US2009221032A1PendingUtilityA1

Entropic bristle domain sequences and their use in recombinant protein production

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Assignee: MOLECULAR KINETICS INCPriority: Jul 11, 2005Filed: Jan 22, 2009Published: Sep 3, 2009
Est. expiryJul 11, 2025(expired)· nominal 20-yr term from priority
Inventors:A. Keith DunkerVladimir N. UverskyMarc S. CorteseJames Mueller
C07K 14/47C07K 2319/35C12N 15/62
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Claims

Abstract

Compositions and methods for recombinant protein production and, more particularly, fusion polypeptides, polynucleotides encoding fusion polypeptides, expression vectors, kits, and related methods for recombinant protein production, are provided.

Claims

exact text as granted — not AI-modified
1 . An isolated fusion polypeptide comprising at least one entropic bristle domain (EBD) sequence and at least one heterologous polypeptide sequence, wherein the fusion polypeptide has increased solubility relative to the heterologous polypeptide sequence, reduced aggregation relative to the heterologous polypeptide sequence and/or improved folding relative to the heterologous polypeptide sequence. 
     
     
         2 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a mammalian neurofilament protein. 
     
     
         3 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a mammalian neurofilament NF-H protein. 
     
     
         4 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a human neurofilament NF-H protein having a sequence set forth in SEQ ID NO: 1 or 3. 
     
     
         5 . The polypeptide according to  claim 1 , wherein the EBD sequence comprises a neurofilament NF-H sequence selected from the group consisting of SPEAEK (SEQ ID NO:23), SPAAVK (SEQ ID NO:24), SPAEAK (SEQ ID NO:25), SPAEPK (SEQ ID NO:26), SPAEVK (SEQ ID NO:27), SPATVK (SEQ ID NO:28), SPEKAK (SEQ ID NO:29), SPGEAK (SEQ ID NO:30), SPIEVK (SEQ ID NO:31), SPPEAK (SEQ ID NO:32), SPSEAK (SEQ ID NO:33), SPEKEAK (SEQ ID NO:34), SPAKEKAK (SEQ ID NO:35), SPEKEEAK (SEQ ID NO:36), SPTKEEAK (SEQ ID NO:37), SPVKEEAK (SEQ ID NO:38), SPVKAEAK (SEQ ID NO:39), SPVKEEAK (SEQ ID NO:40), SPVKEEVK (SEQ ID NO:41), SPVKEEEKP (SEQ ID NO:42), SPEKAKTLDVK (SEQ ID NO:43), SPADKFPEKAK (SEQ ID NO:44), SPEAKTPAKEEAR (SEQ ID NO:45), SPEKAKTPVKEGAK (SEQ ID NO:46), SPVKEEAKTPEKAK (SEQ ID NO:47), SPVKEGAKPPEKAKPLDVK (SEQ ID NO:48), SPVKEDIKPPAEAKSPEKAK (SEQ ID NO:49), SPLKEDAKAPEKEIPKKEEVK (SEQ ID NO:50), SPEKEEAKTSEKVAPKKEEVK (SEQ ID NO:51), SPEAQTPVQEEATVPTDIRPPEQVK (SEQ ID NO:52), SPVKEEVKAKEPPKKVEEEKTLPTPKTEAKESKKDE (SEQ ID NO:53), or a combination thereof. 
     
     
         6 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a mammalian neurofilament protein NF-M. 
     
     
         7 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a mammalian neurofilament NF-M protein having the sequence set forth in any one of SEQ ID NOs: 5, 7, 9, 11, 13 or 15. 
     
     
         8 . The polypeptide according to  claim 1 , wherein the EBD sequence comprises a neurofilament NF-M sequence selected from the group consisting of SPPK (SEQ ID NO:54), SPVK (SEQ ID NO:55), SPAAK (SEQ ID NO:56), SPAPK (SEQ ID NO:57), SPEAK (SEQ ID NO:58), SPMPK (SEQ ID NO:59), SPPAK (SEQ ID NO:60), SPTAK (SEQ ID NO:61), SPTTK (SEQ ID NO:62), SPVAK (SEQ ID NO:63), SPVAK (SEQ ID NO:64), SPVPK (SEQ ID NO:65), SPVSK (SEQ ID NO:66), SPEKPA (SEQ ID NO:67), SPVEEKAK (SEQ ID NO:68), SPVEEKGK (SEQ ID NO:69), SPVEEVKP (SEQ ID NO:70), SPEKPATPKVT (SEQ ID NO:71), SPEKPRTPEKPA (SEQ ID NO:72), SPEKPTTPEKW (SEQ ID NO:73), SPEKPSSPLKDEKA (SEQ ID NO:74), SPVKEKAVEEMITIT (SEQ ID NO:75), SPVKEEAAEEAATITK (SEQ ID NO:76), SPVPKSPVEEVKPKAEATAG (SEQ ID NO:77), SPVKAESPVKEEVPAKPVKV (SEQ ID NO:78), SPEKEAKEEEKPQEKEKEKEK (SEQ ID NO:79), SPVKATTPEIKEEEGEKEEEGQE (SEQ ID NO:80), SPVEEVKPKPEAKAGKGEQKEE (SEQ ID NO:81), SPEKPATPEKPPTPEKAITPEKVR (SEQ ID NO:82), SPEKPATPEKPRTPEKPATPEKPR (SEQ ID NO:83), SPKEEKVEKKEEKPKDVPKKKAE (SEQ ID NO:84), SPKEEKAEKKEEKPKDVPEKKKAE (SEQ ID NO:85), SPVEEAKSKAEVGKGEQKEEEEKE (SEQ ID NO:86), SPKEEKVEKKEEKPKDVPDKKKAE (SEQ ID NO:87), SPVKEEAVAEVVTITKSVKVHLEKET (SEQ ID NO:88), SSEKDEGEQEEEEGETEAEGEGEEAEAKEEK (SEQ ID NO:89), SPVEEVKPKAEAGAEKGEQKEKVEEEKKEAKE (SEQ ID NO:90), SPVTEQAKAVQKAAAEVGKDQKAEKAAEKAAKEEKAA (SEQ ID NO:91), SPEAKEEEEEGEKEEEEEGQEEEEEEDEGVKSDQAEEGGSEKEG (SEQ ID NO:92), or a combination thereof. 
     
     
         9 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a phage sequence. 
     
     
         10 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a filamentous phage fd. 
     
     
         11 . The polypeptide according to  claim 1 , wherein the EBD sequence comprises at least one linker region derived from a filamentous phage fd adsorption protein pIII having a sequence set forth in SEQ ID NO: 17. 
     
     
         12 . The polypeptide according to  claim 1 , wherein the EBD sequence comprises a filamentous phage fd adsorption protein pIII sequence selected from the group consisting of EGGGS (SEQ ID NO:93), EGGGT (SEQ ID NO:94), SEGGG (SEQ ID NO:95), GGGSGGG (SEQ ID NO:96), SGGGSGSG (SEQ ID NO:97), and SGGGSEGGG (SEQ ID NO:98), or a combination thereof. 
     
     
         13 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a nuclear pore Nup2p protein having a sequence set forth in SEQ ID NO: 19. 
     
     
         14 . The polypeptide according to  claim 1 , wherein the EBD sequence comprises a yeast nucleoporin Nup2p sequence selected from the group consisting of FSFGTSQPNNTPS (SEQ ID NO:99), FSFSIPSKNTPDASKPS (SEQ ID NO:100), FVFGQAAAKPSLEKSS (SEQ ID NO:101), FSFGVPNSSKNETSKPV (SEQ ID NO:102), FTFGTKHAADSQNNKPS (SEQ ID NO:103), FTFGSSALADNKEDVKKP (SEQ ID NO:104), FSFGINTNTTKTADTKAPT (SEQ ID NO:105), FSFGKTTANLPANSSTSPAPSIPSTG (SEQ ID NO:106), FSFGPKKENRKKDESDSENDIEIKGPE (SEQ ID NO:107), FKFSGTVSSDVFKLNPSTDKNEKKTETNAKP (SEQ ID NO:108), FKFSLPFEQKGSQTTTNDSKEESTTEATGNESQ (SEQ ID NO:109), FTFGSTTIEKKNDENSTSNSKPEKSSDSNDSNPS (SEQ ID NO:110), FSFGISNGSESKDSDKPSLPSAVDGENDKKEATKPA (SEQ ID NO:111), FSFSSATSTTEQTKSKNPLSLTEATKTNVDNNSKAEAS (SEQ ID NO:112) and FSFGAATPSAKEASQEDDNNNVEKPSSKPAFNLISNAGTEKEKESKKDSKPA (SEQ ID NO:113), or a combination thereof. 
     
     
         15 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a mammalian elastin protein. 
     
     
         16 . The polypeptide according to  claim 1 , wherein the EBD sequence is derived from a mouse elastin protein having a sequence set forth in SEQ ID NO: 21. 
     
     
         17 . The polypeptide according to  claim 1 , wherein the EBD sequence is an elastin sequence selected from the group consisting of VPGA (SEQ ID NO:114), GAGGL (SEQ ID NO:115), GAGGG (SEQ ID NO:116), VPGVG (SEQ ID NO:117), VPGFGAGA (SEQ ID NO:118), VPGALPGA (SEQ ID NO:119), VPGFGAGAG (SEQ ID NO:120), VPAVPGAGG (SEQ ID NO:121), VPGGVGVGG (SEQ ID NO:122), VGAGGFPGYG (SEQ ID NO:123), VPGAVPGGLPGG (SEQ ID NO:124), VSPAAAAKAAKYGAA (SEQ ID NO:125), VPQVGAGIGAGGKPGK (SEQ ID NO:126), VPGGVGVGGIPGGVGVGG (SEQ ID NO:127), VPGGVGGIGGIGGLGVSTGAV (SEQ ID NO:128), VPGGAAGAAAAYKAAAKAGAGLGGVGG (SEQ ID NO:129), VSPAAAAKAAAKAAKYGARGGVGIPTYG (SEQ ID NO:130), KPPKPYGGALGALGYQGGGCFGKSCGRKRK (SEQ ID NO:131), VPGAGTPAAAAAAAAAKAAAKAGLGPGVGG (SEQ ID NO:132), VPGRVAGAAPPAAAAAAAKAAAKAAQYGLG (SEQ ID NO:133), VPGVGLPGVYPGGVLPGTGARFPGVGVLPG (SEQ ID NO:134), VPTGTGVKAKAPGGGGAFSGIPGVGPFGGQQPG (SEQ ID NO:135), VPGGVYYPGAGIGGLGGGGGALGPGGKPPKPGAG (SEQ ID NO:136), VGAGAGLGGASPAAAAAAAKAAKYGAGGAGALGGL (SEQ ID NO:137), GLGGVLGARPFPGGGVAARPGFGLSPIYPGGGAGGLGVGG (SEQ ID NO:138), VPGSLAASKAAKYGAAGGLGGPGGLGGPGGLGGPGGLGGAG (SEQ ID NO:139), VPGGPGVRLPGAGIPGVGGIPGVGGIPGVGGPGIGGPGIVGGPGA (SEQ ID NO:140), VLPGVGGGGIPGGAGAIPGIGGIAGAGTPAAAAAAKAAAKAAKYGAAGGL (SEQ ID NO:141), VPGGVGPGGVTGIGAGPGGLGGAGSPAAAKSAAKAAAKAQYRAAAGLGAG (SEQ ID NO:142), and VPLGYPIKAPKLPGGYGLPYTNGKLPYGVAGAGGKAGYPTGTGVGSQAAAAAAK AAKYGAGGAG (SEQ ID NO:143), or a combination thereof. 
     
     
         18 . The polypeptide according to  claim 1 , wherein the polypeptide further comprises a cleavable linker. 
     
     
         19 . An isolated polynucleotide encoding a fusion polypeptide according to  claim 1 . 
     
     
         20 . An expression vector comprising an isolated polynucleotide according to  claims 19 . 
     
     
         21 . A host cell comprising an expression vector according to  claim 20 . 
     
     
         22 . A kit comprising an isolated polynucleotide according to  claim 19 . 
     
     
         23 . A kit comprising an expression vector according to  claim 20 . 
     
     
         24 . A kit comprising a host cell according to  claim 21 . 
     
     
         25 . A method for producing a recombinant protein comprising the steps of: (a) introducing into a host cell a polynucleotide according to  claim 19  or an expression vector according to  claim 20 ; and (b) expressing in the host cell a fusion polypeptide comprising at least one EBD sequence and at least one heterologous polypeptide sequence.

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