'^TOGiiCACCA?xyi'3%GWA&?<?AGAAiGCAaGCAGG's'SQ kGAT'GATTT'TAAATGTAAiAAATAiAATGAGTAT'AGAAG-AkTAAG^i-GkAAGrAGGiTiTAA'xAAA'AiGA.TTiViTTktATTTkG' OG'K}G{VV.rmGCr;GACriGG?LA;AG?uViG^afT-dTG?<;CAAQ^A<7l itOT'i>/^;ATAAGCAGGTr.:i5'T T-GCAGC.AAGArGAACtsCAf^CTGG^CAGAGATAAMGCysTTCfAyAGTAACTTTTGfiCCATfiA'KKAAAA iA'TjAAATCATA'K'A/vC.AAAAfATCG.AtjAAAGTi'AA'GAT'i'G.AAjiGAGAx'AGG'KAAAG'GGATTAGxGTAGAiG G''A7TAAAA.GA:'.AAAG-GTTGTAAG'TGAi,AT'iGAGGTGAA'AG3-:7GGTTsGAATGAA''iGGA'iTATTGATGCA,A AGOX^-ATf.y-.CfsfCCTTlTAC’fOCOR'WS.CAAT.'SO'CCTTCCCSCTC^ACiSCACACATOAArOCAACC ' ' \ 1 w‘ Λ , , A' < w\. 7 < V x O k ' v Ά, O k AGGAAGAAAAGT GAATGGTATGT7.7777 AT AATT TAACGA7GGTT GCAATATTOT? AT AAAGAAAT .ATTTOC^...................................................................................... | 83 |
BMP-K | ATG7TAGGGAG7AGGAAGTAT'GTTAAAAGGTTGG7TGTTGGGGAGGTAGTGGAGAGGAAAGAGGGTGG CCTrrGTTGCGGrtGCrGGCGCGCAiGnAG^T'rKCf^C^GGTiOTCGLfGCafCCCCTi'.Ti.TCGCAGr.Gi:'!' CTGAG'KxyGGTCG’TGAAAGAisTTACTjGTTCCGTsC^GGAAAGCATGTTT.TsGACKTAAACAG^.GACCAACC CCCAGC.A«GOACGCC«TCK:>TGCC:GCCC‘rAa?i.;G,fAG;.AGCTG'?ATG»?:CAGG~AGTCA<y.:TGA';CGGrK AT CACCCrXCCCAOACCACr.OGT'rOdAGA-S’SGCA’.sCCAgcCdAGCAAACACTdTGCOCACrT^CC AGG VTGGA77A77AAT'GA:7GA-AA?xS,.AT7G'','?'GAA?,G'GAG7''GGiGAAAG,GAAGCAAGAA7,TTA'TTGT-TG’AAT''TTA AkAr'rAWA7cAA7GAG%.y7GPj7;'fAATGisf?'''ArGAGGAGAk^C<';'GA'7GlA'r-rGCGtAGAAGx'V7AAGCAAk1A 1 ) \ A < Λ As , < - ' x . Λ, \ i ' ' . X.G v A-A ' v, ' xG ' 2 * λ' \ un » V 'V - , G OAAAAGGA-7YO?rGl<AAGG'AGGk?Kf'K)A'KAAGiVT<S7i7CAGGAk;AGGGAGA<X;Gk:AACGAAGGA'7-rGG·? Gx7TCG-AAGT'GrT:CG'ACTTGATAAX'TV7,Vv4‘GrV>GA;TGTGTGGTAAG.V7AxGATC<i-TAGG:ATAftACAGG:5T':TT TGG.AGT:A.AGATGAAA.AA.A7GAGGTGA'-:GAGATAA'7AGTT7TTG<TGxGTGx7xGTTTTAGCAATG;ATGG;AAGx?'. i5GGA7!?G'G<5--TTCAAfTG.AAGAGTG.AA£TQGTCAAf5GGJW>GAGGiAAG.AGCGGAAi>GGG'CTTiW.:TGCAG ATGTATGAGx-CACCG'GT •K^^Ci'iTO'SACTTCAOT^CrC.TOOSfjl'iSiSAS'ygACTtSG&TTGTiSGC'TiXCC GG AGGT A? GAG GG AT TT T AGTG, AG AGOG AGA A T© GA ATT T TA C TAT7777777 AT GA TA CGA& CTG'C A AT AAAAATGACATT GTT AAGAGGTT'AGT'TAAATCTGT'T ΑΑΑΪ GT AAGAT TCAT AAAGGAT GGTGWi'C AA ' χ\ ' GT tx ’ , Αχ*· Ύ x x s >· k ,%TGGAAWG;riG;iGi7AGGG5;rAAGGGTGTCSGT?£............................................................................................... | 84 |
ΒΜΡ-Γ | A TAG TAG -,· ';·' 'ΆχΤΆ ! 'TAG·' *>' xGGT 7' A- \7k> „G A'' K 77 „xG> ·' \„ A' I,', GATGGT*T,:A:GkkAGGAA-GkfGAGGAGGiG,GTTGGGGGAGGGGTk?ATGG'';GGAGAAAATAA,TGAGAGGGAT CTiTGGGAGGTGTTTiAGGGGGTTGGAG'T'TTTCTVATGGTGTGA.ATGVG'T'CGGCCTGfG.ACGGAGACGTT'.CG' GAAA;':.AAAtGGAGGAAGT<5A:TGGGAAGTt'.-AATGATciGA:Ai?TA:T«'?CAAA;A:GAA.;ATAAGGTA<\GGAAGA? GT AACAAGGAAGAG AGA ACGGGG TAG GGAGGGA AGAAACCAGGGACAAAG GTGTG AGGAACTTACA AA iATGAA.GAA-rG7'T-iGA,TAAGAAGTAAGAG,A?iAAGAG'TGGAA?G,AAGAAAAG;AAGATTC7'TA'TTTSAT-rTA AG7TGTPTCA€CAA©GACGTAAAT77ATCAAT7VAGCAxG7GG7TCACg:77.AGT'':T.GAGTGT,ACG,TAG,TG,AA T’GGTT 77777 AAAGAAT AGA AGTTT AAA:, i'7A,-AGG,AAT7'A7TT'ATT 7ATGGGG.7 CAT AAAACG:'A’iC,AAAAG Α,,.-Α,Αϊ,,'λ k.G,-AAGT i k,x,A,.,ko; A, :«xk:k,7,k->Ak, 7x7 A,.,kjx-,k,,xk.k.,Ak?kiT T χ.,χΤΤΤχχχίΤΤ Λ,,Αχ,'ΑΑ'νΤΑ,ΐχΑΤ,,'Τ,ΑΑΤ,· .ik.A, k7LVATTTTT-GA'TGTAAAAAAAAA'G<TrGATGAGGTkAATATGAAAA.>GGkA:TA;TAAAAiGAAi'GVi'GGATT'AGT ':ATTA)GixA.GTGGAAAAATAA;ATGGA«AAAAAAGGTGGW7AAAGiTGAiTATA:TAAAGGTA;AGAA,GGTATT TA)A?A:AAAGATGAAAAAAGGTGGTAAAAG?<TAAGGAA:TxA-AGAAAATAAATTTTGGAAATGATGAA)'.A7, ':A.AT'TGATGATATA*AAAAA.AAG;TGAAi'G.AAC;?AAAGAAAAAAAAAAGC?GJCTKAAAAGAAT?AG,GTCAi'A) G AAA A© A GAT A. A AT. AT T©'? AGGTA A «A TT GAA T GA A ATA AGGTA GA ATAiA AT GAGTTTGTGGATCCAT.· A'GAA7GTA'A.AGAGGT'rTTATAAAA,AG‘.AGA-7AATGA'GGTTTTAATGTAGG-A'AATAATATGAAA'-TCAAGT AATGATGGGAGTTAGCAAT7AAAAA’GA;AAAGTGTGTTTAAG”!T:TA:AAiAT-iT'TTrAi>GGAi!'GT-'TT:VGTGAC < W -1 \ , „ X v 1 4 T x ' „ x ',' ' ,A , , | 85 |
BMP-AP | ATGGTGGGAGGTGGGAGGTGTGTTGTAGGGTTGGTGGTTAGGAGGxTGGGTAGTTGGCGGGxGGGGTGG ΑΑΤίίΑΤΤΑΑΑΑΑΑΤΑΤΑΑΤΤΤΑΤΑΤΤΑΑίΑΑΑΤΤτΑΑ,'ΤΑΑΑΑΑΤίιΤΑΑΤ'ΤΑΑΑΑίΑΤΤΑΑΑΤΑ'Α'Κ.'ΑΤΑΑΑΑΑΑΤ AGG.AAkG'.GATC,:?TGAGA(GAATTAAAt:TTGAAAATAAAAAGGATG'?TA'GGA'-TA:AAxAAAAAr.AAAAAACG GAAAGAAAGGGAGATT.'GTiGG'GGi'.ACAAATAAAT.'AATAAACATGTATGGAAGGAAGTAAGATCAGAGGAG AT GAG GAGA. GGAAGAAT.AAA'GGTTGGAGAGAGGAAjGCAAA CAAGGAAAGAATGTGCAA'AGATTGAAGA \ ' X ' G A ' „ X V ' χ. ' x , „ | •33 |
2017200239 13 Jan 2017
2017200239 13 Jan 2017
NAME | SEQUENCE | SEQ iO NO |
ΟΟΟΑΟΟΟ-ΟΟΟΟΟΟΟΑΟΟΟΑΟΟΤΟΟΑΟϊΟΟΟΟΌΟΟΟΟΟΟΟίΤΟΟΑΟΟΟΑΟΟΟΟΟΟΟίίΑΑΟΑΟΑΟΑΟΟΑΑΟΟ CY.OO^GOAfjrTGAOCTCOOTOOTCCOOOCOTiACATOOTiAGAryOiOOiATCOOO-iOOXOAOTOAyyO'OCAiyOC^CCTCACCreCiCCAi.;ACCACa.’i?T?GGc\!Af^?:»i;CAGCCG?jq-CCAACACTP?G'V,CM;CTTCC;’.CC ΑΤαΑΑΚΑΑΤΟ0ΤΤΟΟ.ΑΑΟΑΛΟΤΑΟΟΑΟ/ωϊΑθ:7Α·?:ΤΚΟΟ:ΑΛ£ΑΟ{Ο\ΟΟ0α0:ΑΟΑΟΤΟΟ0ΟΟ?Ο«ΑΤθ:?Α .ί7Ο:ΟΟΟΟΑ·ΐΟ0(.'ί:Α0;7ΐ;Α:7:7ΑΟΟ·Ο0ΑΟ;::ΑΟ;?·?·0Α:7ΟΑ0Α\ΟΟ0·0::Α:?ΟΟΟθ::θ0Ο0;ΑΑ-Α:7Αί7;','?'7Ο;Αί7':--Α rOCT<5 OAOOAAAC ΑΑ,ΟΛΟΟΑΟΟΤ'? CCAt OAOCOAAO OAAOATO-?ATOaAAOCAOAAA.ACnOfiCAAOSO OC.AAOOi70AA.A.O7iyOCC??70.A’OOA.O.A0707 0-.y?y.ACACi'.AO;>0 00-:y'7-OAAOCAO-AAOOCAiAOCAOGOOO GXAAGOTOVAAVjT-CACOCOCOCOGTiYOrOCGO'iWAC'iOCACAOGQACACQOCF.ACC&OSGAOOCG't' I. ( A ‘ - ' t - G ' V , - 1 1 -. , < ' s \ 1 'Κ>?;»0”,ν>ΰΑΚ';Α^/^Λα7τ^7'0Αύ^\ϊΑΑ:ΑΧχ:Αΐ^ί'ΐ·.νίΐ^.σηττ·30ίν;^:^κ:<^ν^Α O:7O0?A000':A700f:770A7'O'O:07A7;OA7xi7A0G00?O7000i'0OA0i?0?-7'O70,A:77:r7':7i'OG-0O0C07A-A:700O:70COi7:TAAf07i:;Af7Ai:CO,rOTO?AC«a’.7707CT'OCAGOOA07?if;f7iAOOf70AATOAOTO7!A,i‘!TGOOGOOi'.CCO Co0O:7:7A0C770:7C700O0?AC0O0C;7Oi7GA.OA?>0C:C:OO000-77C.:AOOC:O'.0(7::A0-A:7A0:7AAT00A;A0O AACCACGCGAGayOGOA7G,OC07CG7roCAOC?0?,OOAy<COOOCG?G.O-A::OCOOAAGOCAOCCOG'7A:0 CCCi'3ACAGMCTCAC't‘OC-rA'iCTr.OATOCTr^».Ci??i3-«3AC<5AiS&AmWl.AOOTWAl'yAA^«3AACT o ca ay aoa too? to a αϊ agog o ay ooc .y-i co co no;y a | ||
8ΜΡΈ9 | a A A -- a a a -, A A \ :.'00:7000:7000;-700·.700000007αΟΟΑ;7000:7α70:7<700:7:77;00000:707;0:.ΌΟΟ:700077·70:7'::ΑΟ:7ϊ:00 07A7A00.777000000A0007:00'?007AO-OOOCO:7:70,'77:7'7-0,A:7::A'?:7'700:700070?-Ai70?7.777i7.AOOOA:'A7 Cnr’Aon.AOGOIACOOC'GOf.XWCGGCOOCOACOiTOOOA-O-ACOOGOAOCOnAOGOiAOTOAAGOOGAOOCG'K; CTOAOYrOCCOOC?>OACCOaCOCC?TOiTAOO-OC-;A7A«OGAGi70CAOOa\OOAC7Ci'OOf;'OAGCTr.£GC;\OC A07AA-7AAGOC0007:;GGAfGAACrG?CAGAA?-CGAGay:yGAAAGC.AACeC?sGAiy?o:OC:iOCOOOA.AOOOA A0'TT7.-:AAT7.0CCA0a:7A<0C.A0'TTT7Ya7?'0'7T0A-;a;c0A':y0:n'rcsGC'r7O'T'C0CO.0AACi\0ArGi7A>\0A OGCOTOAGGASAC AAOAGCA-'y'i O TOO A 7CAGCO ΑΑΟΟΑΑΟΑΟΟ': AOGAAAOOAOAAAAOOGyCAAOOO 7-GAA-Ao0O:7.OGA7'0OCC0O-7OAO:7:A77AO'7O’7’70OACAOC<AOO'7::O;:7'7:AGA-iO:Af7A?i0iy:7ia77:7A<7:7'O0-'7 AA^a, , ,.?ανλνΑ' Η Λ'Λ’ 'a7Aga'.G,,?a.G< 0A7o-AAa A.,Aa -7 1 OOTCOAAO7G0GCO7xCTTa7.V7XiZiOAi7AO7ViOG0OOOTC'CAAOAOi>OATGO-OAGOATAA,.7:CAOi?Oi7O0 77--.-:-7--.. -..-G-,-.--.-a’77::<7Oaa, A--...77-.-,.. a a A?i ;..ΑΑΑ<?ΑΤΑθΟΟ·ν.Λ.λ-ιΤ'Λ <A77,;\GTAA\.O .- i'tXai-A-7. A\i -7----. .7-.-GA-AAA Oi7C07aO'OT-7TC7CAOy\.AA7,GAO:OAAA7iCi:;TOAAOO-7?OAOC?07.AAAO.Ai7COaA-A7A>'7;>:7CTT;aC.7i'OOAfi •7TGT;aATA0:iiGA;7OGTT'G7T7A77TGGA0'7'7CAi;a7?:.Gi7TOG;7C7G':-?x7s'?0:-<\Oa7OOTTOa7:iA7'?f>-70'G 0-yOGCr-rA7CAOGCC7'C7TA<7'rOOAAOO-7COGCTOOTTO'7-TCt7CCT-7-<Vyi70-GAOOATOTCaCGaCGACO .A.AAC.ACGCT “TCOa.ACAOA·.'. OGTOG'TCO A-r-77 0AAO'T-Tn0CC,\0AAA0Gay0CC7aa:yi70--Ay0'TCT0'T CCCOACAG,AACCCAQC::o7:iAOCOOGA-?GC::o:y7CGOOGAOGG3AAOGA7GO,:yGOOGOA':7(AAAGAACG ..........·- -...........................:....:...................- ·............................................................................................................. | 91 : |
BMP- £10 | < aViG,--70o'^C.’-a?„..n.i?rACCA.G, tOO: A 07'A Av. a a? 707 700---^07,70-- -iO‘7:7 ϊ 00 TTCCGGAGCTC-OGCCG 0 .07:7 7077 7000 ¢70000000 7000 ¢7 GOO ¢7 Οί7ί7Ο CO 7. CATC 00:7,0 00:7? :7007aOOA:7000:7'70-:7000:70--''?:70AO 07000000000,:7:7770^77007000:700:7:^0-0:70070700:7000 0:7:7.0:7:7.:,.-0:7.00,.0:700:700-770:7000:7000:70.070700:700.0(70000077:70,7-.:7::7:7000:70.0:700-7000:700:7T7\7GOi7GGO(7i7Ai7O>C7OA:7GC;77a:7:77O?AOGO:7V,G-7i7?AGi7r7OO7:-O0AA;?:7GTO?;?77:7:7Ai?;7'?T'-?:7Ai>:7 .A7GA.Ana.A7 0'?T7i7rC.AAOA.A0’?A::0A-:y,AliAC0AC'!O07?.A?G--Ai?0Ai7C0G;'A?0AT-vC'?Ti'.0'TT?O'.T-r7-A Α·ΟΤ·ΐΌΤ.Α7Ό·?!?ΟΑ:?ΟΟΑ>:70Α·77Τ·7'?Α'ΪΟΑοΟ'?Οί'·-ΟΟΑ:7-“·ΟΟΤ7 OAGOOCOOCCGAOAACAGGayCAAG?· :77 0-»' - ' ' ' 0 ~ ' - ' G. - 'a '' * 7.7 -.-0 - ' *- ' G A : A ' \ Ό ' : G -G - a -, GAAAGOOOO -77^-^-000^00007:007070^0:7000000700707:000:700.^0:70000^/:000:7000000-0 0:770070'ι7?:Α7000:7ΑΟ:'7007,07;Α:7Α-·ΑΑ(7αΑΑ:707'07Α7·?0:770'.07αΟΑΟ:\7<?Γ7:ΑΟ;7{7·?ΑΑΟΟΑ:70·?07'? ΤΟ7Α70·7ΑΑ.ΑίααΑΑΑ:7Α0Α077Τ:.Α7Τ0Α:7ΑΟ0αΑΑ0Οι7!7ίΑ'?·777:7·ΤΑ:77.ΑΑ':?Τ'?Τ’ϊ'Ο:7ι77.7Α·7Α?:7α7>7:ΑΑΑ7-:.7:.07-7777000C'?C':7ACA?5AA:7:7G.G7:7A.G7G?CAtyGC:7A,G7;7ACA:77'.CAGCCG?:.G7':7GCCT?AAG'?CC:7G :7ay7AA:7:70A0A0C:7T0T:7'?AOG'?77:7:7000<7AO-0OAO0'7:::7O-:7O0(70AA00A 0777000017700000:: GOO COGCO:iA'?:77.fAyOOar7 7AA:7-'ny-?*.0'?‘:'A77A7t'7 7-7TAAOTOCr7CCCTOG''?Ai'iACC?'.?CTOa.iA'<CC,70A A.AiyOATOC-AA77AATOCA:yOCCr'yay7 0C.A;'.OiC7'-A-:y-A.CA?COCA:yOA.A:yO'?TOCAA;70t7:77-G;77'G'?i77i' -0‘7 ,7A ,ΑΆ0α ,77-7.0-770-07,0, o Av\.aGA-GAA?--, ' 701.7: :,7mv,7a 1 A ΓΟΑΟΟΑΟ A': 00 77GT-7G 070000-7700070 7 COO 7 AO | 32 |
&ΜΡΈΚ ...................... | AOOO TKGCCGGOACCCGC'mTCTTyrTAGCGTTGCGOGTTGCeCAGGTCeTCiTTGGGeGGCGCGGeTGG :700:7000:7000000700-0-00007070070:-077700:70000:70-0::000:700-7::700:7::0007070::77007:7000 C0OA{?G7i!?;7-70C0OA:?;7OA;7T'?00t7!700O0o:7:70o:7Ci7A0::A00'?0C0O0:70;?:AA?:.0A:7?\7:o-::0i:A:70 c70C7O7OAGO77A:7C:C;7i7:A.;00i7(7:7O0c;7OA;7.0'Y.;c-?A0A7::i7000A0;:O0A0OC-A::?C-AOi;0;7.777;C;:'O7:o C0C7AC::OOC::OCAOACC7AnCC7OOO:7t:A:7i\-7:7O::A:7:7OAO:7:7f7AOOi7-A?i0A!.AC;OOC77C-AOCOA07i7?-0O S.TiyiAC.AAOC’iOOiyG^.A.yA.ACO.AOCAGA.A.AC-yAO'TOyCOAA.AACAACCCrtGAiyAOTCT'OCOO-OAAO-OOA AOOor.O-AOCCOCACOg.AOO.AOTO'? AO-:y?«;oeACCaOACCOOC-SOCOO'?OCCO.AO.AACAOAOOC.AAOA 0C^'0'ri600?0AACNA0A0CA000TCC>O’»CCAA\A0':iAA’i\A00-7:A0OA;AA'rC“7A-ASA0C0GCA7Os0N3 0-A V ' a< V aG G V-, -.- * -v - >O -., ο?θ'θ77οοοο:7Αο:7'7θΑο:7θο::τ;οο:7οο?Αθ<7θ:7θοοθΛθθί7:7Αθ7Αθο:?Αί:Ααοοο-Αί7θθΑοο:7Αθοο:7·? G0a7G.G7:7'}GG:7CC:?:70O:7GAGGAGA7GO7A:7CG':O0G:iO:CAAGA,0:7:770:00077700-000070:7.770-070:7077 00ϊ7Αθ:7ΑΑ0Λ0Ο?,Α0Α0Α··.7:70Ο770:7Α0ΑΟΑ0Α.:7·:7θ:7<7Α0·000057Ο0-Α?>000?α:70:70Α’7ί7Α00ΟΑΑ:ΑΑ 00:?::Α0:7:7000ί70Α;7ΑΑΑ?χ0Α;770'.ΑΛ;0:0-τί:ΑΑ000Α7χ00Αί7·ΑΑΑ:;Α0-η0ΟΑ7Ο,00:.Α:0·0?>Α00:'.0Αίί 0000A”.0AGACAO n-yOTay.-: 7-0-77OOO.AC 0007700 77.--000000:7'70i?A?-0077-0Ο-ΟΟΑΟΟΟΟΟί700 CCOO 00 77000 7o: C.A 7.00 CO TO O AOOG COAOOOAGAAOG CO o CO T CO 0 7.7.0 0AACOCG C A 0 AOCTTSayCGA CO A · ' ' A A ' - ... ' | 93 |
w
2017200239 13 Jan 2017
NAME | SEQUENCE | SuQ SO NO |
ΥνΥΑΑΥΑΥΪΧ3Υ7ΥΑΑ5.ΥΥΥΑΤΥΥΥΥΑΥΥΥΤϋΐΑΥΥ'·ΎΑΑΥΥΑ53ΑΑΐΌΑΑΑΑΥΑ3ΤΥΥΑΤΓΑΑΑΥΑ?;ΥΥ ' a ', ’ x ' \ ! x v. ' | ||
BMP-ET | A,r<5GTGOcocGAeecG.eTA:rc''rcTAGenT'rccTGC'rTi:coAnG'rc'CTCc'r<;«;tm;GcncG.GiTGn ccTCij?YCOTC^YTC-;y3Cocx'AGG?XYArTCY:YfX5YrxA7GTi.,GTCGqY:cc«vf:ccTf.to'ri'.'c.cAGCccT CtOACOA!'iGi?Ci-^<jAiiCOAft?TCO.»’'3-n-<X'«OCTX'TCAOCATO‘rTCOQiXTiSAAACAGAGAec;ACC eccAr;cAGO''jAe-g''7CGW',p'W’A:ec<toe'y?toA'iYjC'TArA'!ecTPTA'rcqcAc<!gAeTf?AGoreAA<'ct;v'Xi •x '< V -., Vv. Αί'ΥΥ-Λύ'.ΑΑ'ΓΥ'ΓΓΙΰΥΑΑλίΧΑΥ'ΫΑ'Υ-Υ,ΑΥλΥνΑΥΑΑςΥΑ'ΧΑΑΑΑ.ΑΑνΑΑΥΥΥΥΑϊΑΥη’ι'η'ΥΥ'ΓΥ'ΧΐΥΑΑΥΥ'νΑ ΑΥ·ΥΥΥΤΑΥΛΥΥΥίΥ$ΥίΑ';ΑΥΥΥν;ΥΥΥ<ΑΑΥ·ΥΑΥΥΛ;ΥΑΑΥΑΥΑΥ;Υ'ΥΥΥΑΥ-ΑΥ·?'!Υ.;ΥΥΚί3Υ:ΑΛΥΑ'ΥΑΥΥΥ/ΥιΥ;Α 'YAC.'T'rYAYYYY'X,YAX'YA<A:ArYYTYCYAYYAYYY7to:i'YAAY.iYYTY.iYAYYYYA'i'YAYYAi'YiA,':':Y’YY;Y.iAYYAY: CCAACTCGAAATTf/CCCGTCACCYiGACTTTT-’.X'YXACCisflGTr-YfXGAAiSCAArjATGCAAGCAGGTYG Gi'vRAGYTTTOz'-.TGTCACCCCCGYTOTYA-KiYftoTfAG^CTGCAYAGYSACAYGCYAACviXTAlGA'TTCGT Ο:Υ:'ΓΑΥ;Υ.ΥχΚ'ΤΥί':.ΥΥΥΥνΥΤΤΑΥϊΑ;'Α;ΑΙ'.;ΑΑΥ.Υ;ΥίνΥΐΥ-Υ<;Τϊ'Υ·ΥΥΥΑΥΥ1Αί3;5ν.ΑΐΥ’?ΥΤΥΥιΥχΑ'.Γ_ΑΑίΥΥ7'.;'_ΥΥ.ί:Υ'’Γ TGCACCAAfiiAYiA^CACA-X'tOqTCACAOA'rSA-XjCCATlGqTStC-'rAACrrT'rqOCCA’rqArqiGAAAA ΥΥ.3ΥΑΤί:ΥΥγ·!<Υ.ί-.ΥΑ.Α.ΑΑΥΓΑ;',.ΑΑΑΑΑΥΑΥΥΑ.ΑΟΥ·ΥΑΑΥγΥΑί'.·ΑΑ;\ΥΑΓΑΥΰ<3ΑΑΑ<ΥΑ'.·γτ·ίΑΑ;3ΥΥΥΑΥν ' \ : <> i v v -vv. v, i’GG^yii-ATCACviYC'iTtiisCiCtoqA'i^SAQAA'iq^yiC-CTYPCGkC^XAC'XACAqAT’^YiX^AdC ACrY:ACCCGA‘nGTCCACACCT'rC-GTYCAYC?T?YK7AACCC<v^TAw;!S'CCCCAAACC9!SGCTGiCC YYCGA;;,iv;AW'YYAY-YSCY<YreT<Y,ivmr.Y'YYACY'iY?YX;,YxuvASi0\AAGC-'rrG-YA,.riAAtoXAYAr.''f' A'TYACXACATAGTTATYYAGYGYYYTGYYYG'YYACYYA | 94 |
BMP-R | A'A3GYYAYCYOKAYCYYYYG'YYYTYYAYYAT'A3YTi3YYYYYYCYa3AYYY7YYYGYYYGYYACYYYY'3$ ΥΥΥΥΥ'ίΥΥΥιΥ-ΥΑΟΥ'ΥΥΟΥΥΥΛ'ΧΑΑΥΑΑΟΤ'ΐ'-ΥΟΥΓΑΥΥ-ΥΥΥΥ,ΤΥ-Α-ϊ-ιΥΥΥίΥίΥΥί'ίΥΥΥΥΥΥΑ'ΥΥίΑΥΑΥΥ'ΥΥΥ c :YX YGAGGTf. e ' A AYAYjAYY YY ·' 3AY,Y 'VyCO-.'jCY 0.3' CA Y Γ3Α TOTYCYAYY TOA A AC AC-AGA YCC .¾ to; cecAGCAixoAcoccatiwocccco'?sA.cAi<x'TAOAccT«:-.TS'nxcAQhCACTCA«G'?<AGO%!G*3 ' A-< ‘ Y V. ’ .,- v. t V.A '' v. v. Y - I. Αΐ'ΥΑ.4ΟΑΛ··5Υ'ΓΓΐ<Ζ3ΑΑ<ΥΑΑΥΥΑΥΥΑ;'Α\ΑΑ·.;ΥΑΑίΥχυΥΑΑί·ϊΑΑΥΑ;>.ΥΥςΥ3ΑΧ.!ΑΥτΥ'Γ·ίΥΥ'ν5ΑΥ:>;ν'Α·ϊ·Α AATT'YYA.Y<YYYY:iACY-''YAYAA\rYYYYAYYY-A;'Y'rYAYAY;AY':':YYYAYY,rY3'-<:YYAA;i'Yi;YAYYViY;YAAA:‘Y:; 3 7 Y3 3 ?AG3AAAYAA3AYYA7Y Υ'ί ΥΥΑΥ ΥΑΥΥΥΑΧΤ YAAYA^ ΤΛ3ΌΑΛΑ3ΥΑ3 ΑΛΑΑΥΥΪΥΥΑΑΥ ΑΚ ΥΥΑΑΥ·ΥΥΥΥγννΓΥΥΥΥ·ΥΥ'Γ·ΥΛί:ΥΑΥχΛΥΥΥΥΤΑΐ;ΑΥΛΥΥΑΑΥΤ'ΥΑΏ'ΑΥΑΥ'.'?ΥΥΑ':ΑΑΥίΑΥΥΥ'.Γ.Υ\ΥΏ3:·'!'Α:Υ ΟΑΑΑΥ7Τ'ΤΪ7?<ΥΥ:ΤΥ'Α7ΥΥ0?ΥΥ'ΤΥιΤ303ΓίΧ7Υ£-ΤΟ3Α0Τ7ΥΑΥΑΑ&3ΑΥΛν''':7Α7ΑΛΥΥΑΤ3ΧΟ3ΥΧ;<Υ7-ϊ ΥΧ'ΑΥΑΑΑΓΧιγ,'ΥΥΥΥΑΥΐ'ΑΥΧΧ-ιγγΑΑΐΑΑΑΥΑΑΥΧ'ΓϊϊΐγΥΥΥΑΑ-ΑΑ-ΥΑ-ΥΑίΥΥΥϊΑΥΧλΑ'ΓΑΑΥΥ,ΥΰΥΥΥΥΤ Τ·:>:;'ΑΥΥΑΑΥΑΥΥ!Α.ΑΥΑΥ.Ας'<'ΓΥΧ3'ΥΑΥΑιΥΑΤΑ.ΑΟ'3·γΥί-γ·ΑΥλ~ΥΑΥ>ΐΑΑ':';ΐ·'Τ''Α3ΥΥί'_ΑΧΥΑ'5'·;;;ί'ίΑΑΑΑ ' ' γ -ΟΥΥ -,. 'Μλχ·- ..., ν V 1 Υ 1GTAAG ΑΥΑΥΑΥΥΥΤ ΪΤΥΤΑΥ YYYGAYYYΥΑΡΐΥΑΥΟΑΥΟΟΥΥΥ GAA3GAYYO3AY ΤΟΙ 7033 7ΥΥΑ Υ ν ' -W Υ 1 ' * V » '< Λ V -,Ο ν' <’ AYYIYAlYYYYAY-Y<YI'YYAY!JY;GTY\;:fYYY;'YVY-rYY<YYYY'.YYY'?A?AYA,rYYY'i'i'Yi'YACAiYAY?'YYY--rYiA? Υ;,ΑΥΑΥ;Α4\Υ-'?ΥΑΥίΤΥΥΥΑΥΥΥΥ·ΥΑ'Α;ΥΥΥΥ·ΓΑ,ΥΥ·?·:Γ<;ΑΥΑΥΑΑ3ΑΥΥΑ,ΥΥΥγ·;:-Α·ϊ·'Υ;·γίίΥΥΥ.ί'ϊ.Α:\ΑίΥ-.ΥΥΑ·=Υ' Α-.ΧΑΥΑ3Υ-73Ά3 37-3 ΑΑ77'Γ3 3777-.3373 ΥΥΥΥΑΥ | 99 |
BMP-GS | A7'37T73777;.i3A73777?77'77777A73377'37777Y7;3C73A33 3:.73377337 33773Υ·Υ'73 : ΥΥΥς'Υ,ΥΥΥΥΥί'Υ'.-ΥΥΥΥΥίΑΥΥΥγνγ-ΥΑΥΓΥΤΥΥ-ΥΥΟΥΥΥΑ'ΧΥΥί'.'-ΥΑΥ.'ΟΥΥ-ΥΥΥΥΥΥΥ’Γί'.'Α'ΓΥ.'ΥΥΑί'ΧΥΑί' Υΐ'ΥΑΥ.ΥΑΥΥΥΥΥΥΥΑ-ΑΥΥΑ/ΑΥΥΥΥΧ-Ο'ΥΐΥΥΥΑΥγ-ΤΥΧΑΥΥΑΟΥΑΥ-Α'Υ-'ΥγίγΥΥϊΥ-ΑΑΑΓΑΥΑΥΑΥΥΥίΧΥ YYYA\gr;Ar,Y<cAY..;;Y<>Y-XAQ'rs-CY-YYYY-i'ACArYAYrAYg’<Y-iY-rA'A<Y3YA<AYYfAYiY'%'YYiYAYCYY:to YYYACYY’ACY'YC'AYvACYA.YCQYYYGYAYA.YSYYA.YYYA.'ACYYAGYYiYACAC'TYYY-CYYYvYY'i'SCYACY Α·ΓΥ.\γ·ΑΑΑΥΥ·;ΥΥ;ΥίΥΑΑΥ$ΥΑΥ''ΑΑΛ';·ΑΜΑ'ΡΥίΥ·'Α'<ΥΠ·Α'Α’ΑΑΥΥ!ΥΑ.ΑΥΥ:';ΑΥΑΥ;Α'ϊ’ϊ·γ·ϊΥΥϊ·ί·ΑΑ·Τ'!·'νΑ i'X3rYC'3AYrY7Y37'3Y3OftY7/3733'3A77AY-Y3vA73A7;AYA?,37CA7Y33’3-33CY7i37AX:3A(3A3'7Y33\7,A ·3737'Γ37£ν37Υ33Α7?733'Α3ΥΑΑ33·3'37Υ33737337ί3ΥΧ33':3Υ3'ϊ'.ίΥ3'33ί7:73Υ33Α7ί3Α'3Υν-'3'Λί.Χ3:73333ΑΥ3-.777AiX7'37AA?Vf'?77Y3'7-37iA373;33A77T'7‘T333>vAYY'773':;T37;733?xAA3YA7?O337CAA7<7A77'7;37 ΐίΑΑΑΫΥΥΤΥΥΥ-ΤΑ'ΤΥΑΥΥΥΥΥΥ-ΥΥβΥΑΑΥΤΥΥ'ΑίΧΥΑ'ΧΥΤΑΥΑιΥΑΰΥΑΑΥΑίΧΥ'ΑΥΥνΥ'ΥΥΤΥΐΑΑ'ΤΤΥΑΤ ΥΥ3ΤΥ/ΥΑΑΟ'ΑΥί',ΥΥΥ;νΥ1'Υί·ΥΓΑΑ<3ΑΟΑΑΑ-:ΥΑΑ;Α'ϊΐΟΤΥ'-;ΥΥ.ΑΥίΥ!ΑΥίΑΥΑ'ίΟ'Τ':\3ΥΥΑ';·Α'-Αί'Α'?ΑΓΑ'>·ΥΥ'·'Τ %ΥΑΥ.ΥΑΑ·ΥΑ'ΑΥΑΑΥ.ίχΥΑ·Α'.’·Υ,Υ-''ΥΚΥΑΓΥΧΥΑΤΑΑ·;ΥΑΥΥΑΥΥ<;ΑΥ·ΓΑς-·Γ.ΑΑιγ··Υ'·ϊ1ΥΧ;ΥΥίΥϊ-!·,ΐΑ·ΓΟΥΑίΥΑΑ £37ΑΥΥΑ:3Υ77θ37ΥΑΥΑΡΑΑ'3Α7.ΧΑΧ?νΑΥ73ϊζ3Α3'Χ03ζνθΥ.ΥΛ7Α.νι7Α3Υ73;?ΟΑΥ777·ί'3ΑΑ737Υ;37 ' “ V t t > t 1 - ' - , ·.. -Y VV? ', Av Y γ;Α'Α?γγ.ϊνΓΥΑΥΥΥΥ'ΓΥΥΥΛΥΥΥΥΑ_:ΥΑΥ;γ·:Α'ΑΥΥ·:·γγγ:;γ;ΥΑΥΥ·Υί:ΥΥΑΥ·ΑΥΐ·.'ΥΥΥΥΥΥΑΥΥ'”ΑΥχγ;·;γΥΑίΥΥΑ.3·ΥΥΑΥ·Υ·ΥΑΥΥΥΑ·ΥΥΧΑΥ-ΑΥΥΥΥνΑΤίνΥΑΥ;ίνΥΑΥΥ'ΥΑΥΥΥί'ΑΥΥΥΥΥΑΑ.7ϊΥΥ;'ΧΥΥί'Α>Υ3·γΥΥΑΫ·ΥΥ 77733x7A3A3A377.A3T77'3ATCT-:37A77777T.A37'~7-7rAYGA''3iO7A$33\A?vAS37773?AT3A?Y\GA;v3T $γ''γγ:'7,::γ;?,:αχ·γ..γγ.ααυ: ΤΥΥίΑ-τ ΥΌΥΧ,ΥΥΛΑ | 9S |
BMP-ER | 7733333777: IGYAvY 7-3703337 3':'i -Υ··'ΰΥΑ3ΥΥΥΥ·':ΥΥΥΥΥΧ$ΑΥ·;ΥΑΥΥ\ίΥΥΥΥΥΥΥΥΥ;.;ΑΥ'5ΥΥ5 ΥΥΤΥΥ?ΥΥ’'.0';ΥΑ0ΥΤΥΑ7Υ-Υ:'.0ΥΑΑΥ,ΑΑΑΥΤ·Υ0ΥΥΑΥί3!3Γ.'0·ϊςγ·ϊ-ΥΥ·ί40ιΥΥαΥΥίΥί;ΥΥΑ'ΐ-ΥΥ-ΥΑ<·ϊγ-:'ΧΤ 77GAY3A3337CvT7A3Y7A7:33 73AG77G7O3G7<X7CA7GG'3G37Yi3GvOi7AAAGAGA7AYGYAYC ’.;'YYAYYAAGGArA2YYYYiY;;i7GYYC<'YY';YA;AA''A3YY'A''3AYCYYYiAYi;YA:AimCAY',iCA\X''rYAY-C-YGY-Y Υ χ ’ν·Α3'·.,<. ΥΑ-'νν νΑν?ΖΙ 3<·.·.Αν3ΛΑ37 i3Ai-Av-AYY-Wv7,3,-- :«ί -.-3.3-Αχ, -..3,Α.ΑνΑΎχ3,7-..-3.3: VΑν ν ,ί '33.3, Αχ.3. A'AYYYVYAYYYYYcXYYfAA.YYYY'iYYACYA&AVAYYAi‘YI'Yt'YAfYAVvY.YYYCYYAAYArn'YYYY'rY'iAAA'.'YiTA ΑΑΤ··ΥΥ·ΥΥ'.·ΥΥΥΥΥΑΥ·'.?:·ΑΥΥ·'',Α3β7'ΤΥΑΥνΑ.ΥΥΤ~ΑΥ':ΥΑΰΑΥΥΥΤΥΑΏ:3'ΓΥ'ΤΤΥΥΥ:?Υ;ΑΥ:ΥΑΥϊΑΥΑΥΑΛΑΑ T-''_YYY'TTAYA':AAAY.AA'TAi,;CAYT-IYYY.ATYAYYYAATYYvAYAYY-;YrGAYvA3''Y\YAAYxAYYr.YAY:YAYAA ΥΥΑΑίΥΐΥΥΑΛΑΤΥίΑΥΥΥίνΥ-ΥΑΥΥΑΥΧ.ΥΥΤΤΤΑίϊΑΥΑΥΥΑίΑΙ'ΓΤΓΑ'/ΥΥΑΑΥΥΛίΥΑΑΥΑΑΥΥχί'.'ίΥΑηΥΥΑΥ, 33.3^33333 ·7'33337'373 3.037 CY33T-370A773-3G7 0-7-3737733.3: νΑΑ33':Α':7«Ο:3ί3ΥΑΑί'.ΥΑ733Α7'·3<3;33' ΥΧ'ΑΥΑΑΑΥ'ϊΟΥΥΥΥΑΥ'ΐ-ΑΑ'χ^ΥΥΑΟΑΑΑΥΑΑΟΥ-ΥΥΑίΥ'ΥΥΥΑΑ-ΥΑΥΑ'ΥΑΥΥΥΥΑ'ΥΟΑΥΑ.-ΥΐΥΥΧΥΤ'ΥΥ'Γ jG^AAGW^A-AAtoYGr 3-3Λ3Α; Α„'-.77-3< X 5G7.%όόώ- υ - ’ΥΕ ΈΜΕΫ5 ~ | 97 |
2017200239 13 Jan 2017
2017200239 13 Jan 2017
NAME | SEQUENCE | SEQ ;0 NO |
0000077031.7377017^707031707030777001370307001370100700370700070077003330700 -.'- x I ' X-, - 5 x -, , >-, ' -X , ' < ·. V 3 > οι·3Τ00ΑΑ«7-3;0070Λ07Τ0ίΑΑίο;Α7:ΑΖ>?.θΑ0·3θ-7ί;7θ7003Αθ0θΛχ70Τ0Τ7Λ0:337θΛί;03.':07χ777 TOC30'CA3OATOAOC30.?y3i.'7i?O;TC?'i7Ai5AT0.0-3i3f.;CO77GC7iAG7Ai'J.C7T7T-0OCCA’rOOTOGO?O>A tJ-OOC'ziTCCT-'.'TCCOC'iiA.AO-GAO'O/iA.AciCOTCAOOO-OiAAzteAiO'iOACA.OiXOOAAOCOO-CTTiyV.iTCCOO οτ-Ο'-'ΑΟΟΑΟΟΟΑΟΟΟΤΤ'Ο’,οο-ιΓΟ'ϊοοΑΟ'Τ-'-οοοοί'ίΟΟΟΟοοοίΙΐΟΟΑΟ'-'ΤίΤΑΟοί'ίΟϊ-τοί-.-ττοοΑΟΟΟΑ AdrtoCTA?dC7oci?^TrT?ic?ctoCACto3Ao.5.A-rGCdi?'i'TTTC!CTi?Td!3CWATCATf.TC-Asc?i:cA-7t Ct X Cl ' 0 V i --. 0 ' A< ' 0 ' o , „ ‘ Λ -> -, ' te'-i - < 3000037007307000.0007 TOOXXIOTC-TCGCTOA | ||
BMP-GE | χ·<οτ·„. ,- “GTG'-wS;, T-,'> , x- „τ-”;ο 'ϊα-<- a-' \„a- , τ' AOK;3;7&7<'C'-:xAOC7f3ftoCAOz'iAO«3iAOiX?rOCi73f'?OOO?-7“;7O-rr;ocra3C7T7<:7AGCi.'AGC0'7T C7G3<y3.AGG?GC7GTCC<3AG?TCGAG‘CK:CGGC7G7TO'?CC&?OT7COOCC?0?J\GC3G'OCteCCCOCO' CCTTf ?-AiXOAi3GCCfe'fd;'3-tOCCCCCCt>-.CAl<3eT<30ACCTdTSCiXGOW)ACTCC«G<XAGC«?TGi3 ^7-:7700^001700700073,70-:3307-.30.^0300-30013-30^-00001^000013^13707-1070037370-700070 00000<30Λ7 000Ϊ 1333003.007:30000.00303-07001303030300300013000133707-377-1-3-:33:7773-13 i n ' >'Λ x < \ ' -. ' -, ·, < -, -, -. os-, COOGtTTCA-ACAAAtoAteTOiAAOCte'OOCATTAGOGGATOie'AtAS'G'rAATAVrAiOATOA^GCCi'.'GOOAOCG •„l.-ASl,· a -..ΟΑί0:7:11.-. -..1,,1.,<-J;A:-:iA-;l..l,-G'-:A, :1 Xil„7*,-xl.iX-Vx,3XA,0,1-5^3,7',.-ΟΆ:·.,1χί3θ7.·.ϊ3Χ,Ό,3ΐ.Χ:',.ί.· X 1,,--.,At:A,i :13:OAGTO-?T-T<y.;ACGTG 0703070070703700.-3370133003170030000-30013003300300007777:7 -:AjT<;G<V-.GTOGCCCACC'TnG?^GAOz'.AGCAi:;K;a?TGTCO,7iAeCGC-C?xC':K'IcOGr>TTCT<’;G«TCOC •Τ-ΟΟΛΟΟΑΟ'ΟΑΓ.ϋΑΟΜΟΑΟΑΟΟΤΟΟΤΟΟΟΑχ',ΑΤΟΟΟΟ-ΟΟΟ. 07^77^71330^7700-300. A»3\'sS'7:3«CAAG iyOCOACCOOCTgCAOAAGAO^OAGiiAiXOXAOiiXAAOiC-ACASiXAGCsOAAOOOSOS'iSAAGTOOTC 07 003Α00000370007Τ07ΑΟΟ'Γ00307 0 0 7 CCOACGTrA’toOVGOAAedAGWP?^ 033 ΑΟΟΟΟΟΟΑ - Ί x -. 1 i ' -„ ,. < X ·. u - X v -- ,W'CAG'toC?<i<GrxQCAiA%CCC?M'<tqcSCCrCfc'PXtoKOC-cqAG?Ai;GT<ii.'CCAAOC<iW-OiO<'i-iC 0-707;.?3>00Α0Ο'Τ:3,^Α000:-:.?7Γ,·-;ι··0;70ΐ·-00·ιΟ':'30'3Τ00χό?037:.Α,307·00;Αί3ι30χ··03·77:<370ί0ΑΟ7ό33?Γ 3003:3030370070077733000700000707003703 | 102 |
BMP- GEK | 370070G0700030730070777007000007i:;77007000703G07007007i300000A733700700 ACrOAXTGCOOAV.lCriVACCA^^^ ~CGACGA73G703,OO?:7COAG?77GS'3C-tOOX00Tx'to?!3700A70?70G<3i7O7q3«.3C3GC-'XteCCAC<3 0037073000300:7007007:33 Oi3-7CC7AC37.3C700AC070TAOOO'3i7G07007700000A000700 ΑτθΛθοτ;’Α-θ<ντ<;Αοχ\ν.'Αί;Α·'ηίθο^^^^ 3:7x33033337-700 ro03,Ai.3330-o303.7.0A.33303!700333C33733G7;A0003iO'-330'r’i-f7-3 7707:0,^-7-:-0 tO- '1 > ' fc 0 --, - , CGec<.-:i;>qGCi\Ar;AACTecTCCT'rcc.v:cAcrtXAATC\ACSTOiiMy;^v7.~A.Ti’ftA'X;ccGCOAca:00:0707-0-:730.:77 7000-00370303:00:70303-7000.03007:00:7700737300-3330300307:071.730:37000:7070077003007030070707-00713370301-.71.37317700707330007.30017730.33730:7:0:777707 itoTGGAAG^OZtxOCteXCCTOGAiAGViOAxyOf.'AGOiXC-GTriTCCAAGC&OGAOG^OOxOOATCTO-tGriO^GCC· TGCAf.CAi'JGACiSAGCACAiX’tOii^CC'CAOA'FCCO-XCCC^qCWrgXCA-KCiSGCCACdA'rGdC.WS O-3;703-77,Oi7-77O'7AO:--A>3O-0.A030A3G'?’.30OA’.3OO-7Ai-O0AC7'.Ai3C3'3i70OA3-O03iO0’333A0i'0C-3' - ‘><'G 't -''-θ': G .. ' „„ , V -' ·„' -.-0 V- 1 1 A > » -, ‘ 1 -. -. V. -. -, - X iWX:A0GCeATC0TGCACv\C”CT;X?rCteAGCT<UVKkAA0C0i.rxAA;TMX;TCr,y;c'AAGCCTTOTTGC0-C er;CC?.-r;CGAGCTG<teCGCtoATCTGCO-'?r;C\te;TACT:iCGACCAr,yUAtTCCAACGTGSTC<.3X<Ate\C)AA<.‘T 37:30003037:3730077033000700000707300703 | IOS |
BMP-JP | 3700700000:3030000000707707030077007007770003007007007000::000003:00700 007-73, ·77·7ΟΟχ':3ΟΟ73χΐΧχΟΟΟΟΑί;θζϊ33377Οζ^Οί:’.0 0000700707^000000007037001-3300007 07 0-300 A G'3?i'. ¢73, AO-7 3-7-077 CO AC, 7 'lYjOOPO 7 07 70APCS 7-:777 COi707 70 >-A ACO-GAG A 700A. 7C •.','CCAOCA-:'A'iOACOCC’:'vCC'.O3CC'.CC-7CACACOO7A<TAr,'C7O-r?-TCOCAO;'jCA7'i'CA':'jO'TCAGi0COOi3 G ' G,'O'to' O' --. - * li-' -, '' O'·' > x -Oto- v - \ 1,»\ ' A v A i. , x-,-G , ,. - -, X 1 ' V - - 0-:07707/17-7-7000^03730000,07 773017:0^00-777^707-.00-7-:7770173337777^70000070-.100^7--1730:00,00 3 GG7'G7A,GG?GGsO,teAAAGC;GGT7'i CG?>7 GAGOCOOvrTiO'iTATi'rA'rOOWGi XAAX iGeAGOGTGCAOOAAi'; 00.0.3-73(:000/-.7701:770330:1:30^^0,-1-3777700.000000,00-3700-0730-.70000,7707071-.7:0007:700 GA3«G7'O77O37O:TCACOf.'CC3,f.:'yC:TGAT3?CGCi7GG3O7GC3CAfiGGAO«C<-.<CCAAf.'CATC5GAT7OC;7 03,703^33357000003.077173,3.33:303.3303.333573^70700^33-,0.303,70:7730037.33003007077 770307A. !:-'OAAi3AA03.7A0070G77 .AO A-7?-7AAOC--7i7.A 77007.31773..37,-777-5 Οι-.Ο,'Γ.Ο-ΙΑ'ΐΟΤΟ-ίΌΌιΑί' ik3f3-:7A'G3GXG'77GA!;AA,A?---.AG3i3AA3AACG7G33-..3CGAAAC’Ai7Ai'-Ai7AGG;3-iiAAAC-3i3G'CTA,AG'Ti3;'G'-i3 ' s A a \< τ 0' 1 A G x - , A ·, --. < i- ' , i- -i ' ' X 0 Λ 0 X ite v. < „ X , V .I-·· 3300Λ00003770700303007 703,770303;77ATC3AC00003to7370700xX:33AC37',73;3.-7 37300 OATOG.AXtAG.iAA'-TXCAAAAGGxOVrGti'eiOG-xTiOxGTAGxT'TG/At'G.AtAV-TA'XA-'G'OVTGxGGAi'.iVri'iAi'O'.Gi'OOA' A70AGG37A77G77GTG373GG7TG7GGG?G70077GA | 104 |
BMP-JR | 370070700000300000707077073000770070077170003007::07077000700000000700 0770107'! CC-003.-0;;’'-iO-'7.',CO--'3:',.?,7-OAa--0770-7-700--A70C:37C:370GGO':70t37i',c07i73'iA70CAOOC77 •.7ΪΟΑ0. OAOOi·? C-OJiC-AOjC03007-:30.37-1-3 07:300700'0GA0CA7G770‘0O7'37OA,AA-3.A0A.0A007.A0i3 .....:...7...„.... Q '2 .......So......o-0:7' <; ... | IQS |
2017200239 13 Jan 2017
NAME | SEQUENCE | SPQ ;0 NO |
71, 0^^--.,:,0,01.. TOO. 7:,-Αο.Ο.Ο.<.--..ΟγΟ'-'α 7..1...0.0,0 :010.7000000070000.:0:0007.07000:0000-700001-10-0-10:00-:700:00000-::00:-000007.1-07-1--0:07107-0:--7:-0 /νΟΤΤΟΤΑΤΟΟΟ'ΟΑΟΟΧ',ΑΟΧΧ.ΟΓΤΤ^ΑΤΟΑΟ’Ο'-ΤΟΑΟ,ΟΑ.ΟΟ-οΟΟΤΤΟΑΟΑΟΎΤΤΐΟΟΥ^ΟΟΑΟ-ΟΑΟίΟΤΟΑ'ΟΑ'ΙΑ ΓΟΟΤΤΐΑΟίΟΛΑΑΟΛΑΤΑΟΟΑβΤΤΤΟΟΑΤΟΑΟΟΟΑΑΤΟ AATOTTeATYi^ATOATAAAAOCTOiGAAOlOiO OCAAOTOOOA.AATKOOOTCiZOiOAO'OAOlAOTTTTOOlOOAOlCOOOT-TOX^OjAA^CAGOATGCAiOKOAfiGT'?.: 0:0Α*.0:θΟΓ'!0ΑΤΟΤ0Α0:οο:00:0ΓΟ70Ο.7000<3Τ07:Α0:ΤΟ0Α0Ο.Ο0·ί:θΟΑΟΟ<:0ΑΟΟ0Α'1'0:·ί:θ'Τ700Τ <30TO!:A00700)OOOOOTTOOoAO;OAOO.OAOOA--'A'j-03T:':':COAOOAiOOOA-TO-TTAO:GO-:!AAO;OA:AOOCTT tb'A 7 7. < 7* , . '·> < 7 » 7< . ,.7 0 7 '7- OCCCAiCCTC^dOhCAANWAQAAAAAOC^AAOCOMAOAC^CASCGOiMAoXCC’H^AC'tCOXS CTOeA^;>C-A<.7vCCCTTTC7A<V;?'?<OZ>CTTCAOTGACGKXa'>GTGqAATCi>OTCGAT'iAT,rCCTCCeA OOAtOlG^TOViCOlO-G'rTT'OAO'OOAlOY^OZG-si.lAiOTOOlOCOlTTCOO.SOOT'.lOACOArAOAiGOTi^iATiXAAsOC .ΟΑΟΟΑΟίΟΟίΟΟΤΤΟΤΟΟ.ΑΟΟ-ΟΟΟΤΪίΤ-.ΙΤΟΟΑΟΟΤΐΟΟ-ΤΟΙΑΑΟΟΟΟ'ΟΑιΐΟΑΧΟΟΟΟΟ'ΟΑΟ,ΑΟΟΟΟΟΟΟΤΟΤΟΟGO OOAOAOOOOOTOOA TOOT Α-ΪΟΤΟΌΟΤΟΟΤΟΤΟΟΤΟΟΟΑΟΌΑΟ.ΟΟΟ 70000:0007.00707:: AAAOA:OA7 ., ' ’ '- ' -,- | ||
BMP-2$ | atgotzoco. iooaot e-.k'.-c·;:·'·:. τ-?'·· -; :::005 ooo too 5t<-o-oo:.oo:--o05 0o-':oooooooooogo:tt ΟΟ'ΙΧΟΤ'ΓΟΟΟΟΟ'ΧΟ-ΟΟΟΟΟΟΟ-ΐΑΟΟΟΑΑΟΤ-ΟΟΟΟ-'Χ,ΟΟΟΟΟΟΤΟόΟΤΟΟΡΟΟΟΟ’ΐΟΟΟΟ-Ι-ΟΑ-ϊΟΟΟΑΟΖΟΟΟ-Ο o )- >7 ,' 0, -. 7 - , 0 V. 0 --. is. s'-X GOlOOOlOiOGoiOiOGOl'O.-GTOlGTOOOCOOOTAOfOl-OlOOAGAOOTTGiATOOOOAGOAlAOOrOAGOTOTiOOlOGOG· οο·ο.οοοοοοοοοαοαοοτα:οοο;τ·ϊοχαο:α:<;:·:-α:αοοοαοο·οο;αοϊ:ο:οο::αο-0·0'-οοο:-ο:α0:οτοοο:αο0: 070::0::005 :75-7:;-G.OA:;AA:70COA<.OAAA.OOAG50;:;GOAAACA:OOOOi;:GAOA57:::050'!';o:'A;Or.OT7:0 Α0'θ?0·0;:00'000Α0·-:>::-Αΐ;0Αβ-Τ'ΤΤ.:νΑ:Α.00Τ0ΐ0·:0:Α0ΑΑ0ΧΤ0Αθ0-ί0:77::'00Α00Α;:ΑθΑ'ΐ'00ΑΑ0Α ΤΟίΟΤΤΤΟΟΟΑΑΑΟΑΟΟΑΟΟΑΟΤ^τ.ΟΓδΤΟΑΟΟΟΑΑΤΤΟΑΤΑίΟΤΑϊ^ΑΑ^Τ-χ.ΟΑΑΑΑΟΟΟΟΟΑΟΟΑΟ 0000-..00 5 00.-0000: 0000OTCXOO.AGAOOT'l-rOC'A.OOCOAOOOOTOOTOqOAT'OOfOAATOOiOAO'OOOO-TGOl v ->' a'a 7 v. ,,-r x -' - ΧΛ-. ' ) a \v- -<· .o x i 7170ΑΑ70:7700 - 17ΑΟΟ.-'ΌΑΑΑΟΟΑΟΟ ίΟ. <? Γ OAAAA-Xv'A i ο1 ΑοΟ0ΓΑΑοΌ AGv 7 ο: 'r-:ocA<'-:-AAAOovoAAACAOAoooozAr-':A<OAO>AAAA<A;;--:o'Zzr-:oo:AOzrA:x-T'r:oKA':o-CATOiA-ro.o,AAAA <Α:Ο.-ι,Α·'Ζ51·., 1λ·. -..Α.ι.·α-^οΑ0--.ϊΑ-.·:«>,αΑ<-;Α.0-' .ο07Αι,·'·ο.·0,:0·.7α.Α010οΑο.·ΑΟ,·0.οΑ:ΑαοΟΟΑ·7Α:,ΟΑ·0,7 ι,ο. A-.., 0:-05:00000000000-0-1-:-7:-1000:1-100:005-00000:-00000:-00010---0100:005-00070007505-7-::0501000 .:·'Αΐο;<5θΤΑθθΑ:ο:οοο5ΤΤθθτο:οίοοτοοΑθθΑτ::ο7·::ο55::οοΑ.οοοΑΛθο::ΑθΑ::Α5ΐ::θΛ5·:ιθΑί'.οο· 000.::0000.:0:05005:100000-05-70:0500.:::::^0500000::000::0070500000::000::007:505::000 GO CGA 0 AGA ΑΟ. 0 0 .00 5005 A 705 000 0 Τ 05 O7AC 5' 1' TGACOO ΟΑ Α55 ΟΟΟΑ ΤΟ, 0 70ΤΑ7ΤΟΑ Α0.&Α AT AO CA00A0A5 005 OC'i'OAOAOOT ? 0500077 0005 ΟΑ | 106 |
BMP-JT | ΟΤ^Τ-ΟΟΟΟΟ'ΧΑΟΟΟΟΟΤΟΤΟτ-ΐ-γΑΟΟΟΤΐΟΟΤΟΟΤΤΟΟΟΟΡΟΟτΟΟοΤΟΤΟΟΟΟΟΟΟΟΟΟΟΟΟ-ΟΟ οοτοο-χοοοοΑίΧτοοοοοοο aogaagot ¢:0000:-:000:01 7τοοοοοοοοοοοτ oatcocogcocs θ'τ<:ΑθοθΑθθτθο-τ<ΑΑθθ<·Αλθ·ΐ-'Γ·'-:θΑ>·:ο·-':ο:/:οοοΓθοτοΑος:Α·χτ·:οο<:ο·οτο:Α-$ΑθΑθΑθθΑο<?ο;Αοο οοΑΑ··-;:-ο·Αθο:οί<οοοο<:-το<:τϊ'θ<'ό>·:Α-ΛΤ;τ:ΑθΑ·:-οο·ζχΑό:ο:τ·οτΑΑ·οο:ο'Αθ'.Α-οτ:τοΑοο·ϊ·ο<Αθοοοοο GT07ooa7o:oGAGAc-OAOx>::G050oOxOAa?.:oiOOAx:AOA'OGooocA.ooA07GK:oGCAo:roiT<:c-A07 ΑΟΟχΑΑΟΑΑΤΟΤΓΟΟ-ΟΑΑίΙΑΑΟΟΑΟ'ΟΑΟΟΟΟΑΟΟΟΟΤΟΟΑ^ΟΟ-.ΑΟΑΑΟΟΟ.οΟΤΟΑΟΑΤ’ΓΟΤΤΟΟ'ΤΤΑΑΤΓί.Α ι Ο 7 ' ( <. ' ' Ο'--,, ' -. 0 ' ΓΟΟ'ΓΟ ΟΑί'ΧΑΑίχΟΑΑ,'Γ ΑΟΟλς-ϊΤ'Γ'ϊ ΟΟΑΟ ΟΑΓΟΟΑΑ''ΟΤ·ΑΑ·-ΟΑ'ΟΤ·ϊ-ΑΤίίδΑΑ·ΓΟΑΤΑΑΑΑΟΟΤί'ϊΟΑΑΟ'«.0 OCAAO'rOV-AAATTCOCCCOOAO'CACAOrTO-AC'.'-ATCAOA'AOTC-T-TOCio-OAAOCAO-AATOCAAGCAOGT-CO OAXAQT'T-;'-.KAAoOTCAC<?CCOO-C--iOT:’AAT-'AX<3-tOGA<:o00:AO-A';CAAA<:CACOCOXA<':0'iVrCGAT-i'C0:-'r o-':oiooA.AO'ro'0!3'':CA!:ccX'-;oAOCAAO-AAAooAA--:coo;'::cTcoAAOAOiA<;\A':ro-r::«oooVAA,AOAOAo:OTATCi 3 0:0,:,0,0.0,::,-,:.¾ 1\?.'ΌΑν.·Α-..Αι,-'<.,Τ--·:.-<.·?ΑΑ,Ζ,ι-.-Α<ΑΑΆ.ΆΑ:.-.-'<.·Α,0,Αν ΌΑ<0·-O'A:.-'.l ΑΑ-0.Ό 5-'1· α 0:0:0.1.,07.:·-.Ο0Ό,·:·-.:χ:7θ <:OOZ:?iT-eZTrO-AOCAO,XV-0:0:?X.V-?OCACi;TOiAAOCO-ACOAi:AC<AZOAOAO€0?-OCOAO:0000ri-A<A070<A\0 0-n0TAA0«iJA07iCC0TTT07ACG70.XUTCTTCA0;T0Aa?y0'A'A5TfX5AAr0ACTO3A,i:?AT50OT0.0O\ AOOO':-orAT'::'ACCOOTTO-OA<'OOOO.A'TOCO--..:OAT<T-OC-TOCT-TOiOTAOTCAACOOACAOATKAA'rOOAAOA: ΑθοθΑχ:οοΑΤ'?οτοοΑο:Αθττ·ΐϊθοτο·θΑθοττ?ν?<:οΑΑθίο:οοΑΑο:·:οΑ·Α:ΐ-ο<:οο:ΑΑΑοοοζ.:οο·ί'θ'7<::ο' ΓΑ3·Ο:'ΑΑ0Α0Α-30'Γ0ΑΑ·ϊ·00ΤΑ·00'Γ0ςΑ;ΤΓ00·ΟΑ0Α0·0'Τί3Α0-0Αο0ΑΤ'ϊ'00Α0<ϊ0ΤΤ0··’'Α'ϊ7ΑΑ00Α0Χ·Γ 0 Τ0 A 00 AO A TOOT TO TO AOAGO 5 500 go 05 ο-τ co οτο ο | W7 |
BMP-AB | ATOOTGOOTCOOACOACAXOTC-rOC'i-OOCCCTOOTCCTOCCOCAOOTCCTSCTOCOOOOAOC-i'OOTCO AC'mG5OO00OAGCi50GGOAOAAC0V^AG7rOGCO6C7OOC5O0TO:i-G0O0W0'-O35OOiiOO-CAOOO7r -,,0,0·:Αθ·0:Α.Οί—,0,:0, .< ΟΤ,,:0.,0:ΛΪ),·<<α5Ο,’:ζΑ07 Λ 0:0-<::0x0v'01:<7i’0i-Tl-.O:iOI0OZ 1,-:..-1,-1.:0-:.. :A::OGGC:OoA.O:<.:<*:.GO,Av.O, Ο-ΟΤΤΟΤΑΟΟΟΑσζΟΟΟίΤΟ-ίΑ^ΟΟαΧΙΟΟΤΛΟΑΤυοΤΟΟΛΟΟΤΟΐΑ'ΧΟΟ-'ΧΑΙΟΛΟΤΟΟΟΟχΑΟΑΟΟί-χΟΙΚ ΑΟΟΤΟ'Ο'ΤΟΟ'ΟΟΟΟΟΑΟΟΑΟΑΟΑΟΤΟΟΑΑΑΟΑΟΟΟ'-Α-.ΟΤΤΟΟΟΟ'Α-ΐΟΟΑχΛΟΑ'ΤΟΟΤΟΟί-ΛΟ-ΟΤΤΟΟΟΑΟΟ .aO'-aaoa;aa'TO<'0';y:;oaao;aaotgoo<aaao;aoato-oo;-:;o-.aacaocaoocgocoootcoottooa;\ooto TOOTO'OAT<:A:ocAoo<AAA<:iAO'TT<:A'TOA.o.:TTOoocooAcoci:-rO.Ao::-TO'?TcooooA:;oAOATOCAOo-A 0-OCOO'TOOO-:AAO.AAOTCO-3o:C7'ACOOCCAOOCO.i<:rO'AAOA5 0TAC'OAiAATCAT-OAAOOCC'OCCACCO COAAO 5000775 COCO 0700005 0000 OOTOOAC-ACOCGOOTOOTi'OlACCAOAACOlCOO'CCAOA TOO V - 0' C\ » - , ' , , > 7< 0 V 7 ' , 0 00'1>00ΑΛ:τ-ϊ0'''?:00Α00Τ··χ;7Α0Α0ΑΛ00Α00Ο00·ΐ:·0'ί'ί-,·0ΑΑο0Ο0ο.Α-00?ϊ-Ο000ΑΤ0·:00:Χ00·ν0·00 'ιοα-.'ΑΟ·:-:αοοαο<·;;αοοαο:ι-α3<:'οοο·ϊο:·οοαο3τια-.'ΟΖ3<-.ι:··<:·οοτοοτοο·ιαϊαοαττο<:οοοαοοο'.το<ιο.ααο; OZlO-CAZO-.C.-eZl'lTOACAAOAOACAO.TAO-OO-GCACiAAlA-AGCACAA'iOAOCOOAAGO'OOC'-mAAOTCOT:-: 00000000000000000001000000000-00000.:0.0000000::000::000::00-7::000:07-1--7:1:000000. .770OTAGOA:,7 :7?7:00:,T?007.CiT:77:TG CTO-77:7:00050:Τ57Α;7.775οΤ7Τ:070Τ -,/ ' 1 · - 3' r O [5 ο o - ,,,7 7 7 7 O' ' ' ' -- , : ' t ' ' I - 0 -O'C . ' ' ,, X ' - 7' ' A , ' x- '< -- 7 -- ' | IBS |
5'
2017200239 13 Jan 2017
NAME | SEQUENCE | SEO ;O NO |
BMP-BO | A TPCP P<P Cl PPGA GC AGATGTPTGG TCP GPCTPP TP CTGP CP CAPGTGPTGGTGGPPGGACP ΆTCP PC ACTPPTPT.PTCGACC'AGTA'AGiCPCAPPPGACP'rPCCGP;CP;GPCPGGTG’A'JCGG>:AAX:’.r’.PP:PAGGP;\GGPTT CPCCXP'.iACPTCCAPCCPPC^PTTCGACCPPCCPPTCPTC'TPPi^TpTCerSCCTCitePCS^f'oopCCP'.CC ΡΡ'ΑΓΡΐΑΡΡΟΑΡΡΡΡΡτίίΡΤΟΡΡΡί/ΡΡΤΑΡΑτίίΡΤΟΡΑΡΡΡΡΤίιΡΡΡΡΡ'ΚΡΑΡΤΡΟΡίΡ'.ΡΡ,ΰΡΡ'ί'ΰΡ A5'PCPPTCPPCC'PG?jCPACAP?xCTP:P'P.ACA.PPCCP’PCCPPCC:'.:-CCAAPACPCTPPCG'APC'?'?'-'.'CAPC AGGAGG-A.A'iGGPTppAAGAAG’ApCGCPAGApAP'PpOGPAAGA.PGA.pCGPPCPPTTCTTp-PTGPAA.PpPIG PGpPGPAPCPf.GApGpA.AGA.PTPGATGA.pGPGPGG'GGG'-GPTGGAi'jGPpTPGpGCiGAGCA.i'jGTppi'-pP?' A pi P»' ' ' λ P --.i · ' ' o' --X ,-P P ΡΡ,ΑΑΡΤΡ'ΡΑΑΰΤ'ΓΡΡΡΡΰΡΡΑΡΡΡιΡΡΡίΤ'.1ΡΤΡΡΑΡΑΡ'ΡΡΡΡίΡ'ΆΆΐΡΡΡΡΑΡΡΑ.ΡΑΑΡΡ·ΡΡϊ·ΡΡΑΡί.ΑΤΡΡ <w'p-<x <,P PΆ <ΑΡλΑ:· 5 '.^Ck.P't.-PTi-P,.<.1-Λ CA-ί?'·.·.-·ApA Λ <ΑϊΑί-Α.·Ρ-ΡίνΟ·ΑίΆίΡΡ--·:'Ρλλ.ΐΐΑ.·:1-.·ΛΑίΆ.·ΛΐΌΐίΑΑ, Pp P'GC PCGPrPAAPTCGGCCivGCICXTPrvPACAACCAPCATPPTGTPCfP'ArGGPiGGGP-.GGPG'syf'GTCTPCGTPCG TPCGCGAGGAPGAPCACAGCTCGT'CPCAPPPfGCGGGPCCGPA:?'AGG''.G?xAPATTCGGGCAGGGT'PGCAAG 'PCCPACPPCC'APCAPAAi;ACiA'rACiP'.PCt;PPPxGPPCAA?PACPPxACPPxCiy:rCAA'PCCPP’.C?rAAC'?C':?r.iP G'A'teA.AGGGTA'A.GpCGPTGTAG'G’APGCGTTGrC'GGG.pGAAyGC'iAAG.AAC'GA.rpTp-GATCGTGPPCGC'A: CGPPP:PPP!pC-iO%GPPAGW'A'.GCPr'PpCCGCTOPPCC-APPAC\'APAAC’T<ppiPC€At''CPACPPCS';'iA'iWG PACGPCG PP'GGAAC TCCG'APA-iCAGCA.AG ATCG G'TAAGPCCPAPi'C-PGPGTPCi'CApCvAGGGAG'rGCPG CA Ti'CPPPGGPTGTACAAPGAPCAGATPPPCGTPCCGACGG'GGAACAAGp’AGPAGPACATGGTGPG CP ' i ' p ' | W |
BMP- EBB | ACGPTGGCTGGCAGCAGAAPTeTPCTPGCCC'iOCTGCTPPCCCACGTGCTGCTPGGCPGAGCCGCTGG APTCPTGCCPGAPPCCPPCAi'.WAPAAAPT'tCPPCCP'rGCPTCCT'PiPPPCPPCCTTCCACCPAGCGT'T PCCACCAPG'CPPCCTPCCACCTPGiP':PTCP<;GC?G-C'?PTCCA.TG'T';'CGCPGCPP,ACCAG<P'.:CCCC;\CP GP TP PT A GPP AGPPGGTPG'GP.'. GPPG GT AGA. TpC TpC A CP TG T ACGPPPGGPA CTPCPPAG A PCG'T pPi ATC-fGGPpGGPPGpG.pGA.pAGA.pTGGAAApAGGpipGpTpGGPiAGPPAApACGPTGCPPP'ipTTTGPAGP APGApGpAATCCGrCPAAGAAC-ippiTPPi.CAGATGO'TGGAAGAGPJAGPPGiGHGT'iC-iTTTTGAiAGP'rG pppPtGA'VPPPeAPCGP\Ar3.APT:i'CATCAPCPPPPCPPAGPPPCA'GCP-GT-pCCPCGAPC>A<3Ai-GCAP<GA CPCGPTGPPCAAPAAG tCCTCPTTPCAPCATPGGATCAAPAT Ci AGPAGATC ATPAAPPCGPPPAPPG CPAC>PGTPCAGP';prPCCPP/:pPAPCCPPC':PPCTPPPPPAPCCPPCP-PCTPA.;\CPACi'P\PCCPTCPACAP'PC <C\P/rPCPTPCAPC'IPACCPPTPPPCPPPpTPACA;<A'P\PPCCPPAPPPCPA'-PCPPA.;\PPA'CPPCP'TCT PiPTCiPAACPPGCPPPsCPTGPAPxCAPAGPTPGCPPCiiTPP'CPPGCCCGPAPCPPCGPATC'ATPCPCPCPC - , < ' , x - - 'X ' PPAPGAPCCpPTpiPACA.ApAGAG.AGAAGPPGCAP/GCPAACpACA.A.GCAPC'GPP-APPGCiC'A'GAA.OTCPTi' pPppAippGGPAGΡΡΡΡΤΡ'ϊ ΐ-ΡΡ'ϊGPAGP-r;'.'P GPpACPTGPPCPppAGf'pACT'PGATPGTi'AjGCPCP-P PCGyPTApPACP/prpTTCP»APpPPPAP«CCtoAppGPTCCT’;'CCPCCPGArAPGCP<?ACAPpAACCCPAPC < ' - X - top K , , , ,- <-w x ' x v -.,. GPCGACTTPP-CGTCGGGCCGATGTPCPrATC'rPTAGAGGGAPGAGGC'GPCGiPGCCGPiAGCCPG-Ai'AACACC ACGAGPACATPGCPCpCPAAPPCTPPGGGCGTPGGTPA | 110 |
BMP-09 | ATPVCPPCTPPCACCAGATGTPpGCTGGPCCTPPpPPpGCCCCAPGTGGCCGCCGPCGGAGPTPCtGG ACTPGTiiPPCGAGGTGGiiPACAAGAAAGG TPGGGrPCPGPCGPGGGTiTGPPGGPGCGPGAGPPAGCPGT C'PPiATPACPT':'A:':G-TCPCAGTTCiAACC5T;CCPPTCPTG'PPP<A'PC:-TPpr:-CCCTCAAPCA;PPGCPPCAPC PGPG'CPApGGApGGPfjGPTGPp’;'.ppCP?ppAPGPTGGApGP'GTSf'pGGPPi>PAp'.iCCpGApAG<PPTGG Α'ΓΡΡΡΡΡ GCPC GPP? AGP AGAp APW5G AAAGA.'PPGCGAP'S CPPGGPCCAACGCPGP'GPGP/ATP PTCGAG:' -G V,»' i ' -- ' ' - P „ 1 A- 3' -.A ,. i ' - TCCppC.pPpCGPAPPPPPPxQAP'pppAP'PAC'PTP-CGGCPPAGp-iGGAGiGTGAP'PCGCPAPCAi'iATCGAiPi'te CTTPCCTGGC?PAACAACGCCTCPCTGPACCACCPGATPAGCA,pT.'TACGA.GATCAlCAAGCPCGCPA'CCG GPAAPP<P;GP'A?TTCGGCG'AP?-GPPCPPi'GPTCP;AGACPPCP;PPCp-A';?PiGPGGA,ACGPCPG''.G\PATCG GAGA;GPTTGSAGGTSAGGGGTGCGSTCPGGSAfSiCGSAGCGPCGAGSGpPSPSSCAAGCA!SGPTP:GST iA'iTCrGAAPGrCGGCGAGCTCGGP'.PAGxAAGGAPGiiGSPGTPGPPAPCGGPGPGPrrGPPGATlGTGTiGGGTP'CP: 'TPCACC.AGGAPGA.P-CAGAGPTGGTGPCAi.P>.PCPCCACGCCCPCCP;GGOAP«TTCX'rGCGCG«TPGCXAG CteGPApppCp'AATAPAAGAGAGAGAPpPGPGApApyPAppA^A'PCAPASPpAGPpiGAApCGprTrGAAGPPCP-p G''A'sP-AAGP'PPGAPCGGCTGTA;'.G'APGAPTTG'PpCP>ACG;'PppGC'APGA.AC'GA.iP'iGPA'iGAP'C'PPG'GCT,A -? v i -.» -c' p< i -p' P p' : p ' , p -.0 -p «v,'' -. AAP:GAr;PGPAp!ppAlGCAGi<CGPto'PACAiAPTPCQG’Ato5GA\AGtoAGAATPG'CPAA<3PCGT-'A;'PP«'X;PtoPC CAPCPACCPPTCGPCCATCPPPCP'PPP'PTACAACGAPCiGPACGPCCp-APCPPACPGTG&AC/CA'PPAGP AGGAPATGPTGPTCGAAGGPTGCPGPTGpCPGTPA | HI |
BMP- 929 | ACGPTGGPTGGPACCAGATGPCTGCPPGCCPCGPTGPTPCCPPGGPTGPTGCTPGGCPGAPPPGPTGG AGTCiPTGCGPGAPGfGTGPPCAPPxAGAAGGTTGGPCGGTCCPTCCTP'P'GPPGGrACCTTCCAGCGACCGTT TlCGAPGAPCCGGTGTGCGACTCGGPxGGTGG'PGG'APCCGTCCATGTTiTGGGPGGAAGGA-GC'.GCGCP-GG GPTPpTAGP'GAGPPGPGP'jG'APi'.'GPPPGTAGAiYjGP'PGG'iCPTGPA'TGGpC'GPP.ApTPGOPAAGA.pC'G'PpP; AT GTGPTPG PCPCPA.pGAPAG-A.pTGfiAAApAGGPiiPGTpGCpisGPPAGGAC'CpTGGP'pP’pTTP GPAGP APGtePPTTG-TCcGi'CGAAGtoAP'GiAPPCPAP^GA'iGCHCAAi'iAGPAGGPGiG'C'rGTTCGT'CiTPPAAGG'iG, A Gi G' - η -i P ' G ' ' ' < , - s x- - , GCCGPTGPPCAvAPAPVPiCGTCGTTCGAPCATCGPATCAA-PiVrGTAGCAGATCATCiTAPPGGPCGAPCG CPAGPTPCAiGPTGCCGPGPAPCPTPPC'TPPPPP-A.CAPCCPPCP-PCP'PiPPCPACi'PiGCCPTCGAPiGP'GC PPA5TPCPTPCAPG'GGACGGPPGGPC'TG;AP'PAGA'PPC;TCPGCPPPiGGCGGAPGPP<\G.PP<AGTGCPTTGT GGTGP;AAGTPG0GCACCTGGAAGzT'_;AAGC?\GGPCC,TGTTCPAAGCGGC.APGGTG7GGACCTi.'G0PGG:CCC TGGAGGA.pGGOPAP>PAGA.--A:’PGpTGPP.AC'A.TCG;GPPGPPP'OPTGPTiPSCATP-ppGCPAGPATGGpPGP3 G,PPCAPCCCPTC,P.AC AAPAOAG,.AOAAOPPGC.APGCPAAPPAC'A.AGCA.PC'GP,AAOP>PGCTG.?5APTGCTC - ---ί ί -T tG , Ά- ' - G ,g - ¢- , | H2 |
2017200239 13 Jan 2017
NAME | SEQUENCE | SEQ © NO |
4 },k k x k k k w k N >k Ik k k X k ^k | ||
v x k x^Ikk k λ k k x > v \k k v x k k l x x v c i ti kw x n e > e < e Ύ xx< ^k k'*”’ k | ||
BMP· | : X.r'.ViSAAArt'A'^.;'’ -s. ,’Χλ'1'.^. ,Xk •.'X-k'.'V-k.vΑ?,-Χ,Λχ·.ν,Ας a,α^αΑ-χ,•Α,ΑχΑί,ν.'-Ανλ;.v;.->xvre,-k ,·\«ν.·ν,Λ;.χ»<·> .Λ· 5 <> k k Τ k \ Χλ χ λ χ Χχ X Α - > X | 113 |
L > χ > c X X λ X X3 X * 3 X λ 3 X k < | ||
k X £ ί - v < a V. 1 V - X XV \ V v | ||
t < V χ „ < k A Vk ‘ k k k k k -k %k X | ||
Ak k k < k \k k k k < 2. k k v k X ,S A, k | ||
k J X 1 k k vkk a> kk kk t k k kkk 3. } 3. k v k | ||
V “ ( <. ” ' , ,, \ - , \ , ' | ||
c - ~ -i' x ” <' c t ” ' c \> > - , ' , | ||
XV. X X V X X - v XX . X X -, χ. «..‘-..-.•x-tik. < ' .-x.-Z:re-.k?-y ·:· kx.-k. k.k>;k-,-..-..k.S:c;?k..-χ^\·:«·>.\.-.'χΚΛ.··νΐ·-:ί·'><1··ν?··;Α:«· .< <<?.'-k<k.<.k.:xx>Ak.;-..-;?x.'s.,i χ.\..':·Λ;:·ν5. >. ;·:>χ>· | ||
x k k \a -k k * k* k k k k k ki | ||
> -k k -k -k G -k f k -k -f- k X kk -s a x a t -’ ! £ t > X X k \ 3 \ X X3 k 33 | ||
λ V* k ** A ίχ( - k Ί < A kk j. kkkk X X kxkk k χ k k \ k kk k k re k χ k re re a, re k V k A ~ kk A X k X kk Av k k X -JX k kk x | ||
> , ' , „Y , 1 , , ‘ < » , ' , , , , C ' 1 < -. ' k A t kk kkJ-k A k k k k V k k k | ||
' crec ic x ' - ' - , i c ' re i c i | ||
..,,,c ...,,....Λ.,re: .,,,,:. . . rerere. ....... .a. re · ..«re.___i .·Λ·.χ. t.i-.k.;.-;.·->%>·.->,·;·{<..re. : lx.', k.k;j..xx xre.xvx ;·-.·>ζ\·;χ.«;·.:-?:·..ν>\·>··;.:ι\·>.kk λ χχχ j:m«. ....... | ||
BMP- | V A. χ Λ - χ Α,-,χ -k X A χ A SA X X A χ k ^ίχ χ t χ xx kx χ k χ Λ- \χ k | 114 |
OAK no | χχ < ' t > χ P X· ’ C > χ ' C ' x· k χ ' \χ' ' ' k ' 3 X ....................s k'A.....k.......' < A............k’ s k *..............A kk i.....x...................χ· | |
SAGA | t .\··ί GAAGCM. CMAC > 1GG C4. 'ΜΑ;Ρ4ΡΑΑΟΑ<4ΰΆΜ: G'l AACc,’ 1GCA·; ’ ACAaGA'C,' GA CCKT ACM4CCGreIACACC'CCGAG'AAG:.MCACG'C,CCCGCi’WG3CC,AACA'i'CC-GCC-GI.rCC'i'3 GAGCA TGGACAAACGGCATGrGCATTAr'CGGGACGGAGGAGTTr.'GCAT'iTfMGAAGGAi'.'A-KlC'iGGTGTTC/GV·.' ATCTCCATCGGCGGGfMreiAGGAGATriACCAGAGGCGAGGTGGGGGTGTAGGTiiTCGTriGGAGAAGCA GGTOGAi.'CGC'rGCGACGACCTGi'iAGGGGTC'C!?:TGGTG.fCCTAClGAGGTGCTGG?sCGGCAGGGAi.'GGGT AOOAC?Ci,'>CTACC.3AG.ACASAOAC<'-nceTOOTS'?CCCAOqATA-?CCAqdA<kSAO,3dC5-0«OAqACA G'CA?MAG'7G7'GCrCGA'lGGG'MAA4GAG4T:'A'M?:CGGC.’rGA'lGlAGGGGGGGAAG'rGCAAGAACAA;3G'GG;'M M > V , re Λ v . ,, . ,.,. ,., - , , ACr-'TGGCCT'MTrt'G'r.’M'iGGCiCTGG.kAG'QAGfGsCrCCGGCC-Gi’AACGAAAGAGACrM'GGeWGAAG-M AGAG4GA'M.4''M'''GGGC4GG4GGAGGAM'3:GGGlc?'''CiGAAP,AAGGGV'CGGAACGAi'.'GGG'iCGACCGAGGG GGGAlGAG'G\:GG;'CAG<G'CGAGGAG'.SGGG;iGGGGGAGGGGGCAGGreMG'rG'i'AGGC-'iX;GGGAGAGGGA AGCGGGAGGCCA4GCACAAGeAK€GGAAGC<'MC''MM4G'7C''A4GCTGCCAG.ire,AACCGCCCTGAGAG';'G' AA0'GC'Xk'?G,G'.\A'V.lGGe'GGGA<SAC:CSCGA'rCA’PCGCCCGieAA?'.GAG'?.4CGAGGCG'i'ACGAG'?GCAA ΟΑΑΑΟΑΓϊί^Ι'ΤΤίΙ-Ι'τΑ'ηΑΑΟ'.'ΑΟΑΙΑί-^ίΑΑσ.ΙΤΑΑ.'.ΧΙΑΟΟΑ,ϊΙ'ΟΑ,^ΑΙΑΑΑίΑί,ΤΟΑΙΪ'ΟΟΛΑΑΓΓ.'Α'Τι'.Κ T0CACCTGAiG'FlCCCCACf.AAAC,7q5'3GCAA0GCCr0CT0G3TG<XCXOCAA>3C2'0TCCCCCATCA0C ΟΤλ'ϊΜΌ'ί ACAAA3AGGAGG?C3GGGGGGGCAAGGC7GAAGGAGGA~GAGGA5A3GCA’rGGGGGTGAGCGA ,cpiiggcmggggtga.................................................................................................................................................. | |
BMP·· QAKSA | A7GGG3GG'iGi3C'GG'rMrG:MGG:MGGGGGGCGGG3GG'MGC';CGGGGGGGCGGGCGGM<3GA3GGCAA GGG'TG':'GGAG'i CG'iGGYMGAGAGGiCi GGGGWiGCdGPA'l'GC'l'G ?MAi’MCGiCTg5GA<At GGG'i GGGG GGGiGiG'!'i-AlGGGJ«;CAGAGGT’!'G^.G.r.'re:GAAlA''reG'GGG'AGGAA,G'AlGTGMMG''I''GGACT:reGTGGGG· | 11$ |
TCa:’rcZKV.Z:*iGTCCGrA-.-CrGCGGAC.GCAG<«\CAAi';AGGGGC-G,S'GGAi\eeGCCG<.-AGTAGATGATr;GA CGTG'T.G:?AAGCGGTACAGC'TCiG'.GAAGTGGAGC,V.'GCCGGGC'GCAAGA'?Gre7Gr.'GGm?re?T':'?;',GG?1 rGtAAAGATCA:GA'rG'?GC;il''rACGAGGAGre.li.GGAG''7'GGi:.?:T';'CGAGA?’.!'.:.GAG?,TGG'fiG'AG';?CAAG ATC7G..'AA7G..'.T.GGGGGAi'AAA.:'AlA,GA7GAGGArAAGGGCAAGGTGGGiGC-'TGGAGATGGGCT-GCGAGAACGi, :','G77Ai.KGGi'.G'!Y.'GGACGAG':'.'7GAAGGIX'7''’.lCGG.'A'ArGAT<:GAGGACG-T-GG'i'3GAf'.1<;G;','i\GGi're,GG~GG V V ,» V ' ,X Ai i, A, , 'Ai^A.kAGiroTG'A'XGG0CCr/i0AAGNi?5G,'M,'iGG&k7GGGAG-iCCAeCAAG?'’;P«A0A«CAa.GG!i-GGA Af?IG?re;GG'GG,M'reGG-MGGCAl.G\GGGG''KlGGAGACGC'A7GArere.cTi-;GriTGCr:GGG'AGG'i-GGGGGA AGGGGGGG7TG'rrre?7GG”A;TTGfiGG?reG':'G'AlGAGTGGiGGGGGA;:'G3GV:.GAGAGAi':GGG3-GGAAG'AA· AGAGAnA-AA'7'GliGGGAre.lA«GAAnAATGGG-rGG-G.A.y,-7G,GG7G’GCAAiAl,i'.GAGGT':l;GAGreGAAGG CGGG.'AAG~GGTGTGACGAAG.GGG;!-.GAGGGACGGCGAGG'7f;GGA':.GlGGGG7GT7,CGG?rGi;r.'C7A:Af:'GrG, AGCGAqAGGCC;V,GCACAAAqz'irt.'Gt;AAGCGGCTf.«V,GTCCAGC'rGCGCTrAGre3GtAG'Gi?rCCCAC?GC GAi'5AAAAGG'rCGGTGAG.AGreGAA<vr-iG.;i.r<re,ACr,7Grrere-C:'A';GAGAGG-rGGAiGAT'.;GGCGGGAAA.GA G,'rCG“GG;'.G'rA<l'GAA,7GG.A?sGGGGGGG'?73C7'7;''r7GGGC<lG;'A;CGi'i«GGACG'l'G-AGGi:\GACGAAGC AGGCGAG'GG'l'QGAG ftGGC'iGA'GGGAiGS •A-.'MAA;C'G?CC-GAGrGAAAG'M;AGGAAGGG;.-AiGG'i GGG'i GGCC GCA C , G , » , <> C ' - , A C v ,-, -M -· νχ>Ά£<('··.;<νϊ<.Α·Λ· k<l.-<?'k^;<:.->k..kA;<s kkrTtrCx kik;;,.-.-:'A',k:k-A-.-kjk;'i'x.>,Ax ................................... ............................ ' | ||
BMP- | ATGGGAGGTGGGGCTG’rGTGCGTGGGGCAGGCAG'rGrerATCTGiGCTGGGGGGGAGGG’rGGAGGGGAA | IIS |
GPP | GGGCC'GCAGTCG-rGGGGCACCG5GGTCGGreireiGGGr;Gixrre';;re?G:AC?C'.lGCGTi:7<;GGAi;'!'GGGTGGGG GCGGAC'W.;C,Cre.;?reZ'AC;',GC7'T'G;AAGGGGAAGi'.'<;?~'7'GG'r«ca.;A?'.ACG'T'Gre,GA,A'iGAre7TGC'?r:.GGG TC COT G A«CC i ¢57 GCGGCG'AGG C GAG C C AGG AC AX· AG GCt'A''G'iYjGA AG’GGGG C GAG TAC&T$ AACG A CGTGG AGAAGGA'GTAGAGG''iCGGAGA.AG7GGAGGAGGGGGGGG''i'GCAAGAS'GG'i'GCGG'.!GG i’GCA(iGA ...U...AL\a>......, |
2017200239 13 Jan 2017
.....NAME | SEQUENCE | SEQ ;Ο NO |
ΑΓΟΟΟΟΑΟΟΟΟΟΟΟΟΟΑΟΟΑΟΟΑΟίΑΟΟ-ΑΟΟ-ΑΟΑΟΟΟΟΛΟΟΟΟΟΟΟΑΟΟΟΑΟΟΟΟΟΟΟ'ΟΟΟΟΑΟΛΛΟΟΑ OOOT'00?xOOOO-0000:AOO?xOO'iO,VxO;OOOTOOO;kOOyi-t-;AOO::ri:OOAOOO.OOO-00;OAOO;OOAOOO;;'xeGOOO? &’.X;ACTCCCr?TACCGA<LACAAA«A-?i'TTCC'TdGTi;TCCCA<:K/!TA:JCCAOGACGA^Oi?-r&3C!AGAq/i OTOxOtojOOG'tOOTOOOOOO^Gz'iAOijOATCSOGTkSO’OOjO-CxAOAOTOOAf.'OAAfiOOx.OAAO^ACAOxOCOfAOA .Οα'ΟΓΟ.ΑΟΟ'ΟϊΟΟγΟΑΑ'ΟΟ'ΟΟΑΟΑΟΟγΟΟΟ^.ΟΟΑΟΟ-ϊ'ΟΟΟΑΟΑΟ-ΟΟχΤΟχΟΑΟΑ'ΓΟΟΟ'ΟΟΟΟΑΟΟΟΟΟΟΓΟΟΟΟχΤΟΟΟΟΟΟ-Οχ ACCTftocdxcrxcq'rGOTfttfTtrrccAAccAccAiTfccTCtoSGCAsxsAA^oAc^oiiOCTGOAAo^ .00;AGAGAOOAOO''OOOOAO\OA.O:iO.OOO:AOOOOOOO,'0-Y'OAik.OAAOC-A'iOOOOxAOOAACO'OOTOOaOOiAOOrA'.· 0 x.- 0 ' 0' A ' o '< ' 3 > * ' > λ x ' v' Λ- i , ( C- , ., -A k·,, kx ' A .. xx” a k 0'AOA-0'AOxO:0000.'OTOOiOAO--OAVxO:T'00:k:AOk;AOoVIOOOO-;-xOOS01'.00:,OC'rOOOOOOO;0:Oi;0'OOOOAOOik;?x OTAOOixx:OO0.-*iAOOA0;TCO.to'xO:O0Z.’x:OO'i'O 00010:-ΓϊΟΟΟΟΟ'ΟΟΟΟΟχΟΐ AO χΟΑΟχϊΤίϊΡχΟΟΟί'ΟχΟΑΟ'ΟΑΛΟΟ .XCGCCATCiJT:k7AGACCCT<>:'rTKOACC?r;?MV?:T?CCO>CCAi':A«5>3':-GC?’.AGCCC'i‘<;CTGCC-TCCCC ACCAA«OTG:ra?CCCATCM^tTGrT<T«C.tokt;GACGAA;JOaSC':TCA?CAACCC:J:-GAAO?ACC.S.C:rA A 0,0 A.·.·;·: Λ·ί ;:.·Γ •/Λ;;.··:· 000000000' ,ΟΟΟΑ | ||
BMP6- SA | AT GOOGG OOO ’! O-.OGG OOGAOGO COT AOOGG COCA 00 OGOOGO OGGGOGGA OOTOTGCA 00000000:005 SCCOC COCCOOTOC-'AiCC^COiAGOOCOOXGCOCCOOOCOOfOOCiOGCOSOoO'SQCAserSCXtoSGCXG XQ 0 k , V 1< < x a * , 0 k V ' x' A ΟΟΧΟίίΟΟΟΟ'Ο.νΟΟΑΟΟΑΟΑΟ-ΟΖν,ΟνΑΟΟΟ-ΟΟΑΟΑΟΟΟ'ΤΟΟΑΟΟ,ΑΟΑΟΑΟΓΥΓΟΟΟ'ϊ'ΟΟΟΟ'ΟΟΟΑΐΟ'ΟίΟΟΟΟίΟϊΟΟίΟΟΟ,Ο XA..k?xA,;,.CxA.AA, kA..k,0.O:k..Ax.-.A-A<x, .i aOAAaW A AA: O<AAAA.-:x.k,XA.A.k>x.· ΑΟ.χ.ΟΑΑ,Αν.τ:,.Α':Α.,Αχ,·':-χΑ:χ,Α:.Α. A-ooAOOAOA:A;;.CA<;':0'TOfoC'To;;.oioo;AGAf,;:ooooc'00':oo'ooof,OAO':oooAAGOO':ooO''Of;coor.;oT'i'C7'.-A> ΟΟΌΟΑΟΟΟΟΟΫΑΟΑΑί/ΟΟΟίΟΓζϊ'ΟΟΟΟχΟΟΟΟ'ΑΟΑΑΟΟΑΟΟΑΟΟΟΟΟΟΑ'ΑΟΟΟΟΟΟίΥΟΑΟΟΟΟΑΟΑΟΑ'ΟΟΟΑΟΟΑ 0'GOOGOOOO>OAO!yA?Y'OGA''000.0000 0'OOT.OCAGOOOO:OOO.-xGOOGOO'OC-OGOOO-GOCO'OGOAOOCGi'.· OOAAO: OOGAOOAOOOTO 0 ”00000 CCGG AOO'YOOOAOOGOOOGCOOao COOOAOOx3AGOAGO:GOGOOO ΟΑΟ'ΑΟΟΟΟΟΟ-ΟΟΟ’ϊΟ,^ΑΓΛΟΑΟΟΟΟ'ΟΟ'.ΟΟΟΙΟΟΓΟί/Α-Ο^Ο'Ο'-ΟίΟ-ΑΟΑΑΟΟ'ϊΟίΟϊ-'ΟΟΟχΟ'ΟΑΟ-'ΟΑΟΟΟΑΟΑ^ SO ΤΟΟΟΟΟΟΟΟΰΐ OAGCOACAOO AGAAAOAGO TO ΑΑΟΟ'Ϋ 0ΑΛ:?;Ο ΑΟ ΟΟΟΑΟΑΊ ΟΟΟΟ GAGGGOG AGO \ „ ·,, -' ; ' - , χ <. ., i- J Ο'ΤΤΟΟΟΟΟΑΑΟΑηΤΑ'ΓΟΟΑ,ΑΟ-'ΟΟΟΟΟ'Α.ΟΟΟΟΑΟΑΟΟΑΤΟ/ΑΟΑΟΟΟΟ'.ΟΑΟΧΑΟΟ'Ο’Α'Ο'ΑΟΟ'Λ'ΟΥ'Γ'ΟΪΟ'Α'ΟΟ'ϊ'ΟίΟΟΟΑΟ Α'-Α·?Ον,-<χΆ<ΐΛ:Α0Υ:ΑΑ?\,ΐ„ χ Α;·ΑχίΰΑ,·ΑΑΟί<Α· GGOa.AGOxxxAO O'.; ΥαΑ,ΑΟ'χ,ΑυΑ; kA.·:. Αχ. ,ί Αχ,Α.ΑΑ ;,Ό.ίΛ: χΟαΑ: τ·'Α·:θΓί;ΑθΑ·ο·ο;\θΑ<;οον?ΑθΑθΑθθΑο:?:θί:το'θθθθ:οτί;Αθθθ'θ·Α:·:;θ'Τ<:·ΑθΑΑίϊθ!ΑΑ'ϊ·οοΑθ'Τθ'.'θθθ'ϊ'θθΑίθ OGOOGOiO-OOOAOAGOiOOOOOTOGiO<;i1OAfOOOOfiO.OOOOAOGGOA0100<\GOO':OOTOAOO'0;’.A>VO:':OrOTTCr.:T ΟΑΑΟΟ'ΓΟΟιΟΟΟΑχ.ΑνΐΟΟΑΟΟΟΟΟ-'ΟΟΑΟΟΑΟΟΑΟζΟ-γΟΑΟΟΟΟίΟΟΑΟίΟΟίΟί'χΟΟΟΟΌΑΟί,ϊ-ΟΑΟΑΟΟΟΟΟίAO OGOOGOAOOOAOOOOOAOOAGOOOGOOOGGOO ?COAO?OOTOOAOA TTAOAACAGOAGOOAA OOO ' V χ ' \ χ'* χ X ” χ „ k χ XX ' X ' 1 i^GACGO/03.G'''A'iG'AO?Y-GcrGGOAO'r!\OG'rOOkOOOOGOAOOOOrOOT'GiO'i-TGOGAOOCAAOC'GAOGOAOGA Ai'OGGGxOGOOiOOY'xO.OO'.O.OixO-'rOOGOOOxOAOOr'OTOA'Or-m'OxkOkOrOkY'.GOGG-oOGOGkiOxO'OAOkOGiOOO'OOxfOOOCO 'XOGiGTOGOGGAArOTitoOZ.-TAJiW^OGCATG'iGOfOXTGtrxTACiTTGSvrriO^WCTxV-ftATC^A'-.^CT ΟΑΑΑΑΛΑΟΑΟΑΟΟΑΑΤΑ^ΰΟΟΟ'ΟΤΑίΟΟ/ΟΟΟΟΟΟΟΟΟΑΟΟΟΟΑΟΐΑΑ | 117 |
BMP6- SL | AOGOCGGGGCOGGGGGGCAGGGGGOAGOOGOOC'OGCOGGOGGTGOGGGC'OGCTGaGCAGGTGCTGCGG 000000:00000-0-000000000000000:.0000000-0:0:000000000.:000:.00000000.-000000000000-0:0000:0 ACO-Of.ooSAGCiOCCOGyCGOCACtiG-BOOAOOCOCOSCGOTCOCOOGAGjOGOX'CCTCO^GT'rcC'TfSTSO G'OOtoO'OGOCAAOACGGAOAGAiOA-GOOGGO.OGGY'OOtoA.OOxikOOO'.GO-rG'O'AOO'COiO'iOO’OGOGjG'GOiOGOCO' 0' <> x' '* ' \ ' x - k k -.χ, x x , -χ, AOCkO'GrYAGOsOGGAGxGTiOOG'riOOOOG'OxGAOGGOOG'GOOGCOAAjCOAkOAGAiOG'rrCOOkiCCOkOX'G'iOGA'OO k.-GOkxAGx,.'Λ χ:·ΟΑΟ.''αΟ,·Οχ.Ολ,-ΟΟΟχ-. Ο.ΟΟ',.θΑ!·.·ΑΑχΑ.χίΑ<ΟχΟχΑ,·Α0.,χΑ,·Ο·;Α.χ:.ΐ !. kAAxxAA.-v.A:A.-:--Ak:Ox.,AkA,,· χ 00'C.'C'rOOOGOrAOOOOOkAOAOOAOOOAOO.'0<OOOI\OOiOAOxO;0’?k>OOGOA'-Oi‘:GOOi'/00''X;0'-00'0;GCiOGOOOxGGOGC TkoA:koOOAAOOkooo-TTOO-005<A:of.'oo?:Aioo':''GOOAO:;o:'ot’:oooooo:'i5'OOOA:OAOO':-JAOOOXOO:o':.'OOAO; ΟίΟχΟΑΟΟΟ,ΟΟΟΟΟΟΟΟΟ.ΑΑΟ-'.ΟΟΟΟΟΟΟΑΟΑ'Τ-Ο'Ο-'ΟΟΑ-ΓίΟ-χΟΟΟ.ΐ'Ο-ΟΟΟΟ.ΟΟχΟΟΟΟΟ'ΟΑΟΟ.Ο.ΟΟΑΟχίΟχΟΑΑΟ/ΟΑ ΟΟ’ΓΟΟΟΟΟίΟΟΟ'ίΥΟΟΟχΟί/ΟίΟΑΓΑΟ'ΟΟΧΟΑΟχΟ.ΟΟΟΟίΟ-’ΟΟΟΑΟΟΛ-ΟΟΑΑΟΟ'ΓίΟΤΟΟΟΑΟ',ΑΟΟΟΟΟΟΟΑΟνΟχ-Οί'ΟΑΟΟΟ; TOO'rO;AO':OOiO'AyO.?“-OOx?'0-”0000..'iO':'?'OA.ixOOAOOOO'0-::-OAOOOOOi?!00-roOA.f'xAA/kk:OAA.?:OOT-Y-0 0'TOOO.i''OOA-OOOATOAAG'00;':'YAO.f'G-OAOOA't'OA':'00,''i'CAOjOxO.AO>--:0-A's«00':OOO'.OY'A's-YO:-,;OjACAO· Xs- .χ ' - 'α λ ϊ e 0 X χ' 0 !\ χΧ , ο·οο<ϊ·θΑθ·ϊθθχΑθΑθθΑ·οθχΑθΑ·-ο-,>'θ·ΑθθϊοοΑθοθ'ϊ<ο<χΑ:θ'ΓθΑ!θοο?χθΑ:Αοοθ<:;?χ·ϊθθθχθοΓθθ'ΑθοοΑθθΑθ θ'οοο:ό-;Αθθθοθ(ο;οοοο:;^θ'-ο-.;ο<ΑθΑθ?χΰΑθΑθοο-.οο;ο:ο·τΑθο·Αθ-θχΑο;:ο-Αθθο;οο:'οθΑθθθ;·-Α·;οοοοοοοο’.ο X. ΑΟχΑχ.χΎ χχΑν.χ'Υ χ:Α'χ:χ, Ο-Α,-!.· Αχ.-ΑχΧ?ΟΙ:Α..ΑχΑ..Αχ.θΑ0:χ:·:Α,-Αχ,-Οΐ-.·χ:'0χ.Αχ:χ. ί,ΑΑ?χ.:^’Ά,ΐ«ΑΑΑΑί,^Ά:,Αχ·?·Ά χ,-;-’.: A .ΟοΤΟίΑΟΟΟ.'ΟΑΟΟ'ΟΑΟίΟΟΟΟΑΟ,χίΑΟΟΟΟΟΟΟΟΟ'ΟΟΟ-.ΑΟΟΟ/Ο-.ΟΟΟΓΟΟΟΟΤΟΑχΟνΟΟΑΟ-,Ο^.ΟΑΟΟΑΟΟχ-ΟχίΟΟΟΟ; ΑΑΑΑΟ.:ί10:00·ΪΟ:ΟΑΟ·:θΑΑί50ΑΟ·0:ΑΟΟ'ΟΟΑ·0'.Οί;θ·<ΥΑΟ::·ΤΟ·ΟΟθΛ?..Α'·-Α'>000Οί>·-.·:ΑΟΟΟΟ·Οχθ;''-:;ΑΟΟί:;θΑΟΟΟχΟ OOGOOGO OAGOiOOOATOGYOOGOAO-i OOTGTOAOOGAOOAOOCOGOOO-GOOAOOOAACOGOCAGOOGA ΑΟΟΟΟχΟΟΟΟχΟΟΟΟΟ-ΟΟΟχΟΟ'ΟΟ'ΑΟΟΑΟΑΟΟ'ΓΟοΟΟΥΤ'ΓΟΑΟΟΟΟΑΟΟΑΑΟΟ-ΟΟΟΛΟ’Γίχ-ΟΟΟΟ-ΟΟΟΟΑΑΟΟχΟ O0000'r000'0'-0A.A0--0k0A0;0:OAA0'O.0i0 0A-r0000to0Or0':OOA0-T0'--00A-r00ACA,-k0O00'AA'i'GO0A-Y-'Y0T .........- -'..... · -...................- - · .0.............·'·.......'.......'.......................................................... | W |
BMPS-A | AOOOOGOGQO'iYSOOGGOOAOOOOOOAGW3GGOOOGOOf5«OxaGOOOGOOO:OOCOGoOOAOO:OOCOGOGO o.:-oo:oooooooA'ooA-.'OA-oo'x5o:o-ooooA'oOkAOooo'Oo;o-o;oooA-AO-.:oA:oo;oooooA-;k;o;ooAoo:ooo:oooooo-: 00^00000000:-:000:000000-.0-00.^000-:.^00-.:0-.:0-0::000:0:.00--010^0-0000^00000-000-000:00000:000-00-:0.0: ΟΟίΟΟΟΟΑΟΤΟΐλΑΟΑ^ΟΟΟΟχΐΟ.ΟΟΟίΑΑΟΟίΑίΟΟ-'.ΟΑΟ-ίίΟΑίΟΟΑΟΟΟχΟΑΟΟΟ-ΓΟΟΟΟΑΟ-ΑΟΟΟΟΟΟ-ΑΟΟΟ'ΐ'ΟΟΟΟίΟΟ. ΟΟθ:θΑθΟθ:'ΑΟΟχ·:ΟΟΟθτθΟΑΟΟ:χ;θ'0:ΟΟΟΟχΟ.ΟΑΟΟΟθΑθΑΟ;;θ'0000'ΟΟΟΟΑΟΟΟΟ'0'·-:;θΑΟΟΑΟΟ?χΟΟΑΟ::θ·Αχ·:0' .AOOAOOO'.OiO.-oOOAOO'OOOO'iOO-OOOA.OOOOOx OO-:’OCO:'0OOCOACOO0AA;k:-rcO'0C'OOi','k:O-0OO-OO0x'-.A0 0''AA?xA.-rO-OOAOAAO'0000'000 00'000'OAO.O'.ACOA--OOAixOOAOOOA'jGOO'i'COOOAOOO;OOAOiOO;AOOOOix Χ 0 Ό χ χ -Χ' ΟΟΥ χ-χχ ' -- χ.1 -χ ΑχΧχ \ '- 0 ' ' χ· O'- ' Ot | η δ |
2017200239 13 Jan 2017
NAME | SEQUENCE | SBQ ;O NO |
7GAOGGGOAAG9GO9A3OTGOO9CG9GGAG9O9GGAGi?GGOGGC90-G-rOOGGA0TGA0GAGGGGG0AG <Α\σΑ:·':^;??€τϊ:7'ϊθ.^\σΰί-ΓνΑ?ΑΑΖΑ,>\'ΐθ9':'σΑ·5τ;?Α?<:τΐ·ΐΑ:ΐΌΑ-:\Αθ':νΑ·;·ϊ''·Α·9'Α?,Γ?<Ζΰ?,σκΑθΑΑ 0?TOT'?CCOTf.OT3AOCO«»i?A9f.;z'.-33A{'>3.i:?AGTT3.-AAO;T7CAACT'rO?CO'C3A3A;3'TG3.K3'fAAOtOx:--r03\'K rf^TGACGGCTCCAOAATTi'.'CGCATCTACAAffoAC’rGTC’r'JA TOGOGOGT'33TAAOAAO:OA3-&0'?-33'3 CTTA?<\A«CATTTATCAA&K:T!rzV?A»?!AGCATCAGC?teA«AGA.C5CTf;ACCTCT'rT?TC'rTGACAir 9CO'33x3iA97 3-373000-30 0A3xAA3.-'-AO‘3O3'-.3.3C'3'.0’3»33’3'-3-30.79373-9.0003903.900093-3.--7300---3:013 'n'CgOAi'.yC-rACAiyCA’VAACArogggCVTCAdg'i OA900O9033A03AG003.000A03090003003.0 A x.- „<>' ' e V* 3 ' ' V i ’ O3,AAZ3-A':33'0-rGk0GG'-£O9A373rGG'--,33AG93xC0AGk93'3i\0O9-:COAG0GG3iGO9COA93A9G0fo:3iCO--39 r'TatCTCTACfXAGTCCCrSGGACGTCKCGaXlOTCTCCrfoTGC'rTCiiCA'^Ai'ftACAGCACIfOAiVlTi·; AuV\A<;Zfo”C:iGfo\GOA^x<iCAT;AAGX::TCTFTr7rGA9:x,f?CC?teGAeCTGC£?,A:iC-G<.'SG9ACTi’ZJATrO\T •^?:CAC?C.?PiGfteJCT?vK?CTC'?C?Pi?T»CTG,fC!ATCJGAG;>ATGCTCC'r'rf.'CCTi,7OGi'!3'GA:JCA'rOT':-i\ AC'T'?C/»CTiV»Tf:?1TOCCATTC?^<'?;C?.CC?TG<;?T?«>C?CK??:JA^CCCC«?te:TATOTC<CCCfiAACro 'W3i;yC?GCOqrAACt.».AgC'3'AAA-r\yCCArC?CgO'TT''^’TACKvrClA-rfiS.CAAeTgCAATCTCAWCt ' - ' ,1 | ||
BMP6-B | AG09C00GG0'30300;39003Z3009GGA3GO09G0-rGG'TZ9-000000GG0O3G00G££-09A0O3GG-rGk:00 ' ,. < x ' x-x A C -χ,,χ-.χ-, fVCCGCGGtVFX'CCrAAXIcrfoAaKiAGCACCKCaKXtGiCGi’COC/foTCrKte'iCQrfoCT'rCCTG'fA/.' CCi»CCCCeA;CCCCTCCACCGCCT-?C.<V.iVxGCCGCAG;CCCaX-CGCTCC^»ChGCAG£.kASGAGi;ii':c ActeAGCAGCAGCAGCTSCCTCG •'.to«;iGz>GCCCCCTC-C>?rAGOn9riCTfA'>A!'.:-TC<y;C<5CCCT<'.:rtTCAT\:'! CTCj0ATf,?GTACAAC-0CCC,nd4CC-XOTA0AACGA-XAC0ACO3r»0O:3TC<3GACg«00ArsSC<fC&CC& GlCCT0<3<i‘.CCAC<5AAGCAftC<>AGCT’'^T<:cCA-X0i3XX;0CAiSC<S3CCCSg<>-3GC<3CCGO3CACiX0<; rCAA.CZXXVAGArAl'C'riCSG'AXdCCqGA'iCiGQCA&CGGGCaXCdlCCateC’J'GitCCkqCGcqCAQ GA>;foQC%CCTTCCT-CMCG?!Cq'’;QQA'rAT--AiSC?<roAGGT7'rGTO%eCCi'G<3'’.'GGAV;e<.'GSiCAAGCA ί ' ' -.,. \ '' 3 39k -, ' x 1 k -1 -, 'Κ?σΓθΑ<τχ·?7'^θΛ^(^τϊ€”9«τσϊΑ'?ίννθ9ί\σ?9τοϊ7ννκ?Α;οθχν;^τ·ΓΛΑΛ<ν^<ν;ΑΑ''.:τιτϊ cfrTA'i'CAG<?;iATTA?^Aq'rf.i--\\czv'z;Aqf;<\'iCACA:ACAGA'-A\e--x:z'GAOG-AArT-?,rrGT'i'GqAAAC CCGTirAAG'TAtXAIGCCTGZ^GA.AGAAAGCTfAXtTGaAATT-AGZiCA'i'CACGGCi'.GACTAGGA-V.iC'AzZ.rars TTGTCASCTC^CAhCATriACATOGRGCTOCAGCTi.SSGCGTCGTGACAAGGGi'Tt'XGh.GTCCACO'rCCAC CCi7CGAgC<?'XA-XC-e?AGT‘X!GCAf,tejAC<5-:3CCC'-'’«COATAAfjCAi3i:CCTTCATS<34rgOCT'rTC?T gAAAqygAfteGAGCTccAaiioqriCAccA'rgAOv'rcAgccTCgscccoiprgccgACAAOigAO'rnfjTA ' , i l x ' Xs x * X X X >· xx - -, X - - A ' Ί -. X - 4, -. k * TCCACCeAAGqrfo'TATGCTrZ.’C.V-iTAG^TCACatAQAAKfGCCTT^CCKTTaiCTi^TCA'XCTGA ACTCAC:AAA'rCj:;TGGCA'nGTGCAG?>C:r'IG€A''iAACrK?TG:rr?-Ai?->GtoiVAGATT':':GT?-AC-GGAlGi': ?-AKrr-r;eeAAeTAAGo:'A,v-T;x?AA^^^^ ' ,- - ' '’-'-- 'V | 120 |
BMP6C | t ΑΛΑ-Α.Α-;··'·;-;·Ά·..ί:·Α.A-GAAGAv?,..:? a g: α·λ,αα\.Α·..-?·α-'·ϊ\:Ά;Α';;Αι·:γ'·ϊ·:;-;α.:α.. C.CCCC COCO G0T003GC C-X CCTTGCCCOCTOCCSC'SQCCOrCGCXCC^S’StSGCAgr^SCl-OSftO’S Aay'iCGCiOA-xcccGdccocAf.GOAGCA’yf.CG-cco-GcqTcgc’ccgS'j’rgc'te'f.TcriGocT-icc'raTAc Z <>' -fox ' »V -A > kA' - * λ k -. -x- xx” kx. 09G099G'G''3G'k:099'-£09AC0G-CGr9Z.9A93s099O1ZAG0Ox'90Oi'i'Z00”3ZGOG0A0Z'i!0i;9i00Axi03.93Z Jiqx;>qG\AGCAGX7AxZ:txOrzC7-CGCGGArteCCCCCei<X:<.WGi:CPiCTCxVxClGi'GCOCCCGTC'?TCATi·; G:;'<yjAX'k'TA17ix:ixACC-<.:CCTGTGCKk'GGACAu\CA?A£''k';AGGrA?GGGGGC?iG':'A;AGrA;GGACxi'.GGCAGC.f:i -'.ίΤχίΖΤ'χχίχΟχΓΑίΐαΑΑ'.ΊχΖ-ΟχΓί-Ο-ΖΤσΑΤχσ-ΖΑησΖ'ΓίΑΧ'ΓΟΑΑΟΓΖΑΖΟί.'χίΑ&ΟίΖΖί^σΟΓΖΑΙΑχΛΪΰΟ·:·:· TCAACCG-ZAikkte0CGTGC?-:kCA'ZxCCx;G?'.?xT<;GxZA5fteGCGGCni>:kZCf>:'.:CATGAx>:'”’.x:':ZGCk:-Cik,G ίΐΑχΛο.?ΑσΑ·?τοοτχΑΑ<'ΑΑχ·:·;.9ί;χ;?χΖΑ·Γ·;ΑΑ?χ:?χ·κ.;ΑΑΖτ?τητίζ;'χΑθχ£κ>·':;-τοζ^Α?ΑθΑθ?χΛθχ:-Ζχ - 3 A ' '- O' ,. ' ' χ ' X ' ' x'x ”lx 1x0 xx' 0-3',Ο'ΟΑΟΟ'Ο-Ζ'Ο'ΟΟΖχΟ'ΖΑΟ'Ο993,03 TO ·7·30Α3Ο9?Ό'33£0-373'·3'?-·03·3ί003.300'3'·3Ό!3\ΑΑΖ-3χ0903Α3·0'3'30' 9'ϊ TAG GAG'C ATEiAiC'-W/i CTTSCASGAgC^TCAGCACftOAGAC •feT’SACC'rdCTTiW-S QOACAO £''X?grAOTA't<if'AiCcrUAdAAqAAQ<.yAtqo,:-iq<5AA‘i7'i'G&CA5CAfxqfo;«C’rAGCA?-'j-<'--Os%rsQ 'rTrteCkA'?:reGACAOCrkTAUxCATGC-GGC4TCriOCm»Qi.OTCGK:AGAAi?SCATCGAG:iCO'fox:Ti;CAr: ccecGAc-ca';t\Aax:c;coTGx;G?xC-M;?xC9Gcefte,i-AGGrfoAi\kx;ACCCCTTCA'i-GOTCc-c^'i\.::s'T CAAtSGTGAGTtory.KteCCACGTGCGCACCACCAGf/TCAGCCTCCA.GCCGGCnxteGACAfteAGAGTCGTA AOOGi:'?kGTAOCCZ>GT9OO3.0::G3G6-3G7.'-OGC7k:-x;TOTGG.AGOGG£O3GZx0x3'OTGG.AAiO37:;G'3GT0x3ATi0G 33..A3C33;00T3,O39G33.>303T3;.009T03’A-£OTi.3?xO'3'ΟΟΟΟΖχίχΟΑΟΌ'ΤΓ,ΐΖΟΑΤΟΟΟΑΟΟ.ΑΟΌΟΟί'χΌΟΖΚί' 7x00T3.0'3737 0f330'O3,T030000T0 3iOA0'O.3'r03T:03307x?'.O09'O09T0 093A0T03A90O3.9Z-C3.TG3 s.TfjCAAZCAACCACGCCA't’n'jTGCAfteCC'tTd-.iTTCACCTTS^-ASCCrCCAGrATOTiXCCAASCrO iZ x. 0 - - lx > - x > < X ..- I x GxO ' X- 0 ' x'x , 'x >' X -- - | 121 |
ΒΜΡδ-0 | A'OGGOGGGOOmOGGGGGGAGGGCOO-OGOGGGTGZOiOGGGTGGTGGGOGG-AZZ'WAt-GGAGGTGOAGGGG G;GG-r:eGGGGGGZGG-Gk;GG;ZGGG:iG<ZGGGGGZGGGG;GG!k;7GGG;ZGGi':GGGZ;C-'-Zi'A;GC-AGG'ri?Z-:'f:;Z;k3GG AGCA1CGA'rG-ACAG.xZG-ZGGG'Zi.\GCxkG.txGGZxGGGGCGGCGGTi7GGx?GGAGZGG!t'GG'ZGGG:Gi'Z?'?GfZTx:-'i';kG Ci'ZGXra.;CTCAAi;ACGCA<AteC;-VxGGiAT'!AGATi.’<;^GAA«fAAfP!TCTTGTCGGteGCTiX;GGCTCCCGGA 900733.0093,0093 ¢-33,0303,0030003.3-033,0 ¢:33-,00900073-χ73·9Ό<303ΐ-307χ003730Α;3ί3?5θ0ίχ0Ο ΛΟΟΑΟΟΑΟΟΑΟ,ΟΑΑΟΤΟίΟΟΤΟΟίΟΟΟΑΟΑΟ,ΟΟΟΟΟΤΟΖΌΌΟΟ^ΑΟίΧΑΑΟΤΤΌΌίΖΟίΟΟΟΟ^ΟΤΤΟΑΟίΟ Ά Ϊ. *’ .,30 33- -a x' O xx x v A-- 3 ks'x, ' xx | 122 |
2017200239 13 Jan 2017
I NAME | SEQUENCE | SEQ ;0 NO |
000(70 000000077770077OAOOOOOOOOTOOOOOAGOOOOOOOOOOOOOOOOOfOOOTtOO7700000077000 '(A.-00(,,:,7:. 3.0, χχιΟ',/,.,χΑΐ-a 7.'(3:,:,7:3. (,,:,3..3,:,,330:., i.3j3i3^\3>3,3x3A.x::.x-<O?x,i.:,’A χ,χ.7:.3,Αχ, ,5 7,31-3,(,,030,:,3.3,3,0,3, 00.0007-:7:7000370-5-:70000/:3-:,370,3:037.0(-00,:-0,3-(7:50:337-3-00730(373000((-0000,37003-000.00,3777-,7:-,3 ,07500075(775000000^.00730750000,7730000:0775-1003707700,00000000700707:,3077 0770073077330,7-00,77075,70.0077377,0000-00037,0.775,7007500,0000-7^50730750000/,0.003777700:07.70000 ViTA'i CAQCaW a rCAGTi ¢00 OCA:'50 OOCA-KCAOOAOOOAOiSOOOOOAOCOOiOOOOOOOOOTSAOSO ¢0(300,0000AOO333^0i73re,AOGAA0GCOG0,0OGOAO(reOi3O0AO0A0('O3370O0OA<teA;3(30Oi'O30,3(0 Ι07σΓ0Αθ·ν<0ΑΧ'Α<ΧΑ\ΐΑ?ΑΑΤ:χι0'0·':·';ο:;\Αα0·ίΰΑ<?;Α/ϊ0:07·0ιΑ0·.?^0ΐεαΑΤ3ΰΑί0'ΐ0θΑ0η';-Γ0Λ<? ' 3 ' , x x Ax- 0 0' ' ' v.Vw'‘ x o' -, >3 v i C,V^533075,5x7075A33<57i'3537A7.x7007;373?07:37A.07'0075037A707'3?07;:'70i7;i7770757.'7;3700,03??J333i«',0370i07700 A. J,’,,GA.'Λ’1,< 00.3,-(.,3,Οχ?θ3<:.,5.:ί-ΐ: Αί/lx, xi χ::.ί(„3·,χ,:„:-,5·,':, 3, m.x.G:,- xxO.Ox'x, .7,3,/1( .,/ ,3.,3,0:./:(,,'./:3, i 3,/1,¾ /-'.O: /:.00.:7000:(000370750,:0:/0/:0:::0000000073(0500,0005737,303300::0:7:-(300 0003700330.03377:0007-37 (-50370000-0000:-50300:7/-137077000000:70::(00/-:01:-533/,370003:0:(3700003700:370:7-0.30:0700/7570053¾ ».O33CAA:',0AAi?0AO00000O:'5000A0A:77000:>5T00OCCO0A075AAC7XC:3AGOA00?-0!770CAOAC00 'r00'('0((73000COi:0O:5?xA0'i0A:3'(00O?,0-0'rCOA-('OCO:3(i%07.'0-O75,30i0A(iAA00AAOi,0,;5OOOOO3O ' ' -. ' ' | ||
BMP6- AOHL | ΑΓΟΟΑϊΟΑίΟΟΟσϊΟΟΟί/ΰ 0007500000 7000:000(0-7 0:307700(37700000:3000000(3:0(0550--5 0000 <·,(,,Ι„’„<.3,:^'«-<.-0(„(7χ,3,’«·3,χ.3,:·θ73,ΐ.,'λ·'33^ΐ,,*„·.χ/„1. (5 x,:.. :,.-3,05,Λ·:,5.-7/,,7,-5 Ο,Χ· .1.3,7,5-:,3,75,3,0,7, 3.,33,3,-/3-,3.-0/-,,:,353,1, .A,i.,3,3,7..3,X,5,03,3, 3,1.,3,3,7:,:.-3,-3,7.-:,ι3,-:,,-:ζ,χχ,χ,θ3,3.Ο1χ3ν3/:<1ν3.-5,-Τΐ,3,7.-3.3,3,/::1:,:/:3,-5, I 3 .-3,0:..3,50,1.-730. 7..1/5.-3, .:7,03. 05737035777.^000750075000^,/:330,,3737753077075,:07500:7:0,7,7555007:0017077^0777:7^77770:-,753.7750077000770 5-075777.-00000770000-750.,:(777530775-000000075005757,7777000773^3:0,00=777-757700:50007507775077770077 Aq'>.G<\AGCAfte^AC^OCT:rC!C'X;AAAi.',f.CCCCTC''f.OqiSi?dAi'te-i3AAO‘te'i,<5Ci3CCCi;'TCTTCA^ ι'.-ΐΟν,ΑίΟΤΟΐΑ',-ΑΑα',ΓΟσϊΟΤΡΓ.ΰΟΟΟΑΟΑΑΟη.ΑΟΟΑίΧΡΑΟΟΟΟΡΟΟΤΓ^ΑίϊΟϊΧ-ίϊΑΟΑ^ΟΟΑΓϊΟΑ 0·ΓίΧτι·?300σ;Α/;0ΛΑ0:Ε^0ΌΜκ·-ί'00;χ000Α0θ·ϊ·^00ανΧ^'0οχ0<>;Λ'0\ΰ0σ?ρ00Α000δ0 ¢¢00000000:00000(:((0:0:30:7^^^^ ' A ,''· x re < - XX ' X GTTi7VO<'-;7eTCGTfA\GCGAC.reA7AC?GjAGt\G'iT:7AA<AreCA?xC'TTAAC<:Cf/AA’i-i\'GKAAOOGTOAGK '5'00/:'005?OOOOX;OAO?Ox'-I-.(0(7-575?ArO--'0(-(..OAOOAOOO-7-Ox7:A'3-05737'7;00-:'':'-::--7'OOi'xiOAOOOAOO-:'T(:‘'>' CTTA?CA«OATTTATCAAfvK:T!rAA»?:AGCATC7A;OACA«AOAC:rC'rfteCTOTTT,i:TC'rTGACAC 000007(7:00,7075(500000000/::730:-,000037:50-7(3(7550300(:00000-37,0:0777-:0(700(703:00/.00:(370:5037 Tte;iOA:','tCACAiXA'VAACATnGX,CVTCAq0'idA«O3Ti;0TCjACAAgG<3A5G'5A0.?<XacqrcCAC οοοοοαοοο 000077770075-0-5 ooof.APoooo'jocoiT-oooi.OoooAtoo.oocircoo-oooiSi'i^oiiiT-oooco-T Ά A A A ' A : ' X ,,.' ' X A,? χ , < χχ, - 4 A χ % ,. x Λ AuV»A<>\Gv<reGm\GCAA<5vAT;kAGCTCT»T<zreA'G:r^7<:Cr>AGrxeCTCGCxATCGC’AGGAOTi’^T<'.b\T 'χοοΑθ^?Λοα·;νΤ<νκ?σκ:νθ?χ?:ΤΤ«ντ:?ϊθΑτα.?ΑθΛ^ΑτοοτοοττοοοΑί·:'Λ·ο<:;?·:ο<Ν\τοΑα.·ι<ν\ Ατοο?,(\ο^Α{\οονχσος?θΑΤ’ίί?κ?<;Αν·Α<·σττα?ττο^χτϊ,ΑτθΑΑίΧοαρ“Α;τΛ'κ;τί.χ;οο\ίΐΑ<:α:- TGCTGTGCCOCrvACTAAGCTrAAi^GCCATC'-TCGGTT'C'rTTAeTT'rGA-PfrACAACTvCAATGTCATTCT ' , x ”x ’ X, X I ” | 123. |
3MPG- RK-KR | 0/0: ':.00-07¢--05000000000-/- -0¢0-/(0: (-.000(50000((0000000000(700:1=(000000(7007:000 00030000000(7--000000000--,-(7-0-5057,000(7-7--.0:7700-77((500000=0=5050:-.75700000077(7-770,:7-0-000(70:-,700: XX- :, :, x xx .A ., o ree re < - 1 X 00M0:7A300O0xkOAA700A0x0A:7?xA0300OO00AiO0A:7?xA0O?x0A-O7'r'rG'<0!707iO,0-i0x7-7A':C's‘000'7,0A O57GO5? 03ΌΟΟΟΟΟ 00000000x000000 07-,770 0(7 ¢7.000 (700000000 00(700(700 OAGOOOGOG 0.00-0 070700:07::7777^007700-7707000000077107--,0000007-:-7-000:70700,70777001777077:7-:707700000707:0000 0-A700:00-~00;7i:AA70;0:7O'T007::000770A7:'.:'.A:777007;,7070,i707:-7700007:00,70i?7000Ai7;70770;70Ci7 0077·?7Τ7ίΟΟΟί70Α770ΑΑ077Α7,07:Α7,7·:'Τ·00'Τ·Οι7'77Λ77:·070·00707Α07:0770ί700007?0>:7000707χ·:0.ί7000077 TOA,707'707AA7,A770;7T0077700f.:C7?OOOAOi7'7007i7AOi077;07070f77???007:00,7i700-,7077,7007:-OOOA7; O00AO077O77 0-7'OC'7'O,77,O'O.A00?-70O,AOO 0-77707 CA-tOAOO’reOOOOAACCOiTiOOGOAGOAOGAOAAGOA o, ooco-tx 0007. οοοαοο-τ^ααο-αοαοαοαοοοοααοοοοαοοοοοο-οοοαοατοοοοοαοοοοοαοο 70:7iO0AOO7S0'SACA-:7AAO-700OO.OOO:7A0Ai70O,AO'7'-7?';77-i-7A7iO,:7770i,AO;-77i0'77x?x7A7x0OA7OiiO-7'7iO'7 0-77.70-:7.70,ΟΑΟΟΓίχϊ 000x7070 j ·ϊΑ·:7Λ.0·'7ί7770Α-ϊ0Α:7ί7ίι0Α7777θ!,·777'0\'·777ΑΟί7·ϊι·3'ϊ-·7·ϊΟΟ770'7:'77ϊ77Ο,Α77 0770000,777-70.-5770 07077:77777.V,7;;OA0077070?00707.07777-7G,77rA'77:.7i7707;07;7x777'A707ii,70i7-:-:7770777; ',-1 3,0:,:,,3-. :7(,,:.-A3, Οχ,χ.Ο : /,0(.-,,73-:,,:,(-,3,7, A '-3 χ,Ο:.χ(Α:.(-,,13,3/,,-.: χ,3,'3-(./0,003,3,::,/,.73-(,,:,/:3,7 :-3.0-5.:.-/3 3,7,,03,. 7700770Α:77:θ077,χ770077·03;7,'Τ07070Α07χ37Α773?0077000,700Α7:,;,ΑΑ30Α770:70000Α070777ϊ7;θΟΟΓ.700’.ί· 0OA0:OO:35OOO0O0OS(OdA0OO=OOi7OA:OOA0OO057OOA0O0O0O077Oi7O77OS00OO(;OOOOSOOO=Ci5OO : A-OOOOf.OAOi'.f'Ais'iCOOAOiaAO-OOfjGO.'teO-T’teOOiOCAOOO.COOCA-yAO'rArAAOAOOAO^O-AaOlO ,7.ΟΑΑ,0.ΐ:00-07ί 070-07-7.70:3007-7?ΑΟ·Ο377?7 770:7 ·;θ.Α:7·7ί0Ο0<7ΑΑ':7Α00'7Οι-ί:7,77Γ·07;7ί77ί-.3:7ί3ΟΟί70ΑΟ'0??Ο 773000-77(7.00-300,000 3Α·'.:·07·'30:'Α3ΑΟΟ30·!'ΟΟ·770·Ο3:7:Ο0ΑΑ000Ο'00'5'Ο:700Οί'.Ο0Οί,0:7ίΟΑΟΟΟ0'5'·0;?' „·, Α, „ ' „ V ' * 30- 3 - χ, -003θ'·ϊ·:·3:Λθθ:·30£ΑΟ;77.^30'?';0?χΑ-’'·:3£ΟΑο370·'Χ3300373 000770··00,Α·:37ΑΟ,χΑΟ'ΐ'03ΐΑΑ·7:θ!θ07'χΟ'7!·,-7 OAAOAAAOAOiOiOAOOAOOOOOOO ΑΑΟΑΟΟ 0700770ΑΟΟΟΟΑΟΟΑΟ . | 124 |
3MRS- RKKR ADHL «g | 37000070700-5-7777077000000077003,00000700000:700:307-7700077000773-07-:300:3030770000(7 (3:7770(7007700000:700:7:77700000000:77500000(3037007:770000077:-.077(377000(70.0(7-3--77070,:3(0737 = Α0(7Μί03:3ΟΑ(30000000000:Α0ΰΟΑ0ϊ0:Αί3ί3770Οί7ό0ί3:30:0(3ί30;3!3ϊ7Ο'0:7Ο0ί30:0ί3Ο73Ο:573;00'3βΟΑ0' -:700,-0(3OO'7'C-AA(3 A 0(30 00·’37χΟΑ.ΑΟ ¢73730 7^-3 7χ 7 00 ΑΟΑΑί'/ΟχΑ.ΟΑχ'-χ 00 ϊ'0'3'0(,330(3(7 3-0(300,00000( ,ΟΑ 00:^00(70:3-73((00000,00,70-3300-0:700030,0:7-000(3(--00:7(-7033(7:000:70:37-:3(70107,73,.00000,00,00:5(-. Ο 0' w „ t ο ο -,- ο< ’ ' ο Ό'-.χ ' χ' ' re χ . ο , , , ; | 125 |
SO
2017200239 13 Jan 2017
NAME | SEQUENCE | SEO ;O NO |
CiATTATCGGTACAAGGCCCGGTGeGGGGArAAGGACGAGGACGGGGGGI'iTGGAGGGGGAGAGGGAGGA ^ccTCq”cccAcc^scAcccArz:^cr?K>?czxGa;'KiGrrCArAr:cx;ccqca‘;cwxOccGcc-Q\cccGc TCA^CC.SCA.Af^nf’C'TTY'TCGCCCCaTIATCTCGCAfaCGGCGGaAliCT'YCOx^CTCACCAGiy.lCCCAC GACAGCxGGGXT'K’GGC.AACGAGGGG'GAGAGGKTCATKTiGG’rG'b'^GAGxCCTOT'rGGii.GTACGxACAASGA i7';'Ti7'TGCC'GTCGT3'AGGG?G7Af;CA73A7xAG?xGTTC13AG1GrG?,AGT7'AGGCC.AG3xxrT'7i'.3rG?xGt3x;TGAx:<7 TGG'rG3GGGG'5''GCA'3AA3'G'.GGGA''A3'rAGAAGGAG?G'A:4'TGA.TGGG-GAG'ArGTA,ixGA.AiG3AAAGT'r-TT c TA 'A? AGGA7 GT AG G A..AGGG TG A G AGG AG GA TG AiX'A C AG AGAGGCTGACG-G3T TTG 'GGG-GGGACAC χ' X. ' * ' X » - , X - * ' 4 ' X' X XX 'rrGTPACTCCACAAGArAACAAGkA'AICr'tCAGGTOAGjCQTGGTO&CAACCvAA'i'GOrxGTGCAO’AtCGAC CCCGx:AGG7CC<'Z4;3GGCTOx71GGX>CA/;?>GAG<1x:GGCTTAGf.:.AC?x*V3C74Gi3CC?‘rCATi7GTi3GGTT!rCTT Χ.ΑΑΑν.χκ xxAv.x'x x:AxAxGGx.-.Ax;;:.x'i xAX:·,.;· AA-.AxA,A<jx« ίχ.ΑΑΑΑ, :ί , GAGGX.XAA.'xXA.X.Av. AAC.AGAx:A X.'xx A ATroCTCTA.GCCAGTCCGiGGGACiSTGAGCf^GGGClCTGCisGTGCTTxGAGATGAf.’SAGAGCAGTGAATTK AAAAf?A:G-GCGGx'AAGlAfAAAG~GrAGC'WiT;\TGT<5AQTGTx:GGAAAG'4GGTGGxGxGiGGrt:'AAA;ACTGGATCi'Gx GGGAiIGGAAGGiGGTAGGGGGGGAATTIGTGGGA'GG/IAGAATGCT'GG'GTG'C'iGAGG'GGGCiGAGCAGG'GG?- ' -x ' «G xx. x G r G x x'xi '~x ' ’ ΐ G-3GGGTGGGGGAAGGAA.3GGAix3TGG7GATGGGGGGT<G'.;GTAi7GTTGATGiGG.A,1GTGGAAG'GGGAG-GGT . . x . ”... x\„ \ i.x ’ < '. ?. .X . .. ... ........ | ||
BMPS-A RK-KE | ;ATiGGGG®GGTGGGSGGGAGGGeGGAGGGSGTG5'GGTGGGi3GS;GGGGGCTGGTGGGCAGGTGGTGGSG GGCx'CCxIGGGGTGX/xlGGCGGGOTTGiCGGGGTG^'GGGOxi'CiXIGIxS'j'GxlGC'C'G-iXIxIGGCxAGGKIC'i'GxlGGxI ΑΟΑΓΑΧΑ::θ,ΑΤχΟΑ”ΑΟΟχΑΟΑί:θΟΑ·χ:ΑΑΏΤσ?:χ'ίΏχχΛ'ΑΓΟΑΓΤχ':χ;ν3Τχί??·χ>:·ΑΑ?''·Α·Α30Τ·ΤΟί:ΤΚ'Τ·Αί': Gf^Gf^GCTCAz'iGAGGGAiiGAGAAfiGGGGiAGATGCAG/i.AGxGTiGAXCTTGGC>GGGxGGGiGCTCCCGGA CGGAICCCCGGG’CGOGGA.GAGGGGCGOGGAAG'AOGG'GCAGyG’GGGG.OGG'GCTG’GGGCAOOGOGAGGA.ijCAGC' A0GAG>GAGG,a3GAOGTGGC1 GOGGGAGAOGOCGGTCCCGyCA7GG?G3TGG'xAGTG€G7G73COC'CT-CG.-'GCAGG GrGGATCGGx.'AGAAx3GGGC'rG7GCGG7GGr.G'xGAAGGAGG7xGGG33xC'3i3GG'3GGGGG7«''3G:7G'3ACAAGGG.'AGGxC - , ' x' x Xs ',,' Ax ' v.4 ,- , ,x x xx . . , S' xx 'xx x'l X ( x χΧ'Χ'χχΑ x k'> x x x 'Χχ CxACA'GGGGXGrTCGTGzGYGGACGGG-GAGATGGGCA'TGAGGIGxiGG'GGGSCCGGGTGxiAGGAOGACAAGAAx ''.TrKTrGGxl'TTGGTCAGGGiAGAGiGAGAAA'xt’GvGTlGAAATT'rxAAGGGIATGGGlAiiAT’rGG'A'GTGGGGGlAGG •GGGTiiAi.'Gtr.'TOCAflAA.TTCGfsGGiTGTA.CAAGGKjCTfiTGTTA'GGlftGGAGTTT’izlAAAnCf.'Ai’.AfGTTTT CTGATGAGi/AIxTTATGAIzGTC'GTACzGGGAGGAGGAGkGAGAGA.GGACTCGGAGGTG'GTGTT’G'/GTG.GAi.'AG GGi'GCGTAGTATG-'AGCGTCA.GAAGAAGGGTGG'GTGxfAA'ryT'A.ACA'i'CAxGGCx.-'GTAGCAATG’A'sTGGG TGGTQAGTGGAGAGGAGixA.·.'. i-'A3G<x>GGGGGAGGTGAGGGT<3GTGAGAAGz3iGA'GGGAGTCx3AGG'GGG77G ' 'χ ' x x' x x ,-7,--3 ,, x - x Χχ,*Υ. χχ- χ- ,,' χ < χ ' ι Ά, 'χ ' χ'* χ χ'» < 3» χ - χχ' , χ5:', χ „ X - ArGGGGx'GAGX'GAG3GGCAG<>AGGTGGG<73GGGG?GTG'G?,G7KXGGx,GAGAGT.A3AAGAGC3'iGTGiAGiTGx: iVkWGArZfGTGGAAGAGGGATGrxG-GTGTATGTGAGTTTCGAVxGxVGGl^GATi^CAGGACTCxlA'i'ilAT T'rA':AGGGAA,GGGG'A?VA'A7'A';:GGAG.T-AAG''.G'.?A'A4T<X'G'AAX,A7kGAAiGk;TGGGi?7ATA';GG'A'GSTGAKG'!'GA AGTGGACGAATGATGGGAT'rGTGGAGGiGG'TWAGTGrAAGXTGTG'TTAAGGCGGxAG'TA.TGTGCGGAAi'sGGf; TGGT:7~fA7GGGA?A7TAAGi:TixAATGG''3ATGT;7i.;GTGGTT>;':AGT7GT<;A7:'GiA;?A.AG?rGGAA'T<:TGATT”T OAAAAAAGAGAGGAAGATGGTTGGAA.GA'GGGTGGiGGAGGGGACTAA | 128 |
BMRS- ADHL long | ATG33GG GGGTCGGjG GGGjAGGG; CGG AGTAG G Gi? TOG TGGTGG GG<7 GGGG'G«3G’i G'G'GCA GGG'GCTGCGG GCCCCCGCCGC'lCCGGCGf'AxAGTwjGCCGrGlGCOGGGsGGCGGGGCCxiGCGGCAAIC-CAGCTGCG'GGGyGG AC3'GGGGG.AGC3CCQG/.Y.'G<A\3GG3AxAAAG/33GA73GGCGC<GA?GGCAGTC31173GGG3-'GGaG'GC7GTA3 ’,A:Gx,'xAA·. x χ.33,<?Αχ.·.'·.:··Χ, AAx>AGA« ΑΑ«:,:.χΑ«Αχ>,ΑΤΥΑ.ΑΥΑ,\Οχ·Αχ.->-.Α'ί· k.’i 7 7,-.Ϊ 7. GG'.AA, ΤχΑΑί'-χΙ,'ζ'χΑ··:. 7.-7.-, GG7'A?;GXG777GG3G77GGGG73GG33GrGT7AAGAGGT.x;GAx73737G7>G<AG'GG7yG7GA77GAGGAGG'A7G3AGx7AG3 AGG'riGG'AGGAGGAGiCTGG'CXT-'.'GCGGAAGAGGGGGGxrGGGTGiTrGGGAf.'GGAAGTGlf.'GiGGCGG'.'IGTTG.A'Ai G:TGGA-TGT':7TAGixAGGCGGTi;'T'GGGGiG7AGA?4GGAGGA?Gi7;7G':'A7GG7i?GT:?i7GA.GGGf':GAiGA':7GGAi7GA. GTCGTG;GCGG:GA.7:G?GG3GAGGj:7t4GG'TGGT;7GGAGGG;TGGi;G:AGf'G'GGi'''?CGG;7GG;3i:G:'.;GKGA':7CGG1C TCAAG3GCAY77AGCCTGG”G!GCCO'3CGCATCGGGCAGGGyG3GGCGAGTG<3CCGACT<3A.CC7-'.GGA>GGG1«.G GAGA GGG GGT 7 GG TCA AGGA GG 3GG AGATGG'G G A GO AG GT TV OTGj&A CGG GGTGG AG GAGGA G AAGGA 3<' ' 'χ G< A - ' x'»V< ' , ,. \ ' χ A χ χχ> χ. xx rCMT''3AGGG7'7-7GG,Ak7AAT7G?GGGA'CTA:7AA'';A3AC:lG7TxGG-G7'AA'A3GAGTG-G('AA?G'4AGG'.AA„Ai'.'T'r'ixj CT-r7,'7<?AGiGATT';GA'rCA^-:7'7'i3TGAx:AG<:A;7GA-GAGK;ACi7GACxAG7C-A:7;7GG;!.’A7T'i-T7W7TGG':7AG.!V7 Χ,1.·χ·ΑΧ>.χ A-G 3.A'GG7>7j\.7, x 7,ZV,x.,xi77,-ΑίΧΧί7ί:χ-3 7,04.,7 7Α,-;χζ3ΓΤζ xiZ'7,«xG,7,A,;:.7.j7,-7.-7.A7.1z37;7,<:A71 '7.7 x: 1-.:,4.-7: TTiGTGAGTCGACAi.;GATA.Ai?AT«x,Gf7CTTCAGGTGA.G:GGTf:K7G'GACAAf7GGATGGAGGCGAGGTCCAC •GCGGGAxiGGGCAiSGCGTGG^GGGCAGAGACGftGCGTGA'XGArAAGGAGCCilGTi'IAGOOrGGCGG'rGXT GAA.AGTGAGTGAG'.'j'G3GAx7GTGGGGAGGA;'.GA’.3GGGAGCGTGGAaCCGGGGGGGAG,AAGAGAGTGGTA AGCOG.GiGTAGGCAOTCCCAGOACGGGGCGCGGOTCGCGAGGOCG'TCAOATTA<3AAGAGGAGTOAATG-G AAAA<AAGCCTGCA3«GAAGGATGAGCGGGAG3GYiAOTTGGGAAOACC'3rA'OATOOCAG<GACG<3GAG'CAT I'GGACCGA’xG'TGCTA 7'G3'VG3G:xAG'A3TG'iGA.;GGAjGAixGf3;.7G'GG'AG'GS733'.GT3GGi'.'3-A.T177A3GiGA. XX Χύ ΧΧΧ^ X X(X 'A, - χ t V. X . χ X <·ν χ 'KAI'GGTGGGGG/xAGTAAGJGIAGiGVi'GGCATGvYXlGTTGTrTxACGTTCATi'AVG.^AC'TGGriATGV.A'xTIiCT \Λ',χ ' , , ’ χί χ · x 'xx 1 X ' G > . . | 17/ |
BMPS- RK-KR AOHL _________________________ | ATG3CGGGGGGGGGGGGCAGGGGGGAGTGGOTG'AGC'GGGOGGTGGGi7GCTi3G1'GTG3AGGTi3GG7;CGG GGGGCGGCCi'.;eKX’fiGCGfA:GCTGGCCCGGTGGf.T5GGK;Ct'<:;CCG!Gi?GGGGqGiAGGG.AGCTGCTG:GG'AG AGGzGf.GGGAGCGC CGG3CG3 ACAG AQCAOGOOCGGGCGTf\;CGGGAGGCGTGGTi;OOGCYTCGGGT«C GG’.7GGGi'.'ICAAGAGGGAGxGAGAAGG;7GGAGATGGAGAAGG-AG7;TGGGGTGG'G'G3GTGG'AiGGGGCGG& GCGGCCCCGGCGGG'ITGACGGGG'iCGAAGAGCCGCGOGOCCGGGCGGGCeGGGAGeAGGAGGAGGAOG | 128 |
2017200239 13 Jan 2017
DAME | SEQUENCE | SEQ ;Ο DO |
AOOAO 00,GO A8OAO8T000808800?,GAO88008ΟΟΟΟΟΟΟΟΟΑΟΑΌΑΑΟϊΟίΟΟΟΟΟΟΟΟϊΟΤΤΟΑΐΟ X'.L xjOAx X.Oi.xx ?0,,??χχ,0·::.·0-'χ. TGxG.-GxxG Gx.GxGAzxAGAxA i^V-iAy^xGi,Ίΐ ΑίΑ χ,ΐ:·ΧϊΖ:Ο;.Α>Ο'χ,ΑχϊΑ':Α.,χ.χΑ'χ::.^: OTCc-K;dcecc?w:GrVv.ic?teCi:»GTCOTC'ccAfjeoTaT.;AGrccw?ccce^&ceQccocGCM;ccc TCAAi:cr;.^GA«ccTTeTS«<>ccceGri?i7x?K;GC«Ge%eGcroc«^ccccACTGACov;:cc5ei'.:cAi'! <5A8AG-?O8OTTCOTi.7^.8GA:Y;f.8?:AA8ATCAATC?xT8AGCTTTCT8:iO’.O8YG8TOOAG?A0:G;’.CAAC/.:vA 01001 00081001 ΟΑΓΧ;θΑί„<ΓΑΟΑΑΑΟΑ«2 TOAAOTTCAACX'?ATCOOAOAT1 CO TOA8008 8 AGO 'AC010xAOG00800AOr:AI'10000-A'0:',1AO?xA80?'C'”0'1080?,10;OGOA.i'ii'TTTA,Ai'xixx,OOSA.AO'1'5'8T G1TAlG.AG03TTGA30AA0OAS'3.AC,AGGA0G0330AGC,AG,GG?,G«0OC8GACC1G'18OGOA8OK88AGA0 CCCT'yrAa'rA'irXiGrXiC^AvkaAAC-aCigrsGigOAA'ir’ro^gASCACGgCPTC'iAAgCAtVJ-C’iGCViiG 1TO:rOAOTCOAGAO8AT,AACATCGO88iT8AOf0TOAO8'0‘?<'AGi'O-ACAARGO?,TGx':AGiCOiAGOTC'GAi·.' 08ί708«\00000ΛίΑΑ0 οτοο'γοοοοοχΟΑΟαοοοοοοϊ'^αοοαοαα.οολοοοοττολϊοοτοοο^το’γιCAAiJ,GTGA03':OAO'00<;OAOA73GO:OOAOOAO'GAOGAOxAGCr.7Ti:CAOCGx:-OGOO>:.'GAO?x?iOOxG.AO'i'OGT?x ATCOrt.'TCTAGCOAOTOGCA.G'GzAOGTxiOGtSCOGtSTOTOCAGOOOI'SOACjATTG.CAOiCA'OCAOTCAATTO: AAA30AGCO'rGOAAG7:0'.AO-AOG.300lO”A1080?O3T1CO.A?,OAOG'0:8iiGATGOO?-GOAOiG8A1'CAT rGOA{:GOAAGO:AOOArGOGGCAA10A01ACTG.Ar’83AOA?x10:0.i081'100'8A\730-OOOGA.'rOGCOt'GA A'O00-AA0CAA0OA0GC'GA1'O81O.CAG'3’:01O'GO1O'CAGC80i,10.;AGC08GOAG7r'ATGO00GCAAGC0G O x G , < - **A >' “0 0 ν' , ' * iC xC* x ’ OAAAAAAOAOteiGAROAAGOAOG'r AAGAGCT i'OlGGAOGOOGCi ?sA | ||
BMR6- RK-KR long | Az xA,G<xxixAxx-x:XGa.AA:G;-.,GA:.AAA.:xx,AG : <·Α?., : O AG,·,? i'G-'.·, .t xA:'Ox.,GGG--x.,G'Ox-x.xOx'.i GCA-zaGG-Gx OG-x?O· GCCCCCGCOTC'TrKf^CCC^CCT^OCCCGCTGCCCC^CCGf’COCCGCCGGAkknGOiAGCrGCTfiGn'S AG<>GG'GGGAGCC0CSG’8GCG0:AOGGAGGAGCCGCO8C:CC:?CGCCC5CAGTGCTCCTOGGG<O?TOC¥G'P«£7 CO's008010AAOAOGCAOGA8A300GG8AG3'i'>8G?x:8AAOOAGi;30il':31'GOOTOOOGOOA8CGAOG08?' 00080 GCOGGOOO 0300 A. CGGOOiGOAGOAOCCOOGOCOOGO'iGCOOiOOOOOAGCAOGAOOGOO AGO Ai;A:iA80A0!OAGOAGCT6i8C'r8GGO''AAGAGCG000T88G08:AGGA83GA?xG38GG0GGG0830'VAGA';'G 8388c:yrO'rG8A81OA8GC00-1-G300i>08GA8A30;AAO0A00O,0:AG0;00G38\A0AGGG8AA,A<3A0iG0AGCA 0oo8O38GGO80A,0OxOA;M3O0-GAOOAi78AGO0:AGf>:A'-;,CO88AO!88G0r,CG'CAGGOGCC88GOAi;'COii:G TOxOAOOGOGGAOAOOOT'iO'IOGOOrOOOilOAA'OTOOA'rAOOGOOfAIOOA'.'OTOOOOAO'l-itAGOAGGOOGGAO; <A\C.1AOC<‘?303Ar;6''i;A^ACOA6\';OOOAO?zTOO':'C?6R1AAA;!rAOOOOA,A001AAAGOO;30TAO<;.,A--.'KAOi'0'. GOTCOOOAAOrOAAOOOxGCOGxOAOA'.AGAAAOOxOG'rCAAGIOCOOxCOIAOOOCAGGAOOCGOOOxGGGIA'GVOG - „ X , c ., - , - - ,χχ- ' 003880 AGOA11'8AT0AAG801'1A0AG'GAGOA80A80AACAOGOA0'iO30;?,O01O381'T1AA1-38GGCAC' OCO'G8A.S13'5A>G00010AG3A.8A3080800,C8'GOAA8’rl--OAOS80AG080CAO:rAr;Oi''A80l'080>GO * \ x'- “Ά J x ' 0- -xx-< XX --0- x -. xx XX X 8'CO8GA88COOAGGO88O;yiGte8OA8AGA.OGGCO888A8GAOAAO8AQO888'r8G38OOWGCn-0G3'l· Χ.ΑΑΑί.Ά Χ:·Α;:.-Ζκ χ:ΑαΑΟ.ΑΑ·.ΑχΛ.τ'λ xA.A'x. AGGAx.GAGx;3x.-ZA,AAz x χ, χΑχχ:χ,·-.· Άχ;χ,';·<.Α,θΑί, χ·ΑΑΐ,-.ΑΑχΑχ.Ά χ,'ζ x A A30A7G3O8/iOO8A0:O,'8OO?x0G;Af.8330;:<A7OOGO0;-?,'83O0'iAG8i1G'O:A':'A0?,3'iA'::i'G:C;AGOAG8'O?GAA-3i·; x, ' X x ' xx'x'x XX χχχχ,χ' , > A'OO.0OOOA80.GO3tATOO1GO0'0iA8'r<AOTG8OA1G8AG7xA8:;030A:83O0OAiOrOxA?xG;GO?sGA0'G8O:?x ATOOAc’jCOAAOf.teCOCdATTOT'.tOAOACCTTfASTTCACOTiATOAACi'.CC&'iOTATCTCCCC&AACro lOOOfjOGGOOOAAOIAAOjGOAA&OOOCATOIO'.'jGOOOIIOAOOlO'i'GAOOiA.OAi'xGTOG.AAI'GTCAi'OOl' ,- \, ,: ,,-, Λ ,, :,-, | 129 |
BMPS-· B-RK- KR | A'XOOOGGGGG'sAiGOGGGOXXJGOGCAGiOGeoOrGOOOG'AGGAG'GGGGOOGC'OG'iGOAGOOGC'rOCGO ' , ' x ' : ' ’ ’ 0 χ χχχ A ' ' > > χ -χ ' < χ χ AO0:GOG8GAGf.:8OOfA;OOGOAOGOA8':'.'.AO<0'8OOOO!OO;';'3O80A7OO?xO3O0''10':'0iOi;i'A';O83GO3i?iA'··: O;7O?'.;8GO30??G':0:xA8OO?xOA7xAG;OA88OO':'GAOA'GOAGi'xA0;i'GAOAAG33G'AO0:G'OO0'A'O0:r;OO80A7O'80G: ccaooocciKccccTi5CAorA;cc^?<:<w?AGi:cfA7AOirccx'c<y.:cocTf.'iroGi,«oo,AGOAOi?}AGirA08· AGCAGOA:'.rt.';A!'.;OAGi7TGGi73OGO:AGAO;AG77O0A7O30OO'8r>Gf7O;AO3G;'xA';8GO80’Gi7O>::<73Or.;'8C?,1x7 G'3x:'.:GA30'1i73AC.AAOGi7OO80;l'OC80A7OA::A.A0AAAOO?xG;'.;A0'GxAiGO8'.fA7O(AGO8G«A0GAG0:O;AGf8A 0»iC:^G«3Cf.C8ACOAA0CA0CCAgCT!3GTCCCS.-0i'?0'TOS0CAf»C8i5CC<X'~><3i»C<5<:C,0O»CS<XCGC OOAGOOGGAAGAOOGTtOI08:'. 0006 GOA1O3OGC?.00!080GO0GGO38GO0A0308C0AGC80G0AG GA0AGC-88-C8'r8C'>T8A3C8AC08GGAI'03T'3G80A'iOA8888'5’O3GAACO:iGO’’.’GGAO-xAOGACAAGGA Ot'iC'3’8CCO3CG :83000 30ACCAroSAA.GA0t'iGAAG830D.%C’8tA3iXCAS&8’r8C3OAGOV£GA&Cx 100:-80000001810,8:083838700:778308.300-0:000883,8808^7087-00810008838:3^00700:3(0108883 0:88080730000880000008080^830,77.1700718000380000:08030^:0080 00:80,30778-0:-388-3 0088003300307 0001803 0030181001003080000308003000800:0.088801000803080000030000080308080 r8030.i7030';7AOGO:77.A8.i''..3007''A'.;0.110:0,0317A?A730010000r0080AO?xl873'.:>;i:87'x8GOA780O.30G8:00:Af7 08000Α00080.Α'Α';0086ϊΟ'5''8000Α030Α;':θ'0·','00'Γ8ΑΟΟΑΤ1,ΑίΐΟ·Α;Α0008'ίΟΑ800'0100Τ'ί'·ϊ·03'.Γ Ο.ΑΑΑ0·?0ΑΟ7ΟΟΑΟΟ8Ο0.Α00?Ο800Α.86χ:083Ο·'0!'0Α!'3001·08?,.Ο;380Ο(Α'ί00'8Α0ΑΑί;Α.8Α03'ί3ί'8ΐΑ 3880080,8AOOOAOH 0088-868808000880-.3 j1080OA08GC31'CA01A18A017-A830>OAG800A888G ~ A - » Ο' Αχ. χ - χ, , , . i χ χ - χχ , χχ- , ,\ 4 «Λ ,, ,3 -Αχ λ χ χ A - -χ XX ' i:.C80A?A08xGO,88.380:77CA880800A003CO-:8x70188AO,0'808;783?,l30308i03;7A3'30'778?o30070l88070 '8-:73078-:700;?0:08?G'A707:'i'-J8330x70?x8'0880-088000:8-30:688-8188-07?,8i030;600G3i13-0180z38:I,A7-7'-8?G7 χχχ - - - χ | 138 |
BMPS- EE _ | ΑΜίΟΟΟΟΌΟΟΟ,ΟΟΟτΟΟΟΤΟΟΟΟΟΟΟΌΟΤΟΟΟΤΟ'ΑΑΟΙΌΟΟΙ'ΟΈίΟΤΟΟΟΟΟΟΟΑΟΙΟΟ-ΙΑΙΟ'ΑΟΐΑΟΟΑΑ 0j0C-r;?A08Af)880OO:A8O0Ar5A8001A?000T0O!0A0CAA1-0C’rCA0A;3f'80OCT00OA0'?'8003-8i's0'0 ',' ,0.: 'ίΛ , ' ,γο, ', ,3'Ό0χ0θ -,Α?,*- IGi-iGi ,G3,-; MvCO X -- X X - X ' χ χ- | 131 |
2017200239 13 Jan 2017
NAME | SEQUENCE | SEQ ;O NO |
ccTCSA<;?5AatOTi\CACCiCCc;Ac?^\OTCCAcc«cc<?ccGi:cTCC/M4c?/K:o;Kx;o«TCC’iiT<'.teqe4 rfto^GZiTCCeATCTCCZiTT?%CC.4CC;V:CGAftoAC^CCC?lTTTCAiO'U5C<3>CA’rOC?qCTr;T'XCAi\C .ATCTC-7.4TOi::cc<^CArto/!rK:AcreTCACC7WL4ftocm\GCTK<y;ito.TC-'r{!CGTCTCi,7Gcc'/'s':AACci, CGTn0ACCCC-Kr0AC0?YQ?TC^.AGGGCTCC0'KX?TGA?f.^A”0AC«5«CTOGAi.OGCi\CCGito0CCT ί>ΑθΛ<?Γθο·?τ?υ::οοΛθΑ~ΑΑ?χ«Α':>:;^το;ττ·'Αθτοτ<:ο\:ΑθΚϊ<?Λ'ϊοθΛί??χΑί:οΑί:;οοητί?οο?χθ.?'.οίι wwqAj.qTOiCCtccijCCCTOAAqHqAtwj'iOGsKmrgoAC'rccAircAAfjrccAACAACAAocTfjOA AAFGA -CG'toOAA'I'O CC ΑΑΟΧ^ΑΑίΑΑΧ'ΟΟΑ AC A£C AAAACAi'rereG'TAACCAC WXiOTCCCCq?· A‘ v>' A it x O x' ' <' 're- '-AC -re re s A:t:AOAC'ArAA4<i-7CCAimA:A:AGqAvrCCnrCC'5A?qb;*V<.e-CGr<:G^<7A’A1-;toC'rr\;AC<:mtoSC C71^G^rCreTCTto\CO,rexx:.XGX5ZA7?A.A<teCG<JCO.V,A?IGAi’AOCAqC-CTCTACCx:TCGCCAC-ACXA7A AOrAZArCOGCCASAOOiXAtoTCCCAeitoOCAi'OwVAiCCTf.'CC'rOiVAAG'iO.AAC'rriXAASCW.CA'ICAOC Ki<X5;ix1A«CT7toATC'?xTTGCxf.'i7CAAiA'!AO'TACGtA;GACTACGA.G:J>GCCAa.KCGAO!?f;CCCCT:SOCA CAKAtoCGAACACCTfAuiCTCirAf.'AAACCACOCCATAGTGCA^CCC’tGx’TGAAA'rCCiArAAxACTCOA AAA?<ref.o.AAqc<-::TO<AOcsTOC'7A:<>.AAA'Scrq?:;\:c<A;\xTCAGcrrtV>;sOTAr;AArA'iAC;7ACA-ro ' ' ~ < - A ' xs - | ||
SMP9-- ES | rererererereAArcAArrexiAreAererererererererererererereAreAreArerecAA '-. re , x'X , ϊ ν' ' ', re „ <>. , < re Λ ,, Gca^mrGCoa;AOCAC?wcTTCZ\*<^:T<uvmATCT7<?CT<iGaAAi\ccTOZvAitoT'SrmcT!rtx':ToeoG· TCeCTCZKAhCTCTC^SCOTCKtoCrtoCCAGrteeAJetr^caiC-GKyAVtoCCCCCi’AGTACATOATi’.toA ί^ΑΤ0?Αί:ΛΑ·:>:;Ό·?:τ?ί0Α0:·:7·:>:·Α0-?ΑΑ:?Γ0ΑΓΩ,ν?*Α0θ·:;θ0?ΐ;0ΑΑ0Λν0:ντ·0Γ:0«·Α?0'?Τ':'.':'1η;-ΑΑ rfto?.AKziTrtoCATOi?ccAT'r0CCACCACcnAftoAC’r?rcoA-r'!'5OAGAAr.:cAnATCCTCx7Tr;ri'rc?u>c ΐ-ΑΤ<ν:;ΑΊΓ·:;Γ CCftoCACOAreA?iOA1 OACOAfAAOC';TAAre<-rOCrefRWg.reA<YA03TrA-. TQCCAreASCCA •'.•O'rijCxXCA'.C-rirCCACO.ACr.^GA.reO'PXTCCOT-y^OATAAAAOArfjTqCT-OqACOiSiTAC'XaCP'AC'r <>A1AC;rCC<re?ACCQAGACAtoAGACA?lGAC-r'At^O:iA'CCAGQA-f?iW;7AOgACQAGCAA'iO<3iA?s.C!Ai’;A ,- x ' X-x, \ >- - x -A '-,'-. xx- Λρ-χο,νχςϊτΰΰΑΑ'ϊοΟ'? AcciX-.WAtoc-tocgACACAtoTPG^CtoiC'riXO'mccoccwsGC'rrA-CB^ SAxAreCTA ?<A Tf-re-y GCVGTTf.·? ·7Χ.'<\ACGACQ\CTC<A CatoCAACA^GAGACACGGCTCOAiVCTr; AOrereAA1reTreATCrr.-CixCOi,CAAAArAkATO're<rrC;?A1A^;,AAG<AAiTre:7.AAre<;AO:A';CT':'.:eA/.::':OAG:;C C-71GC&>f;TCx'TC'rWACx':Ai\GAG«zix7AAA<A>OxiACCAre5'?««CA'.:-C:tn?7:SCTi\CCx:TGf;CCiA:AAGC-A AOCx.AsTCAi-CCGi.UAif.’TOAi.'TCACACTGOCAGriAAiiCATC.’ACTOA'^'S'A-AACTTCxiAGGAC.A'TCGOC Τ·3Α3Α·7ΑΟΑΓ3ΑΑΤΟΑΑΤΟΑ·7:70<ΆΑΑ3Α3ΤΑ03ΑΑΑΑ3ΑΑΑΟΑΑΪΑΑΑΪ1<Α;ΟΟΑΑΟΤΑΑ·Ϊ<·Α£ re re (χ CCTs'JAACiSCCCACATqKA.CGCCAOCAACCAirOCCA’t <>3ΤΟΟΑ^δΟ«Γ^^1<χ·ΑΡΟΤβΑ·?ί3Α&.ΧΓΟ«3 AG'iAQ^'iOCC’C.AA&CCA't'OC'V-SCgAACAC'ACCAAO'A'iQT'GACCAAiA'AGC'AtOC’iX/iAAAAqGACriAC' ^f;ASCSimC<?Ah£A?iAAAArACCACAA;mftA;AA:A3AlCOAA3qCCA^rA3SA<3gCCGS;rpA.......... | Ϊ32 |
W Short | a rev aaa - a o; >re ere re reo<?A rer re Are .<.a re?ore; A-srere: re-re a 5 -re a re ·λά; · -.-./'aa- reAACCCA-CreCWCAOrSCAGCreA’I'TreACOAA'.rexyCrefXAiCreACCreACrereAGre'.rereACArereTCC'i'OGre'AAA ΐΑ?3Α';??,Α?Α??ΑΑΟΑΟΟ';?3ΑΑΑ?ΑΑΖΑ??;ληηΓ;<^ι---;ο;:^ΐ\:<?3ί\Α;ΛΑί·ΟΑ^-;?ΛΑ<ΑΧ;Α3ΑΑΐ?Α·;:ΐ7ΑΑ' iiGCGGC'T’?AAGACGCACX;AGAz,iGf.OGGrA5ATGCAf«iAGG?xGATfTTf^<7x;G;tGx.’rGG<AA7TACCx:CixC C<AJCCCrOi;reCCCTfA7ACCAAA:TCre,reCAGOCGC?AiCCCC'\A3C«i'.Tf'f'CtoC?reCAA'5MreA-7;CireO\ l>CA5X.?:'3CAGCA0CTr»CCri'.'GC‘Xs.«,'3AGOCCCC?ccC5X!3Ci3ACTSASCrcc«3COCC\i:?CTl-CA'r0C ^XsiT-^OTAC.SJiCGCCClOTf.'COArOAC.aAO.iAOjAGOACGrXjriCgTCGgAOgQqG^reAGgCAGC.s^ re·'rev re re, re - - re - ,,- , ' k re ,,. re rev re ''- a' ire ' ' rerec re- m > a re a x x, - x ACAGGGA-rA-rre? ίΑΑ:ΑΑΑΑ<\3ΟΑΑ3ΑΑΑ\-ΑΑΐ<·^ΑνΑΓΤΑΐΑ,ΑΛίΑ:ΤΑΑΤΑΑΑΑ·ϊ.{'ΑΑ3ΑΑΑΑΑΑΑ·; p';-e.'-re;Gere\7GTA;A3';GAGAACA\\reAGAGTTrere,Ge-re,rereT:A?reAGAAi'reT;-;creAGGA:i‘GAGGT ΟΑΤθ;ιΑΚ«ΟΤ:Α7ΑΟ^ν^ΤΑΟ<Α\3Τ·?τ;^Α»0!Αί?'ΓΟΤΟ'?ΤΑτθΑίΧ;ΑΑ·ΓΤΤΤίί?ίΑ!Α\ΟΑ<Αν’.Α'ΓΤΓ·?ί7 TT/iTCA&AATTTA'TC.V^rK'TTArGSiAiAGCATCAiX'ztoArOIGACTCTGACiCKreT'rT'rG'rTfAOACACC CATGTA0T;iTAAreATCAfAiAf0ViGGCTOGC‘Tf.;GaATT7GAfA'rCACAGCCAC?A4i.7sA'rC'rATOn':-T •rA'AAACTCf..iC.AGOA TASCr.XIOAAGCT'-CAOCV'SAOAATCre'AASCAireGAATrAA'-GTAi'.AAiSCAACA ΟΑ·7Α>Α;;ΑΟ·'3ΑΑ<>3Α;'Α3’.';7'3ΑΑΑΑ·.;:ΑΟΑΑΟ<ΐίν:7ΑντΑΟΟΑΤΑΑΓ;·':;?,<ΐςΑΟ'Τ··:’ί'.Α,'ΑΑΟ·ϊ·ίΑ'>03'··''ΓΟ'Τ·?('.· ft&AS'i GA.7-3 OAGOTACftCAG OCGCACreCAAOATAAAAA-X CA recCGGCACCAACAAAAGAGTCXj AA TCcreT::AAreCArACCAA<!Aireoi‘-3iPA3:AA:AAAAr;7C?re'mCi''iCAAA'ir'A:'Af,ACACsC,AGA3A?re'r:'re'i rereA:Af??i77A<?re?3?vA??A7AAGCrAA.?ire'IGA:73TT':?CAA7rre<G:reOAA'iAGCi',AGi‘reTGGATre'-A' 'OCACCCAAGG-C-CTiAVC'IOCCivATTZreTG'm^OrretoAATOCTCCTTC'OCACTCAftCGCACACA '-ΆΛΑ 1 , x r ' \ re re > ' \,k re ! a ' re a 1 rXKTOCGf/CzAACTAAGC'AAAATrA.CATCTCfASTTC'JTTixC'TTiGATCA&VtoTCCAAroTrAG'T'rCK', i' ', ,t 'Ί„ , ' ', ,, ,' , Ϊ \ ~ ’ ' „'A ,re' ' ' 1 | 133 |
BMPS- SA | xrrecrec7Rrejrere-rerexreA:reArerererere”ArereoA :AGAre:7ii3:.'i:rere;7.rei7i<re'iGG;re ACCAAA37Ci7:3<ArOCA:7A7CArreCAGOC:Aire'';A3C''3:reAAAC,3C7\77'A'jreresArerereA':7:CAA':7're3-A'rere' > x A x. \ , Are' ..- 1 A x x re x <» re - AAx ' » > re re X xxx. xx. Ο1·.·<ίΑΆΐν<;Ο:Αί<,;.<:.·ΰ·:ΐ.Αί Axxl::Λ,<,-,.3 ',:3.:-ϊΑι.,;-1;,-Χ,7,χ,Χ.ίΧΧ,Χ.Χ.'.Χ.Χ,Χ:,Χ.,χ.χ.-.·':1,Χ.Χ.'χχ,7.Αν.ν1..-Α:<·;.νΑχ,·':·Αχ,χ,.3.χ.,7, Axix7re7GreA7AA77AAC'7-3x7Gi3AAGQAf7reC77<7xA-7AAreAiAi'i7xre'.:OAA,7;'7A>:7Gre!7\7CCTG-i'T:7A'3:7 f7rereAre7?7::'7AixAi7A:7Ci7rore'7X1G-7fOxGAAOxiAC'GA73r;7:GA7i7xii7G3';7iA3AA7iGf':re-Ai7A':3Gi7Ai3;7A ΑΤΑΑΤ<7ΑΟΐ7:7ΑΑί7:3Α.Α3ί7ίίΰ7>:7.7χΟ·7'Τ077ΓΑί70Α7Α7χ7Γ«3;'.;χ'.Αί33:73ς7ί';·:<7'··:;;3ί3ί7Αί7:7;'.Α'3<7Α':7ί7<7-.Α' TC-^Af.C’iCAiiOAGCC'FiCrftOCCOCCiASATiAtOSA^SSGCdiSC^P^-CrCCACTGACCAgCOOSC&S GAAi3CQCC'rtCCTCAACGA:7'qCG7JACATGCGY;A'AsAGCTTTO'!YiSAgAi:<X;TO!3AG?Ai‘7GACASt3?»A re -' re <· - re re re ' ' χ ' c' re re*- x* AC' -A' - | 134 „ |
S3
2017200239 13 Jan 2017
NAME | SEQUENCE | SEQ ;O NO |
7TGGTOAG>7G7O7CAGA;v7TCGG0A?<7TAGA/GTGx7:TGTGTTA7GG7A00G7T7GGGGGxA7CAaAC3:7TT CT'TATC7AGC?xGTTATGAAC-TC'AOACAGGAGC?xxCACR7xACAxGGACT>TOkGACC'GG-T70I'T7A7xTTGk!A'GAC CGGTGT:\«<rATCGGCC?GAG.>A«AAitGGTfW5CTf5i;AA'rTTOa,CATCAC&GCGi\CTAGCAA:iCTf;!JG'S; rTOTGACTCCzIGAGCATAACATiGKGCGTTGAGCTf.loGCGTGGTCACAAO'.Ka^GaGTCCAGC'TGGai: CCCCilAGCTOCAGGCCTCft-TGGGCAfiAG!ACi57':GCC?'ri\CGA’izi?.GCAGGCC?'rCATCCTCCCTT5CTT GAAAf^GAOtrqAOOrGCAGCiOGgCAGCAGGAOqrCAGGCTCGSCGCGhGgCCCACAAiTA.QAOT^A » T COCOG τ A. GO GAO 7 GG C-AGG AC GTGOC 77 OGGO 7 C GOA GtO C T-TC A: ϊΑΐ 0 ACAA OGGG AGOG AikT TO » A * xx Αχ,χ' ,. Λ χ, i_' , „ . >i * -> x - 1 ^ΟΑ<'α7.ν/;ΟΧΚ'0%Τ'Χί<«7ΐΕν7ΓΪ«ΟΤΟ^;Αΐ02ΑΑΟΑΑ·νδΟ·ϊ·'ΟΟΤ·ί·ΟζΟΑΟΤΟ?^Οί·χ·Λ<χ·ί;ΑΤΟΑ 7τ'Τ7:<:.^77Ο»Α”0ΑΤ7700ΑΤΤΟΤ0Τ\ΑΟΤχ0Ο:7τΤ·<;:·Τ'Τ00,ϊ7;7··ί'ΤΑΐ··0<0Αί70Τί70Α0'ΐΑΤ0'τ<7Οί7ί7ΑΑ2Ο700' T77C7TOGG-GGCG70GxCT?G\GGT?GGATOGG?xGGTGGGTGGTTI.i'.OGTTGG07rOAGi'G'.G>.i'i7G?,?<7GTi7ATTCT ' -·. ' t' > A xk ' χ ' χ x | ||
WR- Sl | ATGCG'GOOGGTOGGOOGGAOGOCGGAOTGOOTGGOG'TOGTiGOGGOOOGGGGG'OOTGGAGGGOGTOCCXI CjCOCOCOCGGCOOGGGCCrAtCC'TOGCOCC'CTOCCGCOOOOGCOGCCGC'CGOGXAGGCAGCOTAOOC/y'jGC. a.GCv1GOA';OAOCGCGOGGGOGAOG''OAGCAOCC'OGCG'COCG7GOOCG':OGT'GCO'GOtGOOGG7'7'GG7O'TAO >' (' « ιΛΑχ ' v,ώ O'» λ , , χ ' χχ,χ 1 O O' ' χ, G ' ' Vw ' k ' x w ' ix . kx» k Λ0Τ7ϊ0Τ0.0€Α00Α<:ί'ΐ0Ο000\70ίΧΑν.:'00000'Κ>Τί'00Τί'ί7>Α0Τ0;νΧ;ϊ<>::0<>Τί7ί70ΤΤί7ΤΤί7Α··07 \.ak?G-Axχ.Όχχ'χ AGA,:ik.G:k.k,k.Tk.'G'k.Okik.x,kiAi,.A;Tk,k:,AGkji:i'«:;?-.Ak,>:ikik;G'..k?TGk:-kjA-'G-::ikiG'x<<ik3ix':.k.-'k.Ak:.k.V:k OTGOTGOCGGGAC:GGOi7G7ViOGG7iGGTC7TrCG7GAOOGTCX7i;GAGf'GG<7GGCCGG<7GGGC;'.;GK<7AGC<77X7 TOCGAGAC'GCAAGxAGOOTOOT'OGCCCOCGGt'.T'CTCxlCAGCGGCGGCCGA'COCC'OGAGOCxAGCACrCGCxrCAC: GAGGG'70CG'”7GGTOGAGGAG'OCGG;AOATGGTGAAGAOCTT-:;G'AAAA.GGTGG'iGGAGTAGGA.GAikGGO 07 TC7 CGGOCG07GAOCGA07xGGACAAAGA07 0GAGGTTOAAG7-00;TOGGAGAT7CGTOAGOGOrGAGG TCCsTGAC7!G77'7GkGAOAATGGC77GArC.'T,\;;^A;7iOOC'G7-WGT-i?i':TiCTGAiAkiTT-GAAA7G:'!AG:7AAAk.'T'i''iT i A 0* * t - t , Ox ' χ 'G ' ' χ i x ' - ' ‘ A G' A ---0.. ' χ G A A i'x xx'x X ' Gl Αχ '' X ' k < '7'GGT7AGTeGAC.ftGGZi4'AAk7ATOk;GGC'7TGAGGTGAC>:7GTT;rGi-GA.;7.fGkOOGA'.rGGAi7Tk7GA'CGTi7'CA>G GGGGOCxGGGOGACrGGGrGGTOGGGAGACGiGG'OOGGTGiCGi'GVOAOxGG.Aii'CGGTTGATkGGTktGGSOTGTT OAASGTGiSGTiGAGOGGGAOGTGOGCASGAOCAOS:GGAOGGTOGAOOGGkiOGGOGAGAOGAGAG'i’kTGTA : ArOGGTi7TAOGGAG'?GCOAOOAGGrrGG:7C;GOGi;TGGGGAGOiT:G7TG?xOATTOCAACA'Gi7Ax:-TGAAT7x; GAAGCAGCCTGCAGG7GAGOATGGGC7GyA7<Ar:>AOOTTeCAGOAOCTrAiGATOOGAGGAGAQGA-7'CAT TOGAOGx7AAO'AGCGA770G TGCCAA'7 TAC TfiTOA TCA5AG?xA'7GG7 GG'TTGGO'AGGOAAGOxIAO'i'sGiCTOA XX x XX xv x x,x , ,V „ Ax ' ' V > X Αχ x' X X '< x.x' ‘ - ^A G<' “1 »X > < ' x ' ' ix X G x’ CAAAAcxG· ΛΟΑ·7λ7ΑΑΤΑΑ· 77T'CG 3 AAGAGGTTG CGGATOCxWCTAA................................................................. | 135 |
SMF-t' NR | \χ- , -X '< XX -Ax x' ' ' xG' ; ' k ”χχ'χ'χχ x G Ax ”, x A X ' χ 'χ X , x» CG7TC\TrTCG7GOAGGTGGG!'7CGGAx;<O;G‘xGTTi7GGGG;x'Gi7GG,l'’':7GTGGGGG;:GGGi7k7'i'GAwGO'CxGGi7GGT GTGxAGGiiGOGGGGGAOCilAik'rTGGAGTTGGOikOTOOGGAxGCATO'TTGGGG'GGGAACiGAOAxlAOCGACG ΟΟχ7ιχ7??ΑΟίΑΟ'χΟχ:0077?<:-ΟΤΟΓΟΟΟΟΟ'ΑΑΟζ\·';Υ;;7·7·ΑΟΑΟΟΤΟ'ΤΑΤΟΟΤΛΟΟΟΑΟΤΤΑΟί;'ί';7.0χ:θ;·:ίΤ7:χ7 G7 GAC CG'OC GG07-GAGC.7kGC'GGTTOGAOGG!GOGA:GCGAOOGG7xOGGGxxFGAOOGTOk GGOGG7'7xGGGx0G s.'TOAAOAAx'i'GAGTOOA.AOi'AGTixGG.AGAA.AG-TiAGOOOOAAAACAACGGGOAOAGTG'-OTG'rT'TAAT-TTi'x x'} A '< -χ G xx ' c- χ ' xx -, G'xv^''x'i’· 1XC'rrT.AGGA:CAGxA?7rAOG,AO'GT'r.TGkx,iG77xGG€xx5ATrG!xWiTVi7‘''VSiTx\;^xJ..'!T7G'rA7x?!AGk.'TT;GGG!xAG CG7iOt7C73'GOiGVx'X'7CGCG7G77GAGC70GAC'7TTTGG-AOAGGAG'Gi'7GOGO.A'rGAi7i'G'.G7Cxi'x©;CA>GOT'GG' GA;GAOGGT7'OATk7'IG,AGOGGGG07Tr7>GGGxGGG':7Gi'fO:<;i'iG'i7i7AG7xGGOACxVG7;GGAAGGAG'Gi7A'7TGi7T <;GT0x7G.AGTG00GGG>GT7OxO;0AGAxGAGA0GGTGTGTCGx'xA0Af::0CAi'0TTAGGAG7;A0;7.0x707C-rr TGkCGGOAASATGAAAlACGiGGGGxn'CAGAGATAAGGCGATTGGTAGTx'xACTTG-AGOCGATtkATGGAAA?'. GGGiCATG\~OCTOCACAAA?xC>.AGAAAAGCG7A7AAGCCA:AACAG7AAACAGCCxCfAAGCGG77O'f;AA0:1'CCAG G7GTATGAO iCACGG'i'7'3ΥΑΓ0<707 OGAC'3',rG.G”OGCGGOGGO-7GG?xOTGACTOOATTOTxii'70TGO'OG CG 00’GT A-7 GA 7. GG G'7 TO 7' AGTO OG A TOG AG J'. A TO G TC'O'T TOO C AO'7 G A AGOG AC A. CGtGASTOCSA GO AACOA5COOOA'ri07'-¥0'A7?ACCTiOOT-tC.AGO''iTA'iOAAOCCGGAOT'&WTC:CGi.'«AAGCOTCCG07iOG '7'GGG.A-'7k7A.AOi7T;'iAOA.GO'GA.TO'3'G;’;ATG;TGGi07GTAGGA.Tkiii'3730'3\'xAtGS.A.i7xi5'3'GA7iGOxAAA,Ak'iO.AGA GTGAGAAOATO^TiGOOGGAGGOxOWG'rOGGTGGTGArAO | 136 |
BMP- GCR-NQ | ATGOGGGGGGGCAxGAGATGTGGGGI'GGGGGTGGGGGGGGGGCAOOG'GGGGG’rGGGGGGAOCTi^CO'GO 7777 ΟΟΟΤΟΟΑΟΟΤΟΟΟΟΑ«ΑΑΟΛΑΑΟΟΟΟΟΟΟΟΟ:ΟΟΤί?7007G'AkGGGiTGGGGOGGAGOGGi'iGGGGG Oi:0;iG7GAOG,TOO''AGGCG<iAGTGxTGAOf:'A7;Ck7x':GTGGGG7rGGCi7rx:.7'7Gx:.OC'i7TO;'xGG':7AG'GOGGGC'AGi7 ΟΟΤ·ΤΟ'7ΑΟΟΟΑΟΟΟΟθτθ'77ΤΟΟΟΟΟί7χΤ.:'.ΟΑ·7·<;ί7ΤΟΟΑΤΟΟΟΤΑΟΟ>:ΐΟΟχΛΪΟΑ'?.τθΟ'·'ί;700χ'χΟΟΟ'Τ07; 7'.'TG-7GG7GGG':7CGOAGGAGAOAC'3G70;sAAGAGG0G'CO'iCGGOGGOCAAOAGGO77G?GGGG'G-7-TT'GCAfG'.· Ak'GxO.OGAA'7CGG7GGAAGAACTGGO'CGOAG'A;'7A'3'GGOG7'AAGAGOAGOOGGOGGG'-i'CGT7TTO'OAGO-7C x. G * » x Ax G G ' XXX' X’ k X XX Ax 'i ' i’x'OGGGTG ΑΟΟ.ΑΑ^Ι-Α,ΑΟϊΟΟ'ίι'χ'ΟΤ'ϊ GGx>GtG,AGGGOxATGxAAk7xATi7'3‘G.GxGsG:A'iG.ATGA.AO’GGGkiCGAC'GG GGAxAGOGGi-GxGTTCGGGGGGGiGGGOGGrGGT'OCOiGAGGGiCGkGCi'iktG'GCGxGGi'xGxAAC'GGGGGGAkGATiiG 0λΑ'ΤΑ'ίΤ7ΤΓΤ0ΟΑ0χ3777Α77χ0Α7Τ00Α7ΟΤΟΑ·θ:7Α0ΑΤ<Α;Αί7ί700'::θΑί7000Ο;ΑΟΟ0'0ί'χΑ':7ί7Α0ί7<7χ0ΤΤ'ϊχ?7 OOTG07k.1GTOGG':70AG:7TxGGAAGAOAAGO.AGOk;GGtGTGOixAGOGx7GAGx:CGGOG:ATG’rGTGOGTGOG Tx7G.AGGAOG.AOGxAO;CAGAGGTOk3TGGCAGA.GGGCA3GO:C77GOCOGA;7G>AGxAGTi7G;Gx':7GGOATxSi7AxAii x.0 x G'x ' ’>· ' G ' xk A A ' ί- χ ' Ax' G , x 1' I ' < : ' xx x G χ· ' χ >χ ' OOGOCCAGOGGOCGT'rCTAOCGGOAGOGGGAGGGGCCGTTGGGCCTOSAGOGGGACATOAAOGGGACG | 137 |
2017200239 13 Jan 2017
-NAMEl | SEQUENCE | »ίο NO |
44004000040000304340000-00000400034304400000430-4030-0000440003-.0)000000 CCCCACC.VxCOTGAUACeCATC-TryrriTrvim'iTACTrqi'UATOATilCTr.S'^wVtdATr^TCrV^ViilOACA ΤΤίΟΑΟΑΑΟΑΤΟΑΤΟΟΤΟΟΑΟΟΑΟΟ-ΟΟΟΟΟΟΟΟ-ΟΟΟΤΑΟ | ||
BSMP-P- NR-6 | Α'Οοοοιοοοόοοοαοοοοοοό'-όό-οόοάοοοο-οοο'τοοο-οοοοόαοοοοοοοοοοοοοοοοοοοοο-οοχι: CkiA%cif'3%r.oqAOC'rrAsqcit'qc.Asnrca.GiA'r,Ocnqcqdcoccqc-f.%oocci'>ccr,'i;ocA'tcA:i'-nr'~CT 0OSSCdAG0T00tYjr-0CrA''0'i'TC,jA0?'iO0CAtCtC-.?'r0.SidCS03Toe%'>CCTAAHA0AqAG.ACCCr'.Ci'.· e-CCaQCAoddAitqCC'qG^iOOpceqp^Ar.’AGqC'tAqAPC'ri'TtATC-GCASqCAPTCA'SC-TCa.QrX'GOq ' * n o' C' 1-.-0 ' G A ' -. -. A 0 -X 400440447-013033040440040040444034100000444404400030.034030300-4:00303-1 TA AGeT'GGiiGnttGGixCC-GAGAfG-GeTTAGGtr-.GGTGAGGGiGtA'GCdCGAGiGTeG'-'GGCG.dAHGA.GGiTOGA.AOA •KICTTTAGG.WsC^TAOGAgTTT-Gr.'ATry^C’GA/iTTAciTATTTA't Ο,ΑΑΑΊΌ.ΑΤΛΑΟ-,ΑΟ'.ΌΟ,ΟΟχΛΟΑΟ CGAAG-GGCGoV'.Trt'GGGGn':''GA<:GA0AGTG'GTGi5Ar.'ACCAf-0'T'i'ii.ri'?GA’'.''G.Af’:AAGG:GAf'.r.';G'.AGir';'t'0Gr «AAAitTTTTOATriTGACr.Y.CGGGTiGTGATGCi’.irfTO'.lAiGKiCAGAGOnACACGtG’CAACCATOGAT’rC'iT Ο0'Ο00.Α5ϊίΐη0>0Ο00ΑΟΐ%Κη-03.'<Α.Α-0Α.ί<</”Ο·ΐ0Ί\;'0%Α:%1ΟΑ%1'ΐ'%Γ·ϊΑΑ<;Α'ΓΑΑθη.\0Ο·?0'·ϊΤ 'Τ··υ·>.<'·;\Α^·:4ΑΤΟΑΑΟΛνυΑΟΟ'0<3'Γ3ΑΟΑϊ'ΑΑΥΑΑΟ%4Ρ\:ΐ'Τ'ΟΟΰΟΑΟΐΑΛθ·Τ''Τ·Τ'Α3<;Γ,'Ο.Α'Α1&'ΓΓ,ίΟΑϊ'.ΑΑ - - A \ „λ\, a λ ί 000744:, 434040007 00)74007304307 GAS 0340073000700440 04073347 730 0337 0330 GGC<ArrA'rCA'G<rGr.Gt'G'rTAGTCGG1ATdCAfGGfiTGG';X;G'iGG':':GAC:.ttGV,G'G-GAGAGA’f'CG,Ai'GC;'G1Cf: AGCGi.GGGCGieTt'GIGGG^GGAlGeTGdGTGSAfGt'GA-iAGA'.GnCCOAGTGGt'C-TGCCGAi'G'.GGG'n'JiGrG'I'GC OCCAAGTcV.dr?TA?^T<1CC/!Ti?Ta.’iOeTCtTTACTTTOATOACAAG,rcr'aATO?CA'r?CTOAAAaAAT 404) =3 44134-703 f0074404r,07707i: | 138 |
BMP- GBP- NR-6 | 47337 0-3070.r'.’AGCAG-A i-ileC'i-OG-i'-SG'G· :0700'r03'ri300'.:03337 00700713030:30430-7-3- i03 i:-.CT33'-003001'--30733:304i34)3A4.)-::7'7 0-3-.:O‘3-3'-QCO--0’C7':;'7i3i3i:O3':3007'-OCAOO-3400C7t 300.0001-.30003,-1003004.0003000301)0000--00000004.000000100300.4300000--:0100040-0 ' k ' ‘ -- 0' -. ' '0- 0i-'\« - S - 0 -Or 1 V. -, 47370070-00300340040404070343434000003--1-::00-733333434003)00-:373-37 73-0303 4i .1.,:41:.7,.4:4 t i.. Ci., a k?',:,0Ak?r-3,1-.-04,-1.. ι.-00-Αι.,.ί-':-.,Α7. a. 1,4,GO 44k, A Cl .-.401..1,31,.-01.4,7.1 ο-7:7 a ; 7:..70101..-.4, 704700470030407344343770477.40040030034307304307377 003034304341304334 300<300;-:.04AOA4000000COOOC40-0400<;340<00403:004403ACx40040i-:'.A30>:-4:C'OOC4'Ot-Oi 0044000-044:-:.0-0001::00:30141-:0001:-::00-00-014004:400-001-:101000::^:1440040:30.00000004001000 04t)'00O'-i: 043043400000 0-300O-0103404-’334030C03SO0O0C40O00AA00Ai00-i30OOO0O 0-3003440-430,00340-:0003440434):3-000-3000--0::0-004,401:0000400-0-00-03400--00-013050-000.o< ο o- 5,- - o- „ -io -< i o - - 0 a- - , - 1- - < 0^ V λ \ - <- \ - \ Vk 0- - X -- Λ5-- W , - S 'GOiOOAAOOGGOAOOOOGOOO ,40707307077-::-700040070300743341:.3):40700470:4770000004 ooxioco-A.crzrcGr.-cTTr.-^AOo-ocrAjCOOiCOAOot-Uitociont'cccocTrirv-iC-ooccAOATi-^AaiccArA: 440040333:4703-574-.:43433373370-34707347:-),4403371:443740373070)0430377-3770037 3ΟΟ4ΑΟΤ4ΑΟΟΤ?544ΤΟΟ3:ΑΤΟΤ0.Όι3ΤΤΟΤΤτΑΟΤτΤ<ΐΑΤΟΑ3ί^'.ΟΤΟΟΑΑ0·Τ1?0'«Τ?0·'Γ-3ΑΛΑΑΑΛΤ 40403447473307:.,14434:)077370-)4733740744 | 139 |
In other embodiments. the nuele-o acid molecule encoding a designer BMP compasses a nucleic aviO ---ouei e i OiS1· 40' o3 < f% 7th' ~ j , ' t%% in - <% - As' m 96%, 97%, 9S%. 99% identical to one of the nucie:c sc-d sequences set forth in Shot ID NC-s 74-139 or a .$ fragment thereof. Sn other embodiments, the nuefeie acid molecule encoding a designer BMP comprises n *f ckk ,iex -<XK*-»ice I e-tut AV cd'- fTh* Bb <. i>s % , so co , ’M , -%> ·)$%
94%. 95%. 36%, 97%, 98%, 99% identical to one of the- nucleic acid sequences set forth in Table 8 or a Ifagment thereoi, in another embodiment the nurfaic acid mcieeule encoding a designer BMP comprises the nucleic acid sequence of any sequence set forth In BEG It.) ?4θ5:7-%133. In yet another embodiment, tii the nucleic aad molecule consists ol the nucleic acid ssguence of arty one of the nucleic acid sequences of SPG ID NOs:?4-T39.
tri another embodiment. the nucleic acid molecule encoding a designer BMP comprises a nucleic acid sequence at least 40%, 60%, 60%. 65%, 70%, 75%. 60%. 85%, 97%, 80%, 92%, 93%, 94%, 35%, 96%, 97%. 38%. 93% Identical In the nucleic acid sequence of SPG ID NO:?S. of a flagmen- thereof. In :15 another embodiment, the nucleic, ac-d molecule encoding a designer BMP comprises the nucleic acid
2017200239 13 Jan 2017 sequence of ScQ Ό NO 78 in yet another embodiment the nucsein ac-d molecule consti-ie of the nucleic acid -sequence of SEQ ID NO: 73 encoding BMPE.
in anofhsr embodiment, ’he nucleic acid inoiecufe encoding a designer BMP comprises a nucleic add sequence at least 40%. 50%, 96%t 66%, 70%. 75% 80%. 85%. 87%, 90%. 92%. 83%. 94%, 95%
96%, 97%, 98%. 99% identic·»! to ’he nucieic aod sequence ci SEQ IB NQ'8O. or a fragment thereof In another emboditne-ti the nucietc itoid molecule encoding a designer BMP comprises lire nucleic scsd «''-quench ci S,.» P NO %; n vet anof w t-mno-am-nt ο - rtcec m I -'cee le i c innts . >f me nucleic sc;4 sequence o; SEQ ID NQ;B9 encoding BMPG.
in another embodiment, the nucleic acid molecule encoding a designer BMP comprises ά nucleic acid sequence ai leas- 40%, bo%, 00%-. 55%, 76%, 75%, 60%. 8552, §?%, 90%;. 92%. 93'% 94%.·. 95%. 96%, 0?%·, 98¾.. 99% identical Io the nucleic acid sequence of SEQ ID NO:102, or a fragment thersoi, In another embodiment. nuolesc sem moisscuie encoding a designer BMP comprises the nucieic acid sequence of SEQ ID NO: 162. In yet another embodiment, the nucleic acid motecuie consists of the nucleic acid sequence of SEQ fO NG 192 encoding BMPGE in another embodiment, the nucleic acsd molecule encoding a designer BMP comprises a nucleic acid sequence at feast 40%. 565» 60%. 85%, 70%, ?5%·, 80%, 85'.%, 67%, 90%·. 92%, 95'.%, 94%( 95%. 96'%, 97%, 9652, 9952 identical to the nucfsic acid sequence of BEQ ID NOBOS or a fragment thereof, in ariotfiisr embodiment the nucleic acid molecule encoding a designer BMP comprises Hue nucleic asid sequence ci SEQ :L) NO 103 in yet another embodiment the nedefe aod tnolecuie consists ot the ft inc < iCdse-i ex-OSEQ P NO ' > e seed n $ ΒΕΚΝΈ’'·· stna Designer
BMPs urn noUifain otpr-syed as pro-proie-es eempnstog a !ong prodomatn, one or men.
cieavage sites, arid a mature domain This pro-protein is then prot:»st;ed by the ceff-jfar machinery io ysMc a, typically, dimsne mature BMP molecule, in some embodiments the designer BMPs are produced In -a similar manner The prodomsin Is believed to play a rose in the folding end processing of BMPs. Furthermore. in some BMPs, the prodomain may noncovaien-ly bind to the motor® protein and -act as a solubility enhancer, chaperone, or inhibitor In seme embodiments. BMPs may be produced as mature damqins produced directly from or refolded from tnciusion booses, ir: other embodiments, the BMPs are produced via chemical synthesis or any other known seethed for protein production.
In one embodiment, flic designer BMP so producing using chemical synthesis methods such but not limited io, syctheisc methods well-known in the ah, in some embodiments. nucleic acids encoding designs» BMPs are prepared by to’al gene synthesis or »y wie d-rected mutagenesis of a nueieic acid encoding a w-ld type, designer, or variant PiMP. Methods include template directed ligation. POP. cassette mutagenesis, slfe-direcfed mutagenesis, restriction enzyme digestion and ligation. or other techniques that are well known m (he ert (see. eg, Prodrsmc-u et ai . Protein Eng 5 827-9 (19927 Jayaraman a; at., Blotechuiques 12392-8
2017200239 13 Jan 2017
Taitons - a! 3otxl»f 1 0¾ X'?£C < * sc , <0 Kvs\ct 1 MQ< u<?
Cloning A laboratory Approach, Cold Spring Harbor Press. Cold Sprsr,, ^orbor, hiV (2001)), in same emitodirnents, an expression vsotei that composes a per:?.; encoding a designer BMP is prepared, Numerous types o? appropriate expression vectors and suitable regulatory sequences for a 5 variety of host ceils are known in -he art The expression vectors may contain transcriptional and translational regulatory sequences including by not fruited to promoter sequences, ribosomal (.Hiding sites, transcriptional larminafor signets, polyadenyiafion signals, arid enhancer or activator sequences, in some embodiments, the regulatory sequences include & promoter arid transcriptional start and stop sequences in addition. the expression vecior may comprise additional elements such as two replication h systems to allow it to be maintained m two organisms, the expression vectors may be extrachromasomal vectors; or vectors lost integrate info a host cell's genome In some embodiments, the expression vet lor contains- at least one sequence homologous to a host ceil s genome to promote integration into the genome Constructs for integrating vectors are well known in the art. In some embodiments, the expression vector comprises a selectable marker gene to allow the selection of a slably transformed nos! S cell Seleetsoi marker genes are well known in the art and will vary with the host cell used
The expression vector may include a secretory leader sequence or signal pephde sequence that provides for secretion oi the designer BMP from the (tost cell Suitable secretory leader sequences and signal peptides are known in the art.
Nucleic acids encoding designer BMPs may be introduced into host coils either alone or in ft combination With an expression vector so Ihai Ihe designer BMP is expressed from the nucleic acid The method of thfroduction -s '.3rgel> donated b\ the best cell tvpc L\on-p;ary metho Js of transfeettoiviransformakon include CaTCh precipitation, liposome fusion. electroporation. viral infection, dextran-mediated trarisieotion polybrsne-mediafed transfection. protoplasl fusion, direct microinjection end other methods known sc toe art. Nucleic acids encoding designer BMPs may srable integrate snto the
..5 ;lost cok genome or may exist transiently or statsly tn Ihe cytoplasm
Appropriate host ceils; tor expressing designer BMPs include any celt suitable for expressing wild type or native BMPs, including, but oof limited to yeast, bacteria, arehaebactena, fungi, insect, and animal cells. In some embodiments the host ceil ts Saocharomyces cerevisiae or Eecherla coll In some embodiments,, toy host ceil m a mammalian cal; such as 293 -eg, 293-T and 293-EBNAk SHK, CHO (e.g., CHOKt and 0044), COS. Jurkat. NIH3T3, or C2C12 celts, Other statable cells may bo found in the
ATCC catalog Designer BMPs; may be produced in more complex organisms, including he? rsot limited to plants and animals, in one embodiment -he ooiis may be additionally generically engineered. I.®.. (0 contain exogenous nucleic acids other than the expression vector comprising tha designer BMP rHioleic acid.
In seme embodiments designer BMPs an? prodijcecl by culturing a host celt transformed with an expression vector containing a nucleic acid encoding a designer BMP under the appropriate condli-e-ns to Induce or canes sxpressioh of the designer BMP. The conditions appropriate for designer BMP
5/
2017200239 13 Jan 2017 expression are the same conditions knows to be appropriate for expressing naby® pr wild type BMPs, These conditions will vary with tbs choice of expression vector and host celt, and may be easily asoer-ained by one skilled in the art through toufcne experimentation.
in some umeou-mutito, th ? cosigni.r BMPs mav uo uuotk-ij ot wototod utter expression Staudu-d 5 purification methods in-’tude etecbonho;«-do, moisruiar immuncioq-'a; ago nni-nmatr.-qtepos' techniques, including Ion exchange, hydrophobic, shindy, and reverse-phase HPLC chiomotography. and chroreatofocu&ihg General guidance in suitanie purification techniques may be tound in Scopes, in Protein Purification. Springer-Vecag, NV, 3'i: Ed (1934) fhe degree ot purification necessary wilt vary depending on the desired use, and in some instances no purification will be necessary.
d Purification from bacterial wits may result tn th© expression ot 8MPs in inclusion bodies and a subsequent step of refolding in a CHAPS-Kigh sail system Purification from mammalian cells may involve n two step puufieatton via Gellubne-Sulfate and Reversed Phase t?hr®nui|o«rapfiy columns.
in some embodiments. She designer BMPs may be modified covalently or non-covefently Covalent modifications may be introduced io a protein by reacting targeted amino acid residues et the
S protein with an organic derivaiizing agent capable of reacting With selected side chains or terminal residues. Optimal -sites for msdi-icaiiori oar: be chosen using a vanety ot criteria, including but not limited to visual inspection, structural analysis, sequence analysis, and molecular simulation.
In some emnodimenhi designer BMPt may |>® tabued mth d teas- cos etviuent, isotope, et chemical compound. The ianei may be an isotopic label vueb as a radioactive or heavy Isotope in e acme embodiments, the iatiei may be an immune label such as an antibody or antigen in some embodiments. the label may las a colored or fluorescent label, ouch as fluorescein, in some embodimen'f:. the label may be biotin a tag -e g., FLAG, Myc. His) fhe designer BMPs may be derivaf-zed with bifucciiona! agents to crosslink a designer BMP to a support matrix or surface for us« it: purifying antibodies or proteins that bind to the proteins or to detect S binding In screening assays. Commonly used crosslinking agents include hut are not limited to 1,,1histdiazoacetyiri- pnenviethane. glutaraldehyde. N-hydrcxysuccintmide esters lor example, esters with 4- attidosaiieyiic ac-d humobliunisfionai imtdoesiers. including disucuinimidyl esters such os 3.3'di'ii-.iobisisucclnimidyiproplonate), hifuncitonal maleimides such as bts-N- msielmido-l.-3-octans. Other modifications include deamidation of giutaminy! and asparaglriy! residues to fhe corres^xinding giuiamy? 30 and aspartyl residues, respectively, hydroxytation of praline and lysine, phosphorylation ol hydroxyl groups of seryl or thiec-nyl residues. methylation c-t the ammo groups of iysme. arginine, and histidine side chains ff.E Creighton. Proteins: Structure and Molecular Properties. W. H. Freeman & Co., San E-ranctsco pp. T9-BS (1383)). scsityiafton of the N-term-na! amine, and amidation c-l any €-- terminal cutboeV, group touch dciivaticufscn mo> 'mpruvu toe sokto-lrh abs'.tphar t-cirisuod across toe bicod 35 brain barrier serum half-kfe. and toe Ilka. Modifications of designee BMPs may alternatively eliminate or aftonuaie any possible undesirable -side effect el the protein. Moieties capable· ct mediating such efiucts
2017200239 13 Jan 2017 are dittctosed. far example, in Remington's Pharmaceutical Sciences iblh ed . Mack Publishing Co . Easton. PA {i9-80}.
Another typo of covalent modification of designer BMPs comprises linking the protein to one oi a variety oi nonprotoinaoeous polymers, e.g.: polyethylene glycol {''RELV:): polypropylene glycol or 5 poiyoxyaikyleoes. In the manner eat forth In D.S Patent Nos. 4,640,835; 4.496 689. 4,301.144: 4.870.41/; 4./91,192 ct 4,l/9;33/. A variety el coupling ohemistriss may he used to achieve PEG attachment. as Is well known in trie art.
In another embodiment, trie designer BMP comprise*. linking the protein via a CovK-lxxiy linker to a CovX-hody antibody such as, but not limited to, the CovX'bodies described In US Patent No 5,/33.757. 9 and US Patent Publication No. US 2009/099:3130. Such CovX-bodtoe may exhibit improved characteristics. Including, hut not limited to, improved stability uod extended serum half-life:
ihe lenepior binging activity «I designer BMPs may tie assessed using any methods used far S assessing the activity of wild type BMPs.
Ihe alfinity of designer BMPs far one or more BMP receptors can be determined by receptor binding assays For example, affinities for At.K-2, At.K-3 At.K-6, ActRil. ActRilb. or BMPPli can be determined Suitable binding assays include, but are not limited to ELISA, fluorescence anisotropy and Intensify, scintillation proximity assays {SPA}. Biacore (Pearce et as., Stocftato.’siry 38.81 -89 (1999)}.
DEI..FIA .essays, and /dphaScrserG* (commercially available from PerkinBImsr, Boss© P.. lily C. and Cheisky U (2902)).
in some embodiments. Blaodte or surface plasman resonance assays are used. See. tot example, McDonnell. Grrm Opto Cbem, &>oi. 5,572- 57? -2901). Biacore experiments have been used previously to characterise binding of TGP-3 isotorms to thesr receptors -De Grsscenza el at.. J. Bio;.
..5 Chern., 2/6. 29632--29643 (2901). De Cmsconao ef ai„ 4. Met. Biol. 328, 11 /3-11 S3) (2(/33).
In other embodiments, a plate-based Direct Binding Assay is used to determine the affinity of one or more modified BMPs for one or more BMP receptors I'hls method is- a modified sandwich ELISA in which BMP is captured using an anti-BMP monootonal antibody and then detected using a BMP receptorPc fusion protoin
In other embodiments, AlphaServer!:ji assays (Boss® R. et ah. Principles of AlphaScreen’“.
PerkinE-tmof Literature Application Note Rot P4989, t:ttp-i'!1ifeec!ences.periiine!mer,cam' Notas;S<t9690802.pdf (200217 can be used to characterize receptor and inhibitor binding. Fluorescence assays may also OS: used to characterise leceptei and inhibitor binding. For example, ei-f-er SMP2 oi a BMP2 receptor or inhibitor may be labeled with a fluorescent dye (for examples ot suitable dyes, see trie
Motecular Probes catalog) Additionally, scintillation proximity assays (SPA) can he used to determine receptor binding affinity, for example. BMP rscepfor-fc fusions may be bound to protein A coated SPA beads or (lash-plate and treated with S35oahe!nci BMP toe binding event results m production of light.
2017200239 13 Jan 2017
Al
AS
In a particular embodiment, the KO at © specific BMP mutant to a Type i or Type H reoeptoi ear; be deterreinse by using receptor extracellular domain fusions to a human igG-Fc. The receptor can he bound to an ode? sensor using antj-h-jmandgG'Fc sensors, and the BMP can hind the receptor exira<-χ.ΙΙ Μ» co mn n »'uLv to uetemtp B< n> d -όΠ a‘ s Ip text ' sisters a si u pn>j (efifj Bici. ayer Interferometry (Bid) to enable real-time. ianel-free analysis, of biomoieoular interactions, and In ptoyide iitfomuslrou on affinity, kinetics artel concentration. As proteins bind trie Octet sensor the tight passing through the sensor has, a waveiengdt shift that car) be raeasursd with a spectrophotometer. The rate of the shrft is measured as the analyte tends die sensor and when ;t loses tending.
The osieogensc activity of designer BMPs may be assessed using any methods used for assessing the activity el w4d type BMPs
BhiPs promote the grovdh end differentiation ci a number of typos oi ceils, Differentiation may he ' ' V Vi „ > ' ,' 4 > >X '} ) > is X », such as Aldan Bice or PNPP (Asahln-s et al (1095) B>p. Cell Res,, 222:38-4? inads et at. (1896)
Bioohem. Biophvs. Res. Commun . 737 3 ί7- 327: .toriikka et al. ('PisBBs Life Bob 02:23771-2333. Cheng et al. 120335,1. Bone Joint Surgery 35A:1544-1552(.
The cR t«nh bed cartilage dPIwitotlaiinn assay may also ba usnd io momkv activity tn primary ' x'|a Io aia-mptisc amvmmeois wgeder gen·, o? Coosa assays ntav La, used, Bmco uM'Ps activate ifso ~a * ' xx x ' n't n, x ' -- O'* se o i m xVP t>\ C''-,a---Οχ-γι e \ X. - X X x' XX , x' <X ixX, x'l u '3 \ ' X 'x3 .. .1 ,0fx otri ' \ χ» x- -, > m< η <x SO x --, _________ v x 1 -, x m ... , 7„ ... ,x x
Ssteogemo activity may fes mlasaBfbd fh oalf ibased assays such so atkating phOsphatace. BRB'x X A X ' X X a * , * i χ* X ,X I \ i ' <χ!χ' '» < C '
1039-1477 MOlOf.
Osteogenic aeti-My may also he meats sod io viva via mf ectopic hone assays or mamm.atmn bone gioy/th models. te some smoodlmante. osteogenic activity is measured in s son-human primate modus fn-yun mouut wa has·*nswi m I'.ewa et -si Mm' BmTmvcfp·' /< uhB- uTZMOfB»
Methods for evaluating done mess xsno guest--.· wo known in the art and include. hut are not limited in K-ray brihnoiiao- OXA' OoQCT, pCaCT chemical analyse;, density fractionation , \O , , 'iW.'f e c Sx '' * ,i'4t ~ -,n L-y ‘v x\xU'e > ah Wx,t Bone item Res. 13:2133-2-13 · 7777 7) Coe assay for determining cortical hone density is the MIoroCT'
X Xx, X ' x ' x' 'xi! ! ' X, X xl t x' χίΐ 3 '1 x't x' M x \x' ’ Xx' X- ! ;
3canoe mC7o(< (Bcanoe Medl.'s; AC; on a lemur.
2017200239 13 Jan 2017 '·. - e -..' c ,' e η x, ' nV t i xv \i \ growfhi'densitv/strehgtn may be used -o assess ihe osteogenic activity ci the designs»· BMPs of fee InventiiSp,
Designer BMPs oi the present invention may be lormasated ter admin-sfraiion to a mammal preferably a human in need thereof as part cd a pharmaceutical composition The composition can be administered by any suitable means, e.g.. parentereiiy. orally or locally. Where the designed BMPs -s to be administered loraiiy, as by injection, to a desired tissue site, or sysiemicaliy. such as, by intravenous., b subcutaneous. intrsmuseeia? iniraof bitai. ephthatmic. intraventricular Imraerabia! intraoapsular.
Intraspinai, intracls-emal Imrapentoneai. buccal, reetai, vaginal, -nlranasai or aerosol administration, the composition preferably comprises an aqueous solution The solution preferably is physiologically acceptable such that administration thereof fo a mammal does not adversely alfect the mammal’s normal electrolyte and tluin voluma balance The aqueous solution thus cor· comprise, eg , normal physiologic S saline {0.9% NaCi. O.f 5M), pH Z--7.4.
Useful solutions for oral or parenteral systemic administration car» be prepared by any of the methods well known in the pttarmaoeutlcai arts, described, for example. In Remington’s Pharmaceutical Sciences9 {Gennaro. A., ed . Mack Pub . IS90, the disclosure of which is incorporated herein by etei't CxA , omufeions · or indude 'or 0,-,,. fo< !i foiya-\de s - gweets such s-s vfoveteyk no glvcoi otte u of vegetable onqln hydrogenated naphthalenes, and the ii'xs. Formulations for direct administration, in particufer. cun include glycerol and other compositions of high viscosity
R Oispatbe on-te-abu biot “ fos foble ,>oymei- tfou’iiq fet smote tyaxi'i c <tcic collagen, incaSeium phosphate, polvbotyraU, goiytechde, poiygi/coiide ano laotide'giyc&iidc eopoiymens may be useful excipients ro control the reiea&e of me designer BMPs In vivo Other potentially useful
..5 parenteral delivery systems for the present designer BMPs can include elhytene-v-nyl acetate copolymer particles. osmotic pump.s. implantable iniusion systems, and liposomes. Formulations for Inhalation administration can contain as excipients tor ex:-mpie, lactose or can tie aqueous solutions containing for exampie, poiyoxyeihyiene-S-lauryl ether, giycocholate or deoxyeboiate. or oily solutions for administration m the form ot nasal drops or as a gel Io be applied intranasaliy.
BI Alternatively, the designer BMPs of the invention including designer BMR2 and BMRfs. identified as described herein may be administered orally for example, liquid formulations of designer BMPs can be prepared according to standard practices such as those described In '’Remington’s Pharmaceutical Sciences:'1 (suprai Such liquid formulations can then by added to a beverage or another food supplement lor administration. Oral administration can also be achieved using aerosols of these liquid iormuistlons
Aitemaisvoly. solid formulations, prepared using art-recognized emulsifiers cart he fabricated into -aoiets, capsules or toaengen sueubie for oral administration.
2017200239 13 Jan 2017
Opaonaily. the designer BMPs can be formulated in compositions comprising means for enhancing uptake of ihe protein by a desired tissue. For example, tetracycline and diphosphonates (bisphcsphcristesj are known to bind to boos miner»!, panicularfy a! zones of itcne remodeling·, when ,1 oy w>* | a - d x re'^tom , « v ? nuw v <.< o tx, v <9 r omoo ie> ί d I > v il a v delivery of the present designer BMPs to bone tissue Abemsd.ivesy. an antibody or portion thereof that binds specifically io an aocessihlo substance speefecaliy associated with ihe desired target tissue, sued as a cell surface antigen, also can be used If deseed, such specific targeting moieotiies can be covalently bound to the present designer BMP, e.g, , by chemical crosslinking or by using standard genetic engineering techniques io create, for example, an ace’ Isbiie bond such as an Asp-Pro linkage. 9 Useful targeting molecules can he designed, lor example, according io the teachings of US Patent No. 5,081.513.
it ;s contemplated also that some of the designer BMPs may exhibit Ihe highest levels,· of ackyliy in vivo when combined with carrier matrices s »., insoluble polymer rosteces See for example. US Patent No.5,288.563 die disclosure of which n; mcocporated by reference heiein Currently preferred cemar
S matrices are xenogenic, allogenic or autogenic In nature, it is contemplated, however, that synthetic roaieriais comprising ootyteciic acid, poiygiycofic acid, poiybetyrio acid, derivatives and copolymers thereof may also he used to generate suitable carrier matrices. Preferred synthetic and naturally derived matrix, materials, their preparation, methods for forrouiafmg them with Ihe designer BMPs o? fee invention, arx, c,ctn,.>c^ of ηΡι.ηο,οί,Μ,οη arc λΜ, kf\/W.i ,n ti .o art a.!C re> c'o t \ ^,i '. sj,xI * I fa c< x»u* So
P tor example U-S Parers No 5 385 883 in certain embodiments. fee designer BMPs can be administered to the mamma! in need tirereof eifeer alone or m combination with another substance known to have a beoetsclei effect on tissue morphogenesis Bxampies of such substances therein, ccfactcrs; include substances that promote •Issue repair ano rettenemfion ano-or inhibit inflammation or fibrosis. Bxampifes of useful co-actors for stimulating bone tissue grcwtii in osteoporotic individuals for example include but are not limited to. vitamin 08, calcitonin. prostaglandins, parathyroid hormone, dexamefeasona. estrogen and IGF-I or (Grit. Useful cofactors for nerve tissue repair arid regeneration can evinfe nerve growth factors. Other useful cofactors Include symptom-alleviating cofectors, including anfiseptics. antibiotics. antiviral and anfifenga! agents, analgesics end anesthetics.
Designer BMPs are preferably formulated Into pharmaceutical compositions by admixture wife pharmaceutically aceepfebie. noritoxic exetpienis and carriers. As noted above such compositions can be prepared for systemic, e.g., parenteral, administration. pameutarfy in fee form of liquid solutions or suspensions: for or.;·! administration, particularly in the fern? of tablets or capsules: or intranasaliy. particularly m trie form of powders, nasal drops or aerosols. Where adhesion io a tissue surface is desired, the composition can comprise a fibrinogen-thrombin dispersant or other bioadhssivs such as is disclosed, ior example, m PCI’ DBBI'Oud.'fe, the disclosure el which is incorporated herein by reference The composition ffereii ran bn pamted ?< prayed or otherwise applied to fee desired tissue surface
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When administered the phamnacoMicys composition of ftps invention ts typicaily delivered in a pyrogen-free. physioiogicaily acceptable form. Further, the composition may desirably be encapsulated o? injected m a viscous form for delivery to the Site of hone cartilage or tissue damage s ocal administration may be suitable for wound healing and tissue repair. I- vVabiy for bene and'or cartilage formation, the 5 composition includes a matrix capable of delivering -e?i aroteln to She site of bone .snd.'er cartilage damage, providing a structure lor the developing bone one cartilage and optimally capable ol being resorbed info the body Such matncos may be formed of materials presently m use for o-ihar implanted medical applications,.
The choice of matrix mafs.-nai ;S based on biocompafibslity. biodegradsfeiiify. mechanical h properties. cosmoth aopeamnee arid interface properties he panrcuia! application ol the designer BMP compositions will de-line the appropriate formulation, Potential matrices lor the compositions may be biodegradable and chemically defined calcium suilafe, sricaieiurnpbnsphale, hydroxyapatite, polytactic acid and polyanhydrides Other potential materials are biodegradable and biologically well defined, such its bone or dermal collagen Further mainces are comprised of pure proteins ot exhaceiiniar matrix S components. Other potential matrices are nonbiodegradabie and chemically defined, such as sintered hydroxyapatite, hiogiass aluminates. or other ceramics Matrices may be comprised of combination··; of any ol the above-mentioned types of material, such as poiylactic acid and hydroxyapatite or collagen and tncifeumphosphste The bioceramics may be altered m composition, such as in calcium-aluminatephosphate and psoces.smg.ta alfer pare size, particle sise, particle shape, and htodagradabltify. h The dosage regimen wii; he determined bv the attending physician considering various factors wme i nsoeih the a<. I on of toe uei ignor ptotom hex ? tsc tote mciuoc, w meat I'milafion hie ameu» it of bone weight desired is be formed, the site of hone damage, -he condiuon o; the damaged brine, the ο vO ol u ' ouio ' co v d-ii ao> d ts a ; I e < -»eois -ig< \ am I hi< wxor h η -? w iib ct or lime of administration and other clinical factors. The dosage may vary with the type of matrix used ig hie 5 recoiistilution, The addition ol other known growth factors. such as IGF t {insulin like growth factor it. to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of bone growth arid'or repair One method of assessing bon» growth or repair is by .x-ray imaging and/or CT scanning, among many art-recognized methods.
The compositions can be lormuhtfed lor parenteral or oral administration io humans or other ${) mammals in therapeutically effective amounts, e g„ amounts which provide appropriate concentrations of the designer BMPs; io target tissue for a 1»«» sufficient to induce -fie desired efleet. Preferably, tie present compositions alleviate- or mitigate the mammal’s need for a morphogen-associated biological response. such as maintenance of tissue-specific function or restoration of tissue specific phenotype to senescent tissues (e.g., osteogenic bone tissue) or the inhibition of reversal of a fibrotic response in a tissue.
As will bo appreciated by those skilled in if;·? art, the concent! ation of the compounds «escribed in a therapeutic composition will vary depending upon a number of factors, innfuding the dosage of the drug
2017200239 13 Jan 2017 lo be administered the chemical chamotensfios ie g hydrophobicity) of the oempounde employed, and the route of administration. The preferred dosage of drug io be administered also Is likely to depend on such variables as the type and extent of a disease, tissue toss or detent, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the fonnuietion cl the < o>aoc> N k® iidw-oti-H hoU r-^eiffr.i-m> vctr ou* ’ adi 'ν'->f of cn
In general terms, the compounds of inis invention may be provided in an aqueous pfiysioiogicai buffer solution containing about 0.1 to W% w/v compound for parenteral administration. Typical doses ranges are from about 10 ng-'kg to about 1 g/kg of ncoy weight per day. with a preferred dose range being from about 0 f mu/kg to 00 mg-’kg of body weight.
Designer BMPs may be used for any indication that wild type BMPs are useful -or or for any method In which a TGf-β superfamily member can be used. Designer BMPs are capable of inducing the developmental cascade of bone and cartilage morphogenesis and te induce or mediate Smart signaling pathways. Designer BMPs induce greater bone augmentation and repair. including: but not limited to, production of greater bone mass. bone stifiness and bone density that corresponding wud type BMP Accordingly, designer BMPs may be used to induce bone formation in a tissue Also, designer BMPs mey be used io induce piolifonation of bone and cartilage in a variety of locations in the body. For example, designer BMPs may be used io repair joints such as knee, elbow, ankle and finger, for h example, designer BMPs may he useful for regenerating cartilage m patients suffering from arihnfis or other cartilage degenerating diseases. Further, designer BMPs are indicated for treating tears m cartilage due to Injury to addition designer BMPs are useful for inducing bone growth m patients For example, designer BMPs are indicated for use m treating patients suffering horn bone fracteies or breaks.
osfeopcioals. or patients In need of spinal fusion or for repair of fee spine, vertebrae or fee like 5 In another embodiment, the invention includes s method of bene augmentation and.'or repair.
Thus, the invention encompasses administering a therapeutically effective amount of a designer BMP to a site where it mediates: detectable bone augmentation or repair.
In another embodiment, the invention includes a method pf Inducing or increasing Smad expression. The method comprises contacting a catt; comprising Smart mediated expression pathway vatb a designer BMP of the invention.
Dowcnor nMPs --=e -.00--61- of κιπηοιηο ;i <.:< ve-eumenfei vav a.·* c* 6,.11-=- mcc-nogi-11-=70 ami tissue morphogenesis for a variety ol tissues in mammals different from bone or bene cartilage. This mcrpnogenlc activity includes the ability fe induce prolifeiaeon and differenilatino of progenitor ceils, and the ability to support and maintain the differentiated phenotype through the progression ol events that id results in the formation of b-bhO, cartilage. oon-minergiiAsd skeletal or connective tissues, and other adult tissues.
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For example. designer BMPe may be used for treatment so prevent toes oi and/or increase txme mass in metabolic bone diseases, General methods lor treatment in prevent loss of and/or increase bone mass in metabolic hone diseases using osteogenic proteins are disclosed in tJ.K. Paten- No. 5.674.844. Ihe disclosures oi wh-ch are hereby incorporated by reference. Designer BMPs may also be administered 5 to rep-ace or repair boos or cartilage at Injury sties such as bone Cranks, hone frao-uros, and cartilage tears. Designer BMPs oi the present invention may he used tor penodenUii tissue regeneration General methods tor periodontal tissue regeneration using osteogenic proteins are disclosed in U.S Patent No 5/33.878. the disclosures oi wh-ch are hereby incorporated by reference;
Designer BMPs may bs used for iwer regeneration. General methods lor liver regeneration using 9 osteogenic proteins are disclosed in fj.S. Patent No, 5.349,566. the disclosures ol which are hereby Incorporated by reference. Designer BMPs maty be used for treatment of chronic renal failure, Generat methods for treatment of chrome: renal failure using osteogenic· proteins are disclosed in U.S. Patent No. 6.861.904. Ihe disclosures of which are hereby incorporated by reference. Designer BMPs may be used for enhancing functional recovery -allowing centra! nervous system isohenuo or trauma. General methods S lor enhancing functional recovery following central nervous system Bohemia or trauma using osteogenic proteins era disclosed in it S. Patent No, 8,407,060, fhe disclosures of which are hereby incorporated by reference.
Designer BMPs may be used for inducing dendritic growth. General methods for inducing dendritic growth using osteogenic proteins are disclosed m 9.S. Pstenl No 6,949.595, the disclosures of n which are hereby incorporated by reference.
D< j, c i< t BVk may u> iawk for tr xupir ? t< ud e I all'cCi Ge? raf m {ί*>% f-A ί >d ι> ί ig neural cell adhesion using osteogenic proteins are disclosed m U S Patent No 6,860,603. the x.is< osuros x/ -v von -uh xfi, it rep ora ed bv r. foram.
Designer BMPa may be used for treatment and prevention of Parkinson's disease. General ..5 methods for freaiment and prevention ol Parkinson's disease using osteogenic proteins are disclosed in
N,S. Paten- No. 6,606723. the disclosures of which ar-a hereby incorporated by reference.
If is within skUis of art ordinary artisan h> modify fhe general methods using the modified BMPs of Ihe present invention for various, therapeutic uses described above, Exemplary embodiments of therapeutic applications ot fhe modified BMPs of the present invention are further described below
Designer BMPs may be used to repair diseased or damaged mammalian tissue The tissue to be repaired -s preferably assessed, and excess necrotic or interfering seer tissue removed as nesided. by surgical, chemical, ablating or other methods known in the medical arts. The designer BMPs then may be provided directly to tbs tissue locus as part of a sfenle. btoccmpatibfe composition, either by surgical Implantation or injection Alten-ytively, a stente, b-ocompatlbie composition containing modified BMP35 stimulated progenitor ceils may be provided to fhe tissue locus The existing tissue at the locus, whether diseased or damaged, provides foe appropriate matrix to allow ihe proliferation arid tissue-specific diffe’eniiate n of uiooenifor cells in addition <s damaged >x diseased tissue focus. porfi-'.utam. one teat
2017200239 13 Jan 2017 hes been iurther assaulted by surgical means, provides a motphogenicaliy permissive emdfoomenh For some tissues, it is envisioned that systemic provision ci the modified BMPs will be sufficient.
Designer BMPs may be used io prevent or substantially inhibit soar trnsne formation following an Injury, if « designer BMP is provided to a newly injured tissue toons, it can induce tissue morpuogunesis 5 at the tocus, preventing -he aggregation of migrating fibroblasts Into non-tidferenfiafed connect!',- t'-ssue The designer BMP pruterabty is provided us a sterile pharmaceutical preparation injected into the bssuo locus within five hours of the injury.
foi example. trie designer BMPs may he used for profsiminduced morphogenesis of substantially injured live; tissue following a partial hepalec-c-my. Variations; on this general protocol may be used for b oilier tissues. The generaf method involves exo-sing an essentially nontexonerating portion of a tissue and providing the modified BMP. pnsferabfy as a soluble pharmaceutical preparation to the excised tissue locus, closing the wound and exarmmng the sbe at a future date Lire bone, liver has a pEdenlm; to regenerate upon Injury during post-fetal life
As eriiyjper e:<emp;e, designer BMPs can afso be used to induce deribnogenesis To date, the 5 unpredictable response of dental gulp tissue to injury Is a basic clinical problem in dentistry. Using standard denial surgical procedures, small areas leg . 2 nwn) of dental pulps can be surgically exposed by removing the enamel end dentin immediately above the pulp ihy dnlilngj of sample teeth, performing a partial amputation of the cotonaf pulp tissue, inducing hemostasis application of the pulp treatment, and seating and biting the cavity by standard procedures.
ft The designer BMPs of the invention may be used to beat fibrosis. The fibrosis may be located in various parts ol the body and can he of a particular bind, for example, the fibrosis may be located: In the kidney, for example, fibrosis as observed In glomeruionenephntis. diabetic nephropathy. atiograft rejection, and HIV nephropathy, in the liver, for example, cirrhosis, and verso-occlusive disease. In the lung, for example, idiopathic fibrosis (and sn-oimmtine flbrc.-sls|; in -he skin, for example, systemic .5 sclerosis. keloids, scam, and eossncphslia-myaigia syndrome; in foe central nervous system, tor example. Intraocular fibrosis; in the cardiovascular system, for example vascular restenosis;; in the nose, for examplii, nasal polyposis; in bone or boos; marrow, in an andocnne organ, and in the gastrointestinal system.
In oiiii: embodiment, a designer BMP having the binding cftarecleristics of BMP? or useful
3ft modification -hereof (extended half life, increase binding alfsnity for a same or different receptor compared wdh wild type BMP? resistance fo inhibition by a BMP? antagonist such as but net fimited to. Noggin, and the like} may he useful to heat, ameliorate or reverse fibrosis. That Is. as reviewed recently in Weistorshen at at.. 20B3; Frontfots in Busse; 14 4992-6(112. TGFb; mediaies a cascade leading to Increased fibrosis, including. but not limited to. eptihetMi-to-niesencisymai transition. The fibrosis-irsducing effects of ΤίΤΒβ; may be inhibiusif or reversed by BMP?. Bee also t.ourelfo et at. 2010. Nephrol. Plat. Transplant 25'1093-1103 Further, certain fribohc conditions may also be treated or ameliorated by administration of BMPl {see Pegc-rie; et a; . 2010, Reap Res f f Bo) Therefore, the invention
SB
2017200239 13 Jan 2017 encompasses a designer BMP eotbef based on a BMP? ffimiewotk and/or fesorporgiing the -type hand type fi mutations disclosed elsewhere herein, to slier receptor binding end provide & potential useful therapeutic for treatment of fibrosis, in a patient ir! need thereof
A fibrotic disorder may be induced by s number of causes Including; chemotherapy, lor exempts.
' pulmonary fibrosis resulting fmm oieomycin citiorambucii eyciopriaphamids. methotrexate. mustine, or procarbazine treatment; radiation exposure whether accidental or purposeful as in radiation therapy, to? example ofe'Viha! s.) ; <. ¢-- wism ΐ I B? s-mut' ό a m a<>, tai v >·. i iniMhl ot r\u f tul ks.tois < eeiSuUnts such «s cliemicgts fumes, m.'tais vapors gas».? etc, for example ’LU resulting sen: asbestos or coa? dost, a drug or a combination of drugs; for example, antibiotics teg. penicillins., d suiionamtdes. etc.;, cardiovascular drugs (e.g . hydratazine, bob? biockets, otc.i. CNS drugs (phenylofii. chlorproniazine, etc.; anti-inflammatory drugs (e.g.. gold salts, phenylbutazone. etc.), etc. can cause it.D: an immune feoction disnrde;. foi example, chioniq graft versus-hoid disease wife dermal fiorosis. disease states such as aspiration pneumonia which is a known cause of it.D, and parasite induced fibiosis; and wounds, for example blunt frauma, surgical incisions. battlefield wounds, etc , as in χ penetrating injuries of the GNS, ir? a particular embodiment, dissiguer BMPs with improved binding to type I receptor AUC2, such as BMPE. may be used to treat diseases related to-ALK2.
Kits t? Tbs invention includes various fids which comprise a therapeutically effective amount of -a designer BMP pi' the invention along with an applicator and instructional materials which dcsc’ibo use ot tiie designer BMP to perform the methods of the invention. .Although exemplary kits are described below, the cuntcnls of other usotut kits win be apparent Io the feGfed artisan m j,gbi ot tt?u present dfw.fesure Pact? of these kits is. included within the invention.
..5 the invcntK't: iriceev e; kr ler ffc«tme-|f to pre vem ass ot and'or snciuasu sone mass n u metabolic bone disease in a patient in need thereof. The kit includes a designer BMP oi the invention. The fet further comprises an applicator, including but not limited to, a syringe, a bone cement mixing device, end the like, for administration of fee components ot the kit to a patient. Further, the kit composes ,»l i G udo ι ι '-ifen |I -i fng I nth ί > mdinenf ι i>>? v dot ?c fee t \e of fee kt ?o '* >?t o poet it vtf he w mass and/or increase bone mass; in the patient
More preferably, tbs? kit comprises a; least one designer BMP selected from an antibody having an amine acid sequence selected from the ammo acid sequence of SEC ID NOs;S~?3, even more preferably, fee designs; BMP composes the ammo acid sequence of BEG IB NOB3. SEC E? NO, 14, BEG ID NG.36 and BEG IB NO.3?. Preferably, fee designer BMP is BMPE, BMPG, BMPGE and
O BMPGBR
6?
2017200239 13 Jan 2017
The kit can compose any number of additional therapeutic agents for traatmeu· io prevent bona loss attd/or Increase bone mass. Such agents are set forth previously and include toeraperfi; compounds, cytokines, vitamins, oilier members ot the ΤΌΡβ snperfamiiy. among many others the invention also relates to an article et mssuuiacture tog., dosage lorn· adapted for t.v. cr oral adminsS’ration) comprising a designer BMP in die amennt affective to prevent bone toss and/or increase bone biass (e.g., more than 10 mg/kg. at feast 15 mg&y. or 15 mg/kg). It; certain embodiments. the artiste of manufacture comprises a container or containers comprising a designer BMP arid a iabei and/or Instructions tor use to treat or pry vent bone loss and/or increase bone mass the Invention also includes a kit to treat or prevent fibrosis m a tissue or organ in a patient In veuhertof ' K kf oxiudcs o < r sgm r I3”l· e i! < m t ton. The k {r d'Cf e^po^es or aoptea'c' Inducing hut not Ssmifed to. a syringe or device ior delivering the protein a mixing device, and fits like, for administration oi toe eomponeife; of the kit to a patient. f-'urther fee lei comprises an Instructional material setting forth the pertinent information for the use of the kit to treat or prevent fibrosis in the paher-t s More preferably, She kit. eontpnses at least one designer SMF5 selector from a protein having an amino acid sequence selected from the ammo acid sequence of SEQ. OMfe d /3, even morn preferably, the designer BMP comprises the amine add sequence of SEQ IB N012. BEO ID NO:14, SEQ IO NO;36 arid SEO tO NO 3? Preferably fee designer BMP ts. PMPE. BMPG. BMPGE or BMPGEB
The kit cars compose arty number of edditsonal therapeutic agents for treatment to prevent bone d loss and/or increase bone mass, or treat t>r prevent fibrosis. Such aoenfs are s.et forth previously and iudude therapeutic compounds, cytokines. vttomms. other msmbersof fee I Of β suporfemiiy, among many others.
The invention also relates to an article of mitnuloeiure -e.g , dosage form adapted fer t v, or oral adminls-ration; comprising a designer BMP in toe amount effective to prevent hone to&s and/or increase ,,5 bem ut.AY o- f' 4e * v or; ve if f h tr g, non than ‘ mg.t'p U iem-p ' he nM< cd ng kc v IS mg/kg) in certain embodiments, tbs article oi martulacfure comprises a container or containers comprising a designer BMP and a label and tot instructions for use to treat or prevent bone fess- and/or Increase bone mass or to treat or prevent fibrosis
The invention is- lurfher described =π detail by retefenae to toe following expenmentoi exampfes
These examples are provided for purposes of Illustration only, end ore nof Intended to be limiting unless ofbafWiae specified Thus;, the invention .should in no way be consfrued ns being limited to the feiiowmc examples, but rather, shoufd be construed to encompass any and all variations which become evident as a result of toe teaching provided herein.
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EXAMPLE 1
Recombinat;t host C-HQ ceils producing arid secreting Wild typo and designer BMPs worn generated using standard recamblnant DMA pmsedures Ctortdinoried mshin was generated from adherent cel; cultures. Bneby, CHO eels were seeded in medium containing 18% uFBS and allowed to grow to near confluence for 3*4 days. After mis growth phase, growth madiam was disrarcted and the 6- colls were rinsed once with PBS-CME and subsequently switched to a sentohFttee medium supplemented with 200 ugrmf dextran sulfate. 2mM sodium butyrate, and >6mM HERBS. Cells were then cultured for 7 days at tenipefafore of 31 C Conditioned medium was harvested and clarified by using stenfoung 0.2 uM filtration. Conditioned medium was atoned at -20''0 until purification.
5. io order to purify the novel designer BMP mo-eouias from CHO cell conditioned madia the BMP's were captured by two steps oi conventional chromatography arid the results are shown ip f igure g. comprising panels A-D. Only the details of the purification of BMPE are shown herein Since all of the other novel designer BMPs were purilied m an essentially similar manner
CHO conditioned medium ICH-O CM} (pH adjusted fo 8.0 with 1.0 M Tns, pH 8.0? was loaded it onto a Ceilufme Sulfate cofomn {65nil, 2.6 x 12 3dm} that was equilibrated with 20mM MBS pH 3,0. The column was washed with 10 column volumes (CV- of 2.0 mM Tris. pH 8.0, 10 CV 50mM ME-S pH 5.8 and 10 CV of Buffer A (6.Q M Urea, c-OnsM MES, pH 5,8}, The BMPs were eluted wiih a linear 0-1,0 M NaCf gradient over S CV {Buffer F-~ 8.0 M Urea. 50 mM MES. 1.0 M UaCI. pH 5.5}. Upon application of a -''jC u a h ο; ,P- - o< wrl a hr >ad pe to tet'e i -. Pt Pl vC'-s <r < ' id c > no» m -1,» χ;e s-*r of
BMPS was observed (Figure 5.A) Fractions were analyzed by Coornassie stained SUB-PAGE gels and BMP containing fractions were pooled BMPs in fractions wars identified as reducible dimers on SOBPACE Nou-Redueed gels {left panel of Figure 5Bs. The BMP pools from the CeSiulsr-e Builafo chromatography step were further puriffeii by preparative Reverse Phase HPTC on a 10 x ?50mm Vydac IS pm C3 Column (Solvent A= 0.1% TEA. Solvent B- 98% acetonitrile, 0.1% TF7G, with BMP eluting with
5b approximately 321¾ acetonitrile A tracing of the Reversed Phase chromatography step is shown in Figure 5C. The protein was concentrated and acetonitrile was removed using a spcedvac and the concentrate was formulated info MFR-163 buffer via dialysis The purified BMPs were characterized by SDS-PAGE, A288 and LAL. Assay {endotoxins} A photograph ol an Non-Reduced BOB-PAGE gel (left side cl Figure 50} and a Reduced SOB-PAGE gel (right side of Figure 5D- showing the same gel fractions (FI 3 through F13) its shown, A total of 16 BMP designer proteins were porif-eu to essentially the same levels of purity and exprsssion/punfication yields ranging from 0,3-1 -1 mg'f. CM and the results are shown in Figure 3 showing photographsa {Figure S). Brieiiy, wild type BMP2 -W τ; and designer BMPs
SO
2017200239 13 Jan 2017
BMPSE, BMPEL EtOBB, ΒΜΡ0. BMP SdhFL BMP SMB, BMPB, and BMP-EN Me Shown in photographs of a non-reduced ge; (Figure FA) and a reduced SDS-PAGB (Figure 88;, and designer BMPs as (variant of BMPA). aii (variant of BMP A) c (BMPC). hi (variant of BMP!), h». i, f, and g are shown in photographs of a nori-reduced SOB-PAGE (Figure EC) and reduced GDS-PAGE (Figuie 60).
s
EXAMPLE2
SQ«£8QG£.Activity.< dessonerB>Psdemongt^edfis^
Approximately 8000 C2G12 csfts/weil in a 98-weti piaf& were Imated with fhe indicated BMP ant 9 toe dose indicated, (wenty-iour hours post-treatment. toe plates wore processed to measure alkaline phosphatase which is an aft-recognized assay for osteogenic activity. The culture medium was removed, and the plafas were washed twice with C3ieturn.foiaqnesii.fni--frfee PBS 59 μ| oi a-Methylurnbeiliferyi phosphate (4-MUP Liquid Alkaline Phosphatase Substrate Sigma cat. ?? M3168) was added to each weft, οπή Ihe plates were incubated In the dart; el 3RC (or 15 minn-es F-iucrescance was measured on a S Victor iumlnomeler (settings: excitation at SSSnM: emission at 460nM: €W lamp energy at 1120;, 1 second per well Adei the readmg was complete. SO pi of 2>- grotem assay lysis buffer (200 mM Tris-HO. pH 8,8-0 4% Triton X-100) was added Io each well and the protein concentration was determined using toe BOA Protein assay (Pisfce; following- Tie manufacturer's miemplata procedure. The alkaline phosphatase measurements were toen normalized to the total protein concentration (l.e.. fiooromethc it omts per mkrogram ci protein; As shown by trie graph in Figure 7 O2Ci? muscle pre-myoblasi cells treated wtb mult-pie designer BMP moieouies snowed slgnsitcantiy increased Alkaline· Phosphatase acliviry, as a marker of osteoblast differentiation, compared to treatment wi>h wild type BMP2;heavy fine veto smell circles), Designer BMPs oxh-b-tlng increased AP activity compared wito WI BMP2 included designers; BMPA. BMPF, BMPG, and BMPF. Surprisingly. designer BMPF demonstrate;! equivalent 5 activity io that of the wiid type ΒΜΡ2-Ί3 heterodimer (heavy line with squares), which is known io bind both the type s receptors of SMP2 and type if receptors of BMP6 with high affinity. Designer BMPB is the resuit of infrodushon o; the tow affinity type I binding region oi BMP6 ;nto BMP?. The extremely high activity oi ihe designer BMPE molecuie was extremely surprising since it was predicted that BMPE would have low aihmty binding to both type f and type It receptors. Iniensstsngiy. Ihe cfne- designer BMP 69 molecules, designer BMPA. designer BMPF. and designer BMPG. have regions of wild type BMPS that bind the type ϋ (high a-fini-y) nxieplafs of ΒΜΡ0 which have been iriToduc^d info BMP2 (see Figure IB), and these designer BMPs showed Increased activity compered {O BMP2. hut not as high as that of wild type 3MP2.'6 heterodimer (Figure ?).
uZd? cells steMy v> pressing fhr, EMF-mitxmse-etemem tucitorase reporter (dement is from (he lot promoter) were plated at OGGO ceils/wei; of a 99 weii and tiea-ed with the indicated BMP and
TO
2017200239 13 Jan 2017 dos& 43 hours pest treatment, me cells wen-- tysed and luciferase activity was reed using tee Promegs Dusl-Gicrsssay kit.
The data disclosed herein demonstrated mat net only was tee activity of SMPE equivalent te teat of BMP-Z'6 in art alkaline phosphatase assay. It was also equivalent in g BR£.-tucifer<sse assay in C.2C I2 5 ceils as shown In Figure 8 Further BMP? demonstrated approximately tC-20 fold greater activity in the BRE-!uo:ierase assay compared wtte Wild type BMP-2 (Figure di. Thus the results observed in the BREluciterass (BRE-fuc) assay correlated stmnglv' With those obtained in the Alkaline Phosphatase (Alk-phos) aQimv asxuy i j< o „e hviuiii vci > ju ύ; ' IU t c n no h i-n A-po.eit’
BRF-luc ass-ays, are also shown Io Table It) for wild type BMP2 aod the indicated designer BMPs, h Without wishing to be bound by any particular thc-ory, these data suggest test trie addition ot
At.K-2 as a high afitrilfy receptor tor BMPE could be the reason for its Increased osteogenic activity. This Is because an AEK-2 mutation has been found tn cause fibrodyspia-tta ossificans progressiva (FOP), a disease where young children develop inappropnate ectopic hone formafior- Thus, mutation ol Al.K-2 binding Is associated will·· inei eased osteogenesm and may he correlated to the increased osteogenic Is activity of BMPE. Thus, BMPE Is a new- class of BMP molecule with high affinity for tee type I receptors AI..K-2, 3. and 6
C2B12 celts were plated in 6-well hssue culture plater. at a density ot 4 x 10 cells/cm' sne a Incubated overnight at 3?‘ C inside a 5¾ CO-fSfj'X} humidified air incubator After the recovery period, the coituro medium was replaced wth freshly ptepared esfeogome different-afcon medium. Growth Medium containing oOnq/mf i.-ascorbic add phosphate {I.-Ascorbic .Acid Phosphate Magnesium Salt n-Kydrale. WAKO Pure Chemical Industries bat No. OfB-tzbB';}, p-glyceroi phosphate fg-Glyceroi phosphate Dlsodium sab, fOrnM Penfahydrate: Fiuka B!oChem;oa Cal. No 50026-, atvd 100 nM Menadione sc-dinm
..5 bisulfite (Vitamin R5 Bsoma Cat, No. M2518I. I tin indicated BMP was added to the apprepuats wells at the uesited concentration Tbs plates were incubated at 3'P'C for approximately to days with medium tepla-cemeni every 2 to 3 days. The ceils were stained with the Aitzann Perl stain following tee standard published protocofs.
Aa shown in 'fable 9. below, designer BMPE induced nttnereiisiihon ol C3R101-1/2 mouse )0 mesenchymal stem cells to a far greater extent than corresponding wild type BMP? as Indicated by alizarin red staining. That is. as more fully discussed below, at doses; where wild type BMP-2 was unable to tncsuce mineralization of the C3hBQ]'-t/2 celts ig 25. go. and tOO ng/ml) BMPE homodsmer induced strong mlnerahsaiion similar to that of -fie BMP--24) heierodsmer sill as shown <n Table 9. Thus, tee aifcann mu staining assay results fudhe-r cormtatr. the results cotmnod m the Aik-phcv aod BRE-Eic assays as d isefosed pray ios t d y herein.
2017200239 13 Jan 2017
TABLE 9
Tmatmant | BMP2 | BMP2fe | BMPE |
cnotmi | ............... | ......... ·' | ....... - |
25 ng/mi | 7 44 | A / | |
50 ng/mi | r :.. 4 s- s-i- | 4-$- / | |
W ng/mi | - | 7 74444- | .............A-H- ..... 7 |
To determine wfietfror the stranger osteogenic activity observed in vitro by the designer BMPs corresporrced io similar increased activity in vivo, rat ectop-e bone formation assays were poffonntfo. BrlefSy, art ACS {absorbable coiiagen sponge; impregnated with the indicated total amount of designer BMP •n 180 roicroifesrs of buffer was vnptanfed mfo ibe hamstnna of S woo;·: old tnafs Long Evans rats. More spfeotikaily. three 5 mm biopsy punched ACS dises were sutured together wdh non-resorbabie silk sutures The sponges were wetted wdh 188 miemlitere oi the BMP solution containing the amount of BMP indicated in the chart in rig ora 9 fl.e.. 0.1 pg or 0.5 ug). Tits welted sponges were equilibrated id room temperature for 2« minutes The sponges wore then surgically placed into the hamstrings of each rat bilaterally, Each BMP {wild type and designer molecules; was pieced into both limbs of 4 rels. Two v.ev’wi cost ϋηρ: srfahne hre jrorouh· weto --acrsfiOi· ) an. hmm,mure. wwc >iisv <e?ed glared in 18 formalin end scanned by pGT (Scanco Inc.) to determine the amount of ectopic bone present The S amours of hydroxyapatite in miltlgrams (mg HAr present In the limbs of the treater· animals. :s snown In f igure 9. f igure 8A shows fee results for BMP2, BMPE and 8MP2f6 heterodireer. f igure SB shows the results. for BMP.2. BMPG, BMPA. and BMPF. For oach of the designer BMPs, ectopic bone was- formed at doses at witieh wild type BMP2 was unabfe to form a detectable bone mass, in a hesd-toTteed comparison rtf wild type BMP? with d&stgnur BMPF. was. able to Induce ectopic bona to -he same extent At os w;fd type BMP2.6 heterodimer, csosely matching the results obtained in trie srt vuro experiments disclosed previously. Designer BMPs BMPG: BMPA, and BMPF else· demonstrated Significantly higher ectopic bone formation compared to wild type BMP2 {Figure SB). Festets from tide assay are shown in Figure 9 and also presented in Table 10.
2.¾ TABLE 10
Nome | Aik-phos | SRF-iuo | Rat actogtc bone formation |
’ BMP2WT....... | 8 44 . : | :44- | : ' 7 7.. 44 |
BMPA | 4444 : | : .^.+.4-4- : | 4.,4.4..^;- ....... : |
BMPE | : 44 : i | '?'·· | '44' |
BMPG | 44 : | : -aa | 44 |
EiMPD | :/ 44 / | : 4-4· | |
BMPE | t44444 | ΐ- 4- 4 4- 4 · | + ++. + . + .}. |
BMPF | 7; ^444I: | 4-44· | |
BMPG | 44444 : | A-te-ite | < 44444 |
BMPI-i | 44- | 7 44 | . .+, .+. |
2017200239 13 Jan 2017
iBMPi [ i | -t ·< + I 4-4-4- I |
[BMPJ 5- Kwvvvvv'awXw^wwvvxxxxxAvxxxxxxvw.w.waW.v'Xww.w.w.xv BMPO-P T W | |
L BMPS-shprt j ee-i-r f | •'ί··:*·?·:' I 4 4 ... .J : .......' ' ..........i .................. ..... ............,1,1,1,1,1,1,1,1,1,1,1 II ,ι,ι,ί |
BMP Blndhw | EXAMPLE 3 |
To further elucidate the mechanism of increased ’'ducgenio activity of the designer BMPs, binding kinetic analysis of each ol the designer BMPs with a panel of BMP receptors was performed using the (betel system tPor-eBio, Menlo Park. CA). The Octet QK analysis was performed af degrees in |
TBS with 0,1% Tween-20, Samples were agitated at WOO rpm. Anti-Human IgG Octet tips were satumfed with W ugfml of each receptor-human-igGl-Fc fusion protein for FOmin, which typically resulted id capture levels of iris receptor that sire saturated wtthm a row of eight tips, Each BMP was prepared as a ft sevenfold serial dilution (typically 200-6 nM m smgiieate} pins hotter hit-mite. Fact: Reofcpfor.'BMP binding pair was run at least in duplicate Association was monitored lor 10 mm and dissociation was followed for into duffer alone Kinetic parameters, (kon and koffi and affinities (KDI were calculated using -he Octet Data Analysis software 6.0 using a oarlsai binding I 1 model following manufacturer's instructions.
Tbs data set forth in Table l? show that wild type BMP2 and BMPd proteins, each demonstrated 5 fhe expected high affinity binding to type i (AL.K-3 and ALK-6; and type fl receptors fActRsfA. AoiPliB. and BMPRIt). respectively The wild tvpe BMP2.<& heterodimat exhibited high affinity binding to both grotipt,- of type i and type i| receptors, as did designer BMPG, which the type H binding domains A and 3 of 3MP2 have been replaced by fhe domains of wild type BMP6, Designer BMPE showed similar affinity as wild type BMP? for the type It receptors as expected .since no mutations were made tn die type d binding fl regions Unexpectedly, designer BMPE maintained high affinity binding for the type I receptors AI..K-3 and ALK-6 with the type I binding domam of BMPft which has Penn substituted in place for fhal of BMP?, while also unexpectedly binding the type I receptor Al.K-2 with a KD of 2 rim. Thus. BMPE surprisingly gained a very high affinity binding for ALK-2 not observed >n either WT 3MP2 or vVT BMP6.
TABLE 12
2017200239 13 Jan 2017
As shown in Table 13. combining tbs mutations oi BMPG and 8MPE, comprising either proline or arginine at amino acid residue 36 (P36P) reiaiive to the amino acid sequence of wild type BMP2 as set torih in SEQ fD -40.1, to produce BMP-GEP fuiso referred io us BMPGE P36'i and BMP--GEP (also referred io as BfvtFGE P36R}. respectively. produced designer BMPs which demonstrated high affinity, low nM KDs. for all type I and Type if BMP leceptofs including MK-2,
TABLE 18
Psrvptar | 8MP-2 | SMP-S | BMP- 2& | 8MP-E | SWCJ BMP* hMP-b| gER j | |
MK5 | >1080 | TOO | 350 | 2 | Μ» | 2 i |
MAS | 4 | 11 | 2 | 3 | r, i to --- ; | 2 i |
AIKS | Ί | 20 | 0,5 | 1 | L 1 | T L |
ACTRIIA | S3 | 3 ....................:: | 2,5 | 40 | ( 2 | 2 i |
AWtS | 8 | 0.5 | 1 | 8 | 9 O | 0.6 i |
........—.η .............ί | 4 | 3 | 82 | r < | 3,6 |
Thus, the dais disclosed herein demonstfate novel designer BMPs, ouch ui, Lui not itmitud to,
BMP-GER end BMP-GEP. which combine the attributes of BMP-G and BMP-E such that these novel designer BMPs demonstrate high alfinity biriding to a wide repertoire of both type I and type If W-eepiofS, Including. but not limited Ιο, ALK2, ALK3, ALK6, ActRiiA. AcfRhB ano BMPPifA. The data lurther demonstrated that replacing the praline at residue number 88 oi the: amino add sequence of WT BMPS
I S (SEQ 113 NO: 1 > to arginine produced a designer BMP that was as effective as an otherwise identical BMP where the amino acid was: not replaced. These novel osrfeogenic BMPs as exemplified by BMP-OER. provide high levels cl biologic activity thus allowing lower dosing and. In some cases, more rapid osteogenic response, stmngty suggesting that these molecules would provide highly effective therapeutics,
2ft
EXAMPLE 4
In Vivo Osfeogaitic Activity in Non-human primates osteotomy mode/
To further assess the potential therapeutic potential of the novel designer BMPs oi ihe invention, die activity of designer BMPE end BMPG was compared to that of wild type BMP? In an NHP inonhnman primate) fibula osteotomy model.
2017200239 13 Jan 2017
A mld-dsaphysoai osteotomy of the fibula was performed bilaterally with tty? imm blade o’ an oscillating saw ir; adult mala Cynomolgus monkeys gVfaoace lascicuams) with g mean body weight -;sn« standard deviation) of 7 5 + 0 2 kg and an age range ot seven to fen years. A. small intramedullary Kirschner wee was added to the previously described hbular osteotomy model to maintain alignment ot the proximal and ulsfal bone ends for more uniform torsional biomechanical testing. The two major a jvai'tao·, set this model ite II io sp-tity to aiifze a h'eterai ss..d> doyen as a resv ti J the tow medodav ot die procedure and the ability io remove a n io 8~em segment of the fibula containing she osteotomy site ?ot subsequent biomechanical and histological evaluation without having to sacrifice the animal. A 500 pi solution of 0.5 mg/m! of either wild typo or designer BMP was added io a 30mm X 13mm ACS sponge
The spongy was wrapped around the detect following surgery. An approximately 2mm fracture of the fibuia of each limb of a skeletaiiy mature NHP was wrapped in an ACS sponge comprising either a designer BMP molecule si 0,5 mg-'ml dose (250 ug total delivered) or the same amount ol wild type BMP2 in the contralateral hmh. Thus, each animal received wid typo BMP' In one limb and a designer BMP m too oontralatetai iimb.
S In tins model, designer BMPF and BMPG were chosen stone each represents a different class of designer molecule, designer BMPG shows high sifimty for both type I and type II receptors while BMPE binds the typo I lerepter Ai KA? with high atfsnify In addition to binding type ϊ receptors At X-3 and At i<-G with nigh aiflnsiy. Radiographs were obtained every 2 weeks io compare the healing ot the limbs treated with -ha designer BMP molecule compared with the healing of lbs contralateral limb treated with wild type
BMP2 in each animal. As shown in Figures TO.A-fBC. the data, which: include seven animals from each group, demonstrated that the callus formed earlier and more robustly in the limbs treated with each designer BMP (BMPE shown in Figure 10A and BMRG shown <n Figure IBB 1EG} molecule compared te that with bone formation observed in the limb treated With wild type BMP2
Tables 14 and 1b, below. set forth date providing quantitative assessments of -he difference in bone mass and hone volume observed between limbs treated with wild typ© BMP2 and limbs treated with designer BMPF. As shown in ligery 11. BMPF administration resulted m an average of a 33% increase in bene volume {mm'') when compared with bone volume increase in wild type BMPS treated limbs. This gCT analysis included all the nativts bone where there was callus, accordingly, BMP-F 'was much more robust than BME-g ,π the same animals.
MF
Zb
2017200239 13 Jan 2017
TABLE U Bone Mass (mg HA)
MHR | Left BMPB | Rtqht - W? BMP2 | '3 .n<. tease vs Ft 16T. |
’ 5304 | 721.2206 | 600 33 i ? | |
5604 | 581.4153 | 485 766 | '15% |
8104 | oil 4215 | 313.4351 | 63% |
9604 | 524.7777 | 4 74,0543 | 11% |
:8204 | 714.8123 | 638.7811 | 33%. |
17504 | 431 5738 | 406,1264 | 6% |
22508 | C25.7563 | 466.0707 | 34% |
average | 28%. | ||
si.d dev | 20% | ||
aid error | 7.40% | ||
peired l-fesf. | P“0G40 |
TABLE 15 Boon volume (mmS)
NHP | Left BMPF | RioM-W; BMP2 | % increase vs R |
5384 | 307.4342 | 728.8308 | 25% |
5504 | 632,6525 | 534.9825 | 12% |
8104 | 583 9513 | 336 073? | 74% |
5684 | 573,0165 | 507.0014 | 13%, |
18204 | 852.5589 | 531.2446 | 55% |
17584 | 814 226 | 482 0475 | 6% |
22556 | 736.8873 | 528.5033 | 45% |
average | 33% | ||
ski dev | 25% | ||
std error | 9.80% | ||
paired f-fest | p-,0070 |
Proisne at position 36 rsiaifve. to the amino acid sequence of wild type BMP? as set forth in SEO IO ID HO J •55 pntporiedj important ?< ronfemug Noggm resistance and ;.?evdiHg ιι ·>η eased οχ-ΐ-ρο-κ AcfMt? :o 'Vt:J t\pe ΠΜΓ’1 (see s g Wi < ?889<08GT3i) TneuPniA fe assess 6--=- offer* nf mpi.wnq Γ38 with it non-cotiserved amine acid substitution on fee novel activity of BMPGL, P'30 of BMPGLP was mutated io ergininine to produce RME’GPR and osteogenic activity of the two designer molecules was assessed it; vitro. fhe data disclosed herein m Figure 12 demonstrate that repiacing P33 with arginine id -P36R- die not aifeci the binning afiinify of the nevei BMP'-GE designer BMPs. end both BMPGEP end
BMPGER wens as active as E>MR2;5 hefemdimer.
§Μί^βΕΒ±4ϊ%ηΗνο^^
As shewn in figures 13 and '54, rot ectopc experiments show that BMP > L a as potent as BMP-2/6 at driving the formation of ectopic bona at the very tew dose of 0.25 τ o a! BMP when ail
2017200239 13 Jan 2017 moisoulea are delivered or; an ACS sponge Figure 13 shows that only BMP-2Z6 and BMP-GER. hut not BMPF or BMPG; were significantiy more active than BMP-2 at this low dose when the milligrams of HA formed in the ectopic were quantified by yGT analysis
The some samples wore demineralised and scored ior bone forma-ion iBone Score) by histology and these results ore shswn in Figure 'i-E By this method of sconnc, at ihe low dose at 0.25ug delivered BMP-2 has no bone formation, and BMP-GER arid 2/6 had the highest score. BMP-G and BMP-E were also significantly more potent than BMP- 2 but no- as active as BMP-GEP,
Figures 15 and 16 show the results c-l a severe Nitp fibula osteotomy model comparing Ihu activity of RMP-2 and B-MP-GER. In -his mode; a -wedge with and approximate width of 4-6 mm was removed iui each fibula oi thu Nt IF and pui back m place and held with a titanium pm The defect was liter wrapped with an AGB sponge containing 260 ug tots? BMP ar a dose of 6 omgrml In each NI-IP BMP-2 was placed in one limo and BMP-GLH was Maced in trie contralateral limb, i soars ISA shows photographs ot radiographs taken at 5 waexs showing the detect m 4 ot the 6 animals. The BMP-GFB limbs showed signiFcon-iy mom m-busi bone formation then tl-ose with BMP-2 Figure IbB (bn-tem panel of the figure) shows uCT images of the finuias oi she same i animals following their sacrifice al week IB, As con be s«en. ilia amount oi bone lormud is muci? more robust in the BMP-GER l-mbs Iha-t in the cohtraiatefs; limbs ln-;<sited wi-h BMP?
i igu’c “SAG snows tte ana\v!s o' tuew turns comoa'irq hie sPeuclu stillness «no ,'nc bone volume comparing -he BMP-? and RMP-GEE; treated limbs from each animal On average Ihe BMP-GER heated limbs requited 21% more torque io break {Figure ISA). were 24% more stiff (Figure 16B}. and ihe calluses were on average 55% larger {Figure “iSGi than -he contra laferas BMP-2 treated limb. All of these domps: icons hod a p value of less -ban (51 by pairwise analysis. These «Ada show that
BMP-GER induced ffac-iure repair «and bone formation significantly censer and more robustly than BMP-2 :q the same animal.
BMP:GEPjndticed hgf?skinm:dipry!rLittLNHPosattejagmyeienliyte.B5^:G.atjj.Bj<!ld.it)w«rdf>s«;.
To lurllisr assess ihe effectiveness of BMPE hone formation in NifP. ihe ability of BMPE to
Induce osteogenesis in si wedge defect assay was compared to that of BMP?. Figure IrA-C shows radiographs of the bone formation following the wedge defect model In three non-human pnmsies wnere f.Smg/ml of BMP-? was used in one iintb and only 0.5 mg/rnl of BMP-GER was. used m the other limb using a caicium phosphate cement based carder. Radiographically, the heaiing and bone iormabou were equivalent for each of the animats whether the iraatmeni was with tha nigh dose of BMP-2 or ins iowsr
3S doss of BMP-GER Thus oven at one-third the eose. BMPB was equivalent to BMP2 In iiiducing bone formation, demonstrating the greatly increased activity of this dasignar BMP compared with wild type BMP?
//
2017200239 13 Jan 2017
EXAMPLES
Crystei'/tzefter? D;VE’-2 end 5<V;-’-6
Pui'lfied fuily-giycssyiatsd wild type BMR2'8 h-e-eradimer. wild type f3MR2/2 homedimer. ansi wild type BMPO/6 bcnicdtme?: each produced in tcurnniaffan cells, we:e concentrated to 8Ί0 rng-'ml in 10mM eodtum acetate (qH ,).£:<). and crysfasit~nf:on attempts wens penc-mad using a 'moaqu;te' automated, robotic setup at 18cC (TTP LsbTech inc,, Cambridge, MA;. Initial crystelticatfon bits were obtained tor each dunof and the conditions wars sub.seq»&ntlv optimized to quire crystal·· ot goon diffraction quality.
Crystals of wild type SMP2'6. PMR2/2 and BMPo'o were transiently cryoprotee-ted and frozen In liquid nitrogen poor & X-ray diffracnon data collection at -F-o synchrotron sources fill) besmiioe of Advanced Photon Source SER-CAT). Data were processed and seated ussng programs Mosilm/Scala to deduce corntiat crystal lattice type -and to integrate-scale data. The resolution arid und ceil fsammetets are 5 listed as follows· BMP2/S belongs to the space group of P4?.2:2 with two copies of the heterodimer per asymmetric unit: 4 diffracted to 2 8A In one direction end 3 OA in ihe other two. With a unit cell of a~b* 10S.23A c~ 1-38.73A. ct~p~V:80'. BM;':‘2/2 belongs to the space group of P3-: with two copies of the homodimer per axymmetnc und: it dtlhacfed to 2.7A wilt; a unit cell n- a-b:-o2,74A, C”128 3-5.A, ο-β-90'1. y~ l20.· BMP6/8 belongs to the space group of P-3,21 with one copy oi the nomod;;ner per asymmetric d unit; it diffracted to 2 6A will’s a unit ceil of a:-h::S7.40.A. c::85.84A. «::β::9&, y120'. One to anisotropic dtffracbng nature of SMP2/S crystals, the data was eliipstsidatiy truacatsd end anisotropically scaled to preserve contribution of hfgh-resoiufion data, the structures of CHO 8MP2/6. BMP2/2 arid BMP8/6 were determined by molecular replacement method with program Phaser, using E co) BMP2 iPDB accession: IP.EVV; and ,E, coir BMPS :PDB accsssloh: 2R52) as search models. After correct molecular replacement solutions were obtained and space groups confirmed. Phaser-;aicalafed electron density maps were used Io evaluate the qualify of the search models, and regions m question (especially areas involving type I and type II mneptor binding- were stripped from the original model for rebuilding In order to avoid model bias.
The structural models went through ogid-body refine:print, followed by simulatiKl annealing.
positional and temperature factor refinement. Stepped areas were rebuilt using omit maps, and the processes wens repeated along with FIS refinement until the tetinemeni stabilized. The Fnai refinement statistics are as follows. Eos' BMP2/6. Ew-'Rf ~ 0,2231 Ό 2775. rmsd bonds ~- 0.008 rmsd angles * '1.1545: Tor BMP2,;2, Rwnsf - 0 2114/0 265S. rmed bonds - 0.005. rmsd angles ~ Fl.982: Per BMF-X’26 Pw.Rf -0.2170/0,2810, rmsd bonds - 0,000. rmsd angles = 1 182 All torse structures arc- m very good geometry
OS based on Procheck results.
The CHO 8MP2A> crystal structure revealed extensive giycosylation. In particular., the prehellcal loop of CHO-produced BMP2 which Is an important binding motif for type t receptors, Is different from the
2017200239 13 Jan 2017 corresponding region of F cqli-ptorjuced and refolded BMP?. in the presence of giycoayfalion, the CHO BMP? loop has a uniquely loopy conformation when compared Io the same region in bactehally refolded BMP? which is more heiicm iXeitei et a!., Maf BTwcf A4oi S/οι 11481--488 {2094)) The data demonstrated Inal the D53 ol CKO-produced BMP? points towards the receptor interlace while the K54 5 points away fmm the receptor as shown In Figure SA. in E. coll BMP?, the 053 points away from the receptor and the H54 imes up toward the receptor -referred to herein as a nbtidine doorstop''}, stacking aoainsf a prokne residue (P45) on the 3MB? type i receptor Alk3 as shown in Figure 313 ;H54 is alternatively labeled Η33δ) Witoouf wishing to be bound by any particular theory, this stacking could prevent -he type I receptor from fully binding to F ook refolded BMP?, explaining the reduced binding activity of F. coll BMP2 when compared w*th CHO BMP2. This structural feature is illustrated In Figure 3A-8. In this figure, histidine 54 {Κδ4) is numbered as K336. asparagine 56 fN56) is labeled N338, and Ftolj of ALB3 is Shown in darker gray.
As illustrated in Figure 4, tolly glycosylated CHO BfvfPB also has this 'doorstop’’ histidine residue pointing into the receptor binding site This doorstop- His structural motif is a common structural temure a among BMPs {excluding CKO BMP2) (sea e.g. Keller et ai. rvaf Shycf MoZ tSfo/ 11:431-8 -2004? ktofzsch e-! af. EMBO ?S S3?'-4? <2059) vVitoc-n- wishing io be bound by any particular fheoty, it may be that a specific giycan of CKO BMP? is linked though extensive hydrogen bonding with arginine 15 {'the giycan tether' also designated as B2Sb} This giycan tether is illustrated in Figure 4.A arid 4s interaction with the giycan is depicted using doiteo lines between The giycan and tins tether B298 winch is
P aiso referred Io herein ns B19. Thus, without wishing lo bs hound by any particular theory, the giycan tether may serve to stabilize the ccnlcrmation of the ore hokea; loop cl the BMP2 molecule such that the histidine doorstop, if o-horwise present. is snstaad oriented away from the type 1 teceptor interface thsteby allowing the ligand to contact trie receptor to a greater extent than in the presence ol the histidine doorstop, in other words. the re-orlenfaticm of me histidine doorstop as observed in 5:HO BMP? is most likely to be the consequence of giycan tethering, Without wishing to he bound by any pellicular theory, the data disclosed herein suggest that where the histidine doorstop is present, removal of the doorstop in the absence of giyensyiakon (i.e., by introducing a muta-iori that cfianges- the orientation of the His away from the receptor interface'· increases binding of -he BMP ligand with the type I receptor.
Designer BMPE, which eenfame a tow affinity type it binding domain of BMP? anc- a low affinity type I binding domain similar to that of BMPB. shows (I) increased osteogenic activity in both In vitro and in vivo assays' and t?) hat; an unexpected gam of -unction to hind Aik?:, a type i receptor, despite the presence of a low affinity type I receptor binding domain. Without wishing to be bound by any particular theory, it may be that this surprising dtooovr-n v is mediated by mulhpfe hydrogen bonds formed between the giycan moieties and the Bio (the 'giycan tetfiei'} m the type I receptoi-binding domain ol BMPE, This tefbenug inter =1--non ma\ mediate a «mckeei -enrangemem at ihe pre-heli'a: mg.on m Pie ΡΜΐΎ mpiccule that pr-'senh a 5,-topcr bif-mny auibice ton· wpy py pcs-i:o.g Kt-4 pne k.toof~n>p'· awav trom too interface thereby snowing closer interaction bstwsen toe BMP and the receptor In contrast, as Illustrated
T9
2017200239 13 Jan 2017 in figure 4B, BMPS. which also hag a tew affinity type ί blr-iting domain slniiiar io that of BMPE does not bind Aik? because its 'glycen tether' (Rdf 3; which would he needed to tether 4s glycan moieties, is shifted :C loco-ion when compared to fire BMPE tether -PE&S/RfB) Thus, In BMP6; tbs giycan is not lothmed and the doorstop (11454} is ooi positioned away from trie ligand-receptor interface, ftie ylyean x Mho pp^-a's ό a pnmom- on s< ;; jue io v'd 'yen ι μο.ΆΙλ ~9,F > (as <-\emp fe i >.
produced sn CFIO ceils), anti structural remodeling of trie prehelfoe! loop ot SMPs by introducing (et removing· 'giycan tether'' cao now be user:, tor the first time, to modulate type I receptor-binding ability of other BMPs. Therefore, one skilled sn the art, now armed wsth the teachings provided herein, would understand how to mutate ihe BMP in order to position the doorstop away from the rsceptor interface by 9 mtroJue rig mutations Fiat sws g toe 154 away or t?y atA-rfst-g the gwcan xtlt> i so tbat xthvnng mediates the shift In Ho4 and would furihet appreciate toat these teachings can be used to design a BMP with incr-aased (or decreased if mutations are introduced to .swing BSa into the doorstop position; binding to rts receptor or to create designer BMPs with gaimoMunction mutations such that they bind fo novel rgcepiors that they did previously bmd As more fully yet forth heiow the preraent invention demonstrates s bow to use tills novel doorstop;tether design method to design improved osteogenic proteins. Thus, the present invention provides a novel method for rebornd design of improved osteogenin proteins comprising altered receptor binding,
In order to more fully understood what drives me BMPE and 8MP-GER binding to ALt<-?, end to further emc-date this novel mechan-sm of a-fecting receptor binding us-ng trie doorsipp/glycun tether, the crystal structure ot BMP-F. was eoived and compared to that of BMP-? and BMP-t). The key structural findings are shown >n Figure?; IS and 19. As tliustrated t-i Figure IS BMP-B maintains trie ordered sugar of BMP-? white maintaining the ce-i-rai helical structure of BMP-8. The structure- shown in Figure IB demonstrates teat BMP-L. arid presumably BMP-MBB, is different bom both BMP--2 and BMP-b in the entice; region of type I receptor binding Figure 19 is a blow-up companng the area surrounding me
..5 potent-al Bis doorstop of BMPE flight gray; and BMPG Mark g;ayj the dtygtem demonstrates the similarity of the alignment of the histidine end asparagine in both molecules and also shows -he difference In gtycar? positioning and detnoneirafing thsr tethering of the EMPE gtycan by p t 5 phe -ether} which also causes a more rigid conformation ot the gtycan suet- that a longer glycan Is rendered for BMPE by the artaiyst-s compared wt-h die shorter giycan rendered lor SMPfj (in dark grey;
In order to determine if the glycart ot BMP-E is driving the interaction with ALK-2 and Its higher activity, BMP--2 BMP-6, and BMP-E wore treated with Endo H to clip the sugar down to two GinNac units The binding affinity of BMP-E for Alb-? decreased to 400 nM whereas 4's affinity for AI..K-3 and A-.K-G were atilt in the 3-8 nM renge showing the sntoct carbohydrate ;s extremely important for ihte interaction. The activity ot this degsycosyiaied muiarti also decreased significantly. As shown ;n Figure 20 in this experiment the Endo H treated deglyeosylated BMP-E activity shift?; to the right and Is airne?,·· equivalent to BMP-8 WT the EC-50 shifts from 3nM to approxtmateiy 50 nM These data show that the carbohydrate of BMP-E is essen-wl for its activity, and this, shouiri trasisiate io BMP-GEP since it ha?; the
SO
2017200239 13 Jan 2017 exact same region of BMP-8 substituted info BMP-2 with only the finger domdfes dlWeHhg.; Since the carbohydrate is essential for increased receptor binding and osteogenic activity, these results indicate indicates that production of BMP-E or BMP-GER In E. coii. or any other system tacking giyoosyiafion would not produce a BMP with activity superior fo BMP-2 WT,
Purified, folly-glycosylated BMP-E, was concentrated to 8 ? mg'nti in 2SmM sodium acetate (pi ! 3 bi, and crystallization a-teoipis were performed using a “moe-quifo' automated robotic i^-uo a; tS'C fT'IP LabTeeh Inc Cambridge, MAt ϊηιίιΜ crystallization nite were obtained for each dimer and the conditions were subsequently optimized Io acquire crystals of good diffraction quality
Crystals of BMP-E were transiently cryoptcfocted and frozen ;n tigufo nitrogen poor to X-ray diffraction data collection at. the synchrotron sources (ID beamiine of Advanced Photon Source SERC.AT}. Data were processed and sooted using pi agisms Mcsfim'Scaiu in ihe CCP4 package to deduce correct crystal lattice type and to infegrate/scaie data. The resolution and unit cell parameters are listed as follows: BMPE belongs io the .space group of P4$?<2 with two copies of the BMPE in each asymmetric s unit; it diffracted Ιο 2.TA. with a unit ceil of a-h~67.r8A, C-14S.01 A, o--3--y-00'.
The structure ol BMPE was; determined by molecular reptacemeni method with program Phaser, using fully glycosylated CRO BMP2 and BMP5, both determined at Pfizer. as search models After coiiect rrioiaeukt?' replacement solutions were obtained and space groups confirmed. Pheser-celcutetad electron density recipe were us-.ti to evaluate the >..uality c* fee seeren modes and regions iti question fespeoiatiy araas around type I receptor binding and glycosyiafion) were slopped from ihe ougipal model for rebuilding >u order fo avoid mode! bias.
The structural model oi BMPE went rhrouph rigid-body refinement, followed by simulated anrKfii.ng povttru-t ano ten pe-ature lector refinement using pogrom pnen>\ Stripped ureas were rebuilt, using omit maps, and the processes were repeater' along with TLB refin&merif until Ihe refinement
..5 susbifixed. The fit mi o„sfo<.-met it statistics are Hw td ~ 0.2252 0 254b, tmse bon-rfo - 0 505. rmsd angles = 0.339 The structure is in very good geometry based on Prochock results,
BMPE a designer molecule wherein residues 44-30 cl BMP2 replaced by the corresponding region from BMPS, maintains the overall framework of BMP2 while possessing fhe Typo I receptorbinding segment cf BMP6. As shown in Eigure 2? the crystal structure revealed that the grafted segment stilt retains a similar conformation as in BMP6. forming a small helix in the pre-hellcat loop within which ihe ''doorstep H54 points toward the receptor However, without wishing to be bound by any particular theory, it. may be that due to the presence of 'glycan tethers' at Rib and E!10x fEIQS ol BMP-2), both of which form multiple hydrogen bonds with fee third and forth ciyean moieties fp-reanno&e and u-msnnose respectively), fhe extended giycesyiation chain is attached to the protein surface, exactly as seen in CKO
BMP2 fhe tethering of oiyoon chain also dislocated the pre neiica! loop by about 2A, in nuieronoe to the overall framework. Without wishing to be bound by any particular theory, it may be that ihe surprising discovery that the BMP&fike pre-helicai loop combined with the BMPg itka giycoeyiyiion present a
2017200239 13 Jan 2017 binding epitope for the Aik.2 receptor which does not normally interact with es-her BMP2 m BMPO DagivcosylaUon renders BMPE incapable pf binding to Air;?, which tirtderseores the importance of glycosyiaflon in medlaang Aik? recognition tor BMPE
EXAMPLE B ftfermfe Restofartoe i rter to m-eOtofe r tesn.to'k'- to *1 *· v-ci-i- 1 61% irbhtre xto<, pi v mid sc -ore t m activity of ΒΜί-'-GDR or BMB-E these potential tilerageutk molecules were ferther modified to potentially iocrsess their resistance to Noggin Recently, it wee demonstrated that in E coil-groduced proteins.
incorporation oi a G-tomitnal portion oi actlvin-Ά -nto wed typo BMP2 increased resistance io Noggin inhibition, See WO 2010(0392IS at. e g„ Figures 15 and try Therelore to determine whether the novel designer proteins disclosed herein could fee improved even further by incorporation oi acisvin-A sequences ihe Noggin resistance (NR) amino acid sequences were substituted Into BMP-E (SEQ ID NO :2) and BMP GER {SEQ SO NO.37) tc produce BMP-E-NR (SBG 10 NO; 70) and BMP-GER-NR (SEQ
ID NO:?1As shown in Figure 22 ΒΜΡΈ-NR and BMP-GER-NR have equivalent In vitro activity in an .Alkaiine phosphate activity assay compared wrth BMP-E and BMP-GER and are completely resistant io Noggin while BMP-E and BMP-GBR are sensitive to Noggm
To uoderetonb the potential basis fo= the Noggin resistance demonstrated in yiho fey BMPE-NR and BMP-GBR-NR. the binding aiituiiy oi these molecules 'tor iqy type II acfivsn receptor ActRiiB was assessed As shown in Table 18. belc-w. activfe-A Is unable to bint! Noggin but the Noggin resistant BMP-E--NR and BMP-GBR-NR feiud Noggin, but not as strongly as BMP-2. BMP- E. ot 8MP-GER. These data also show -ha- the Noggin resistant BMPs bind the type li BMP receptor ActRiiB with extremely high affinity that is even higher than that ot BMP-QER. Without wishing to fee bound fey any particular theory these data suggests mar BMP--GFR-NR and RMP-B-NR are resistant to Noggin due to their much higher affinity loi the BMP type if tecoptore than diet ot Noggin and ary therefore ahis to bind BMP receptors even in the presence ot high amounts of Noggm f ABLE IS
......................... AetRtfh affinity W).......................................... | |
BMP-E | o.eo |
BMPE-NR | 0.81) |
BMP-GER | 2.00 |
BMP-GER-NR | 0.07 |
Noggin A-Tiniiy | |
BMP-E | t 00 |
BMP-E-NR | 9.00 |
BMP-GER | 4.00 |
§2
2017200239 13 Jan 2017
7BMR-GER-NR | ' 7,50 |
AotRlib on rate (K on) with equal molar ration of noggin* | |
BMPE | no bsnaino |
BMP-E-NR | ho brnoing |
BMP-GER | woe-5-03 |
BMP-GER-NR | I.OTEffi: |
Nit tie to no: change in on raid with Up fo 10 fold molar excess of Noggin |
Although the 3MP--E arid BMP-GER molecules comprising the Noggin resistant portions of aetivin-A damonstrsted Noggin resistance m Vitro, thesis results; did not con-stats to improved in vivo activity f hat is. when the osteogenic activity o;' these BMP-E-NR and BMP-GBR-NR was compared with shat of BMP-E and BMP-GFR in a rat sotopie assay. the NR moiacsdes wars much less potent This data is shown in Figures 23 one 24. More specifically, the Bone Score for BMP-GER and BMF--GER-NR was compared and. at ail concentrations tested (ON 25 pg, 0.25 pg, 0.5 ng. and 1.0 pg'i BMR-GER greatly outperformed BMP-GER-NR as shown m Figure 22 Similarly, Figure 24 demonstrates that BMP-E produced a much higher Bone Score compared with ΒΜΡΈ-NR in -his in vivo assay. Thus, for Goth S 8MP E and BMP GER fne purpottediv Noggin reswlaril versions were much less potent io vivo then their NR (Noggin resistant) counterparts, and in the case of ΒΜΡΈ almost all in vivo activity was lost, due to incorporation of sequences ot activtn -A (see Figure 24 comparing BMPENR with BMP-E)
These data demonstrate that Incorporation of sequences potentially conferring Noggin resistance, while increasing binding for eertasn type is receptors (e g . AcIRtfB ·, did not Increase in vivo osteogenic a activity of thy designer BMP,
Further, although the addition of Noggin did not improve the osteogenic activity of the designer BMPs ϊγϊ vivo, indeed: if appeared to decrease their in vivo activity. thy novel designer BMPs of thy Invention demonstrate greatly increased osteogenic, characteristics compared with wild type BMP and provide potential novel therapeutics lor a wide vaneiy of applications even without demonstrating Noggin its resistance in vitro Therefore, the designer BMPs of the invention provide remarkable novel potential therapeutics demonstrating a greatly improved clinical profile for. among other uses, hone augmentation and repair.
The disclosures of each and every patent. patent appiioatinn, urirt publication cited herem are hereby incorporated herein by reference in their entirety.
2.S Whiie the invention has been disclosed w;fh reference to specific embodiments, it is apparent that other embodiments and variations of this invention may he devised by ethers chilled m the art without, departing from the true spinf and scope of the invention. The apperxied claims are intonded to be construed to include ail such embodiments and equivalent variations.
2017200239 17 Mar 2017
Claims (5)
1. A designer BMP protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 70 and SEQ ID NO: 12.
2. The designer BMP protein of claim 1 wherein the BMP protein comprises the amino acid sequence of SEQ ID NO. 70.
3. The designer BMP protein of claim 1 wherein the BMP protein comprises the amino acid sequence of SEQ ID NO. 12.
4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a designer BMP protein of claim 1.
5. A method of producing the designer BMP protein of claim 1 comprising introducing a nucleic acid encoding the designer BMP protein into a host cell, culturing the host cell under conditions where the protein is produced, and purifying the protein.
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2017200239 13 Jan 2017 ο
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <110> WETH LLC <120> DESI GNER OSTEOGENI C PRQTEI NS <130> PC071685A <140>
<141 >
<1 50> 61/375, 636 <151> 2010-08-20 <1 60> 139 <170> Pat ent I n version 3.5 <210> 1 <211> 114 <212> PRT <213> Homo sapi ens
Cys Arg <210> 2 <211> 116 <212> PRT <213> Homo sapi ens
Page 1
SEQUENCE LI STI NG
2017200239 13 Jan 2017
115 <210> 3 <211> 138 <212> PRT <213> Homo sapi ens <400> 3
130 135
Page 2
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <210> 4 <211> 139 <212> PRT <213> Homo sapi ens <400> 4
130 135 <210> 5 <211> 139 <212> PRT <213> Homo sapi ens <400> 5
Page 3
2017200239 13 Jan 2017
130 135 <210> 6 <211> 139 <212> PRT
130 135 <210> 7 <211> 110 <212> PRT
Page 4
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110 <210> 8 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 8
Cys Arg <210> 9
Page 5
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Arti
Cys Arg <210> 10 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 10
50 55 60
Page 6
SEQUENCE LI STI NG
2017200239 13 Jan 2017
Cys Arg <210> 11 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 11
100 105 110
Cys Arg <210> 12 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic Page 7
SEQUENCE LI STI NG
2017200239 13 Jan 2017 pol ypept i de <400> 12
100 105 110
Gy Cys Arg 115 <210> 13 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 13
85 90 95
Page 8
SEQUENCE LI STI NG
2017200239 13 Jan 2017
Lys Val Val Leu Lys Asn Tyr Q n Asp IVbt Val Val Q u Qy Cys Q y 100 105 110
Cys Arg <210> 14 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 14
100 105 110
Cys Arg <210> 15 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 15
Q n Al a Lys Hi s Lys On Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg 15 10 15
Page 9
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100
105 110
Cys Arg <210> 16 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
Cys Arg
Page 10
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <210> 17 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 17
100 105 110
Gy Cys Arg 115 <210> 18 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 18
35 40 45
Page 11
SEQUENCE LI STI NG
2017200239 13 Jan 2017
Cys Arg <210> 19 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
Page 12
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 20
100 105 110
Cys Arg <210> 21 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 21
65 70 75 80
Page 13
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110
Cys Arg <210> 22 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
Cys Arg <210> 23 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 23
Page 14
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110
Cys Arg <210> 24 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
2017200239 13 Jan 2017
SEQUENCE LI STI NG
100 105 110
Gy Cys Ar g 115 <210> 25 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
Gy Cys Arg 115 <210> 26 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 26
20 25 30
Page 16
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110
Gy Cys Ar g 115 <210> 27 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 27
Gy Cys Arg 115
Page 17
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <210> 28 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 28
100 105 110
Gy Cys Arg 115 <210> 29 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 29
50 55 60
Page 18
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110
Cys Arg <210> 30 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
Gy Cys Ar g 115 <210> 31 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source
Page 19
2017200239 13 Jan 2017
SEQUENCE LI STI NG <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 31
100 105 110
Cys Arg <210> 32 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 32
Pro Thr Q u Leu Asn Al a
65 70 75 80
I I e Ser Val Leu Tyr Phe Asp Asp Asn Ser Page 20
2017200239 13 Jan 2017
SEQUENCE LI STI NG
85 90 95
Asn Val lie Leu Lys Asn Tyr Q n Asp K/bt Val Val Q u Gy Cys Q y 100 105 110
Cys Arg <210> 33 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 33
100 105 110
Cys Arg <210> 34 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 34
G n Al a Lys Hi s Lys Gn Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg 15 10 15
Page 21
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110
Cys Arg <210> 35 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 35
Page 22
SEQUENCE LI STI NG
2017200239 13 Jan 2017
Cys Arg <210> 36 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 36
100 105 110
Gy Cys Arg 115 <210> 37 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 37
Page 23
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110
Gy Cys Ar g 115 <210> 38 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
<210> 39 <211> 115 <212> PRT
115
Page 24
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 39
100 105 110
Gy Cys Arg 115 <210> 40 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 40
Page 25
2017200239 13 Jan 2017
SEQUENCE LI STI NG
65 70 75 80
100 105 110
Gy Cys Ar g 115 <210> 41 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 41
100 105 110
Gy Cys Arg 115 <210> 42 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
Page 26
SEQUENCE LI STI NG
2017200239 13 Jan 2017
Cys Arg <210> 43 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 43
Page 27
2017200239 13 Jan 2017
SEQUENCE LI STI NG
Val Pro Thr Leu Lys Asn Tyr Q n Asp l\/bt Val Val Qu Qy Cys Q y 100 105 110
Cys Arg <210> 44 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 44
100 105 110
Gy Cys Arg 115 <210> 45 <211> 114 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 45
20 25 30
Page 28
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110
Cys Arg <210> 46 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
115
Page 29
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <210> 47 <211> 116 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 47
100 105 110
Cys Q y Cys Ar g 115 <210> 48 <211> 116 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 48
35 40 45
Phe Pro Leu Al a Asp Asp Val Thr Pro Thr Lys Hi s Al a lie Page 30
Val G n
SEQUENCE LI STI NG
2017200239 13 Jan 2017
115 <210> 49 <211> 123 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 49
115 120 <210> 50 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
Page 31
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 50
100 105 110
Gy Cys Arg 115 <210> 51 <211> 117 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 51
65 70 75 80
Page 32
SEQUENCE LI STI NG
2017200239 13 Jan 2017
115 <210> 52 <211> 117 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 52
100 105 110
Al a Cys Q y Cys Hi s 115 <210> 53 <211> 139 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 53
Ser Al a Ser Ser Arg Arg Arg On On Ser Arg Asn Arg Ser Thr Q n 15 10 15
Page 33
SEQUENCE LI STI NG
2017200239 13 Jan 2017
130 135 <210> 54 <211> 138 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 54
Page 34
SEQUENCE LI STI NG
2017200239 13 Jan 2017
130 135 <210> 55 <211> 139 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 55
130 135 <210> 56 <211> 138 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic Page 35
SEQUENCE LI STI NG
2017200239 13 Jan 2017 pol ypept i de <400> 56
130 135 <210> 57 <211> 117 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic
Page 36
SEQUENCE LI STI NG
2017200239 13 Jan 2017
115 <210> 58 <211> 117 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic + ί ζΊζ-»
Al a Cys Q y Cys Hi s 115 <210> 59 <211> 117 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 59
Page 37
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110
SEQUENCE LI STI NG
2017200239 13 Jan 2017
130 135 <210> 61 <211> 139 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 61
pol ypept i de
Page 39
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <400> 62
130 135 <210> 63 <211> 139 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 63
65 70 75 80
Page 40
SEQUENCE LI STI NG
2017200239 13 Jan 2017
130 135 <210> 64 <211> 138 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
<210> 65 <211> 113 <212> PRT <213> Ar t i f i ci al Sequence
Page 41
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 65
100 105 110
Arg <210> 66 <211> 110 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 66
Page 42
SEQUENCE LI STI NG
2017200239 13 Jan 2017
pol ypept i de <400> 67
115 <210> 68 <211> 117 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 68
Page 43
2017200239 13 Jan 2017
100
SEQUENCE LI STI NG 25 30
105
110
AI a Cys Q y Cys Hi s 115 <210> 69 <211> 117 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 69
Al a Cys Q y Cys Hi s 115
100
105
110
Page 44
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <210> 70 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 70
100 105 110
Gy Cys Arg 115 <210> 71 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 71
Page 45
SEQUENCE LI STI NG
2017200239 13 Jan 2017
100 105 110
Q y Cys Ser 115 <210> 72 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de
Q y Cys Hi s 115 <210> 73 <211> 115 <212> PRT <213> Ar t i f i ci al Sequence
Page 46
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ypept i de <400> 73
100 105 110
Q y Cys Hi s 115 <210> 74 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 74 atggtggccg ggacccgctg tcttctagcg 11get gettc cccaggtcct cctgggegge 60 geggetggee tcgttccgga getgggeege aggaagtteg eggeggegtc gtcgggccgc 120 ccctcatccc agccctctga egaggtcctg agegagtteg agttgegget getcagcatg 180
11 cggcct ga aacagagacc cacccccagc agggacgccg t ggt gccccc ct acat get a 240 gacctgtatc gcaggcactc aggtcagccg ggetcacccg ccccagacca ccggttggag 300 agggcagcca gccgagccaa cact gt gege aget t ccacc at gaagaat c 111 ggaagaa 360 ctaccagaaa cgagtgggaa aacaacccgg agattcttct ttaatttaag ttctatcccc 420 aeggaggagt 11 at cacct c ageagaget t caggt 111 cc gagaacagat gcaagat get 480
11 aggaaaca at agcagt 11 ccat caccga at t aat at 11 at gaaat cat aaaacctgca 540 acagccaact cgaaattccc cgtgaccaga cttttggaca ccaggttggt gaatcagaat 600
Page 47
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 75 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 t gt gt cccga caaagct aaa t gccat ct eg gt t ct 11 act 11 gat gacaa ct ccaat gt c 1140 at 111 aaaga act at cagga cat ggt t gt g gagggt t gt g ggt gt eget g a 1191 <210> 76 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 76
<210> 77 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 49
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 78 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 50
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 79 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 get gcct 111 aat cat gcca t gt gt cccga gt at t aaaga act gccacgg 11 gt t cagac cagaact cag act at cagga agaat gccct gt t ggt caac t get at ct eg cat ggt t gt g
111 cct ct gg t ct gt t aact at get gt acc gagggttgtg ct gat cat ct ct aagat t cc 11 gaegagaa ggt gt eget g gaact ccact t aaggcat gc t gaaaaggt t a
<210> 80 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 80
1020
1080
1140
1191
<210> 81 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
Page 52
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 82 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence
Page 53
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 83 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 54
SEQUENCE LI STI NG
2017200239 13 Jan 2017 aaacgt caag 11 gt acgt gg cacgcct 111 aaccacgcga t get gt gege gt t gt at t aa ccaaacacaa act t cagt ga act gegat gg 11 gt gcagac cgacagaact agaaat at ca acagcggaaa cgtggggtgg agaat get cc ct t ggt t cac caat get at c ggacat ggt t cgcct t aagt aat gact gga
11 cccact ca ct t at gaacc t eggt t ct gt gt gagaggt t ccagct gt aa 11 at t get cc acgcacacat ccgagt at gt act 11 gaega gtgggtgtcg gagacaccct caaggggt at gaat gcaacc ccccaaaccg gaat t ccaat ct ga
900
960
1020
1080
1140
1194 <210> 84 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 84
<210> 85 <211> 1191
Page 55
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
<210> 86 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence
Page 56
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 87 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 57
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 88 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <210> 89 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
<210> 90 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 90 atggtggccg ggacccgctg tcttctagcg 11get gettc cccaggtcct cctgggegge 60 geggetggee tcgttccgga getgggeege aggaagtteg eggeggegtc gtcgggccgc 120
Page 59
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 91 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 60
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 92 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 t get gt gt cc cgacagaact cagt get at c t egat get gt acct t gaega gaat gaaaag 1140 gt t gt at t aa agaactatca ggacat ggt t gt ggagggt t gt gggt gt eg et ag 1194 <210> 93 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 93
<210> 94 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 62
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 95 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 63
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 96 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 64
SEQUENCE LI STI NG
2017200239 13 Jan 2017 cacgcct 111 aat cat gcag t get gt gt cc gt t gt at t aa act gegaggg t cat ccagac cgacagaact agaact at ca get gt gegag cct gat gaac cagt get at c ggacat ggt t
11 cccat t gc t ccat ggacc t egat get gt gtggagggtt get cccacct ccgagt ccac acct t gaega gtgggtgtcg ggagcccacg accacccacc gaat gaaaag ct ag <210> 97 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 97
1020
1080
1140
1194
<210> 98 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
Page 65
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 99 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence
Page 66
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 100 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 67
SEQUENCE LI STI NG
2017200239 13 Jan 2017 aaacgt caag 11 gt acgt gg get gcct 111 aaacat gcca t gt gt cccga at 111 aaaga ccaaacacaa act t cagt ga act gccacgg 11 gt t cagac cagaact caa act at cagga acagcggaaa cgtggggtgg agaat gccct gt t ggt caac t gccat ct eg cat ggt t gt g cgcct t aagt aat gact gga 111 cct ct gg t ct gt t aact gt t ct 11 act gagggttgtg ccagct gt aa 11 at t gcacc ct gat cat ct ct aagat t cc 11 gat gacaa ggt gt eget g gagacaccct caagggct at gaact ccact t aaggcat gc ct ccaat gt c a
900
960
1020
1080
1140
1191 <210> 101 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 101
<210> 102 <211> 1194
Page 68
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
<210> 103 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence
Page 69
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 104 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 70
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 105 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <210> 106 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
<210> 107 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 107 atggtggccg ggacccgctg tcttctagcg 11get gettc cccaggtcct cctgggegge 60 geggetggee tcgttccgga getgggeege aggaagtteg eggeggegtc gtcgggccgc 120
Page 72
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 108 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 73
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 109 <211> 1176 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 74
SEQUENCE LI STI NG
2017200239 13 Jan 2017 ctgtccccca tctccgtget gtacaaggac gacatgggcg tgcccaccct gaagaactac 1140 caggacatgg tggtegaagg ctgeggetgt eggtga 1176 <210> 110 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 110
<210> 111 <211> 1191 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 75
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <400> 111
<210> 112 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence
Page 76
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 113 <211> 1197 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 77
SEQUENCE LI STI NG
2017200239 13 Jan 2017 gaggcct acg aaccacgcca t get gegt cc cccaccct ga agt gegaegg t cgt gcagac ccaccaagct agt accact a egagt get cc cct ggt gcac gt cccccat c cgagggcat g
11 ccccct ga ct gat gaacc t ccgt get gt t ccgt cgccg acgcccacat ccgagt acgt acaaggacga agt gegget g gaacgccacc gcccaagccc cat gggcgt g t eggt ga <210> 114 <211> 1308 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 114
1020
1080
1140
1197
<210> 115 <211> 1329
Page 78
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 115
<210> 116 <211> 1329 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 116 at gt gt cct g gcgct ct gt g ggt ggccct g cct ct get gt ct ct get ggc cggcagcct g 60
Page 79
SEQUENCE LI STI NG
2017200239 13 Jan 2017
tgccggtga 1329 <210> 117 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 80
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 118 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 119 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 82
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 120 <211> 1539 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 83
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 121 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 84
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 122 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 85
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 123 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 get 11 ct t ca cgacaacaga t cagat t aca 11 ccaagacc t gt gat ggag gt gcagacct act aaget aa aaat acagga
<210> 124 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 87
SEQUENCE LI STI NG
2017200239 13 Jan 2017 t cagat t aca 11 ccaagacc t gt gat ggag gt gcagacct act aagct aa aaat acagga acagcagt ga t gggat ggca aat get cct t t ggt t cacct at gccat ct c at at ggt t gt at t gaaaaca ggact ggat c cccact caac t at gaacccc ggt t ct 11 ac aagaget t gt gcct gcaaga at t gcaccca gcacacat ga gagt at gt cc 111 gat gaca ggat gccact ggcat gaget aggget at gc at gcaaccaa ccaaaccgt g act ccaat gt aa gt at gt gagt t gccaat t ac ccacgcgat t ct gt gcgcca cat t ct gaaa
1260
1320
1380
1440
1500
1542 <210> 125 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 t gt gat ggag gt gcagacct act aagct aa aaat acagga
<210> 126 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 actaagctaa at gccat ct c ggt t ct 11 ac 111 gat gaca act ccaat gt cat t ct gaaa 1500 aaat acagga at at ggt t gt aagagct t gt ggat gccact aa 1542 <210> 127 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <210> 128 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 128
<210> 129 <211> 1542
Page 91
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 129
<210> 130 <211> 1539 <212> DNA <213> Ar t i f i ci al Sequence <220>
Page 92
SEQUENCE LI STI NG
2017200239 13 Jan 2017 <221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 130
<210> 131 <211> 1287 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 93
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 132 <211> 1290 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 94
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 133 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 95
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 134 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 96
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 135 <211> 1542 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 97
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 136 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
SEQUENCE LI STI NG
2017200239 13 Jan 2017 t get gt gege ccaccaagct gagacccat g t ccat gt t gt act at gat ga t ggt caaaac 1140 at cat caaaa aggacat t ca gaacat gat c gt ggaggagt gt gggt get c at ag 1194 <210> 137 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de <400> 137
<210> 138 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 99
SEQUENCE LI STI NG
2017200239 13 Jan 2017
<210> 139 <211> 1194 <212> DNA <213> Ar t i f i ci al Sequence <220>
<221> source <223> I not e= Descr i pt i on of Artificial Sequence: Synthetic pol ynucl eot i de
Page 100
SEQUENCE LI STI NG
2017200239 13 Jan 2017
Page 101
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PCT/IB2011/053638 WO2012023113A2 (en) | 2010-08-20 | 2011-08-17 | Designer osteogenic proteins |
AU2015202418A AU2015202418B2 (en) | 2010-08-20 | 2015-05-06 | Designer osteogenic proteins |
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EP1571159A1 (en) * | 2004-03-04 | 2005-09-07 | Bayerische Julius-Maximilians-Universität Würzburg | Mutein of a bone morphogenetic protein and use thereof |
WO2008051526A2 (en) * | 2006-10-23 | 2008-05-02 | Stryker Corporation | Bone morphogenetic proteins |
WO2009086131A1 (en) * | 2007-12-21 | 2009-07-09 | Stryker Corporation | Bmp mutants with decreased susceptibility to noggin |
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EP1571159A1 (en) * | 2004-03-04 | 2005-09-07 | Bayerische Julius-Maximilians-Universität Würzburg | Mutein of a bone morphogenetic protein and use thereof |
WO2008051526A2 (en) * | 2006-10-23 | 2008-05-02 | Stryker Corporation | Bone morphogenetic proteins |
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