US2011082283A1PendingUtilityA1

Multimeric elp fusion constructs

Assignee: DAGHER SUZANNEPriority: Jan 4, 2006Filed: Feb 25, 2010Published: Apr 7, 2011

Est. expiryJan 4, 2026(expired)· nominal 20-yr term from priority

Inventors:Suzanne Dagher

C12N 15/62

PatentIndex Score

Cited by

References

Claims

Abstract

ELP fusion proteins, multimeric ELP spider complexes formed of ELP fusion proteins, and methods of using the same. The construct may be in the form of an ELP spider structure complex including multi-leg moieties comprising ELP fusion proteins capable of forming covalent disulfide bonds. The multimeric fusion constructs may be employed in peptide production and purification and/or to enhance proteolytic resistance of a protein or peptide moiety in a fusion construct, by provision of the fusion protein in an ELP spider complex.

Claims

exact text as granted — not AI-modified

1 . A fusion protein exhibiting a phase transition, comprising:
 a) at least one target protein or peptide;   b) one or more proteins comprising oligomeric repeats of a polypeptide sequence selected front SEQ ID NOs: 1-17, wherein the one or more proteins exhibits a phase transition and are joined to the at least one target protein or peptide of a);   c) at least two residues capable of forming a disulfide bond; and   d) optionally, a spacer sequence separating any of the phase transition protein(s) of b) from any of the target protein(s) or peptide(s) of a).   
     
     
         2 . The fusion protein of  claim 1 , wherein the target protein is IFNa2b. 
     
     
         3 . The fusion protein of  claim 1 , wherein the target peptide is Orexin-B, MMN, NPY or Gh. 
     
     
         4 . The fusion protein of  claim 1 , wherein the phase transition protein is selected from the group consisting of: SEQ ID NOs: 1, 2, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17. 
     
     
         5 . The fusion protein of  claim 1 , wherein the phase transition protein is selected from the group consisting of: SEQ ID NOs: 1, 2, 9, 10, 11, 12, 13, 14, 15, 16, and 17. 
     
     
         6 . The fusion protein of  claim 1 , wherein the phase transition protein is selected from the group consisting of: ELP1-90, ELP1-180, ELP2-64, ELP2-128, ELP3-72, ELP3-144, ELP4-60, ELP4-120, ELP5-15, ELP5.30, ELP5-60, ELP6-15, ELP6-30, ELP6-60, ELP7-13, ELP7-30 and ELP7-60. 
     
     
         7 . The fusion protein of  claim 1 , wherein the phase transition is mediatable by an agency comprising one or more of: changing temperature; changing pH; addition of solutes and/or solvents; side chain ionization or chemical modification; and changing pressure. 
     
     
         8 . The fusion protein of  claim 1 , wherein the at least two residues capable of forming a disulfide bond comprise cysteine. 
     
     
         9 . The fusion protein of  claim 1 , comprising said spacer sequence. 
     
     
         10 . The fusion protein of  claim 9 , wherein said spacer sequence comprises a proteolytic cleavage site. 
     
     
         11 . The fusion protein of  claim 1 , selected from pET17b-SD33-ELP1-90-IFNA2bSD (SEQ ID NO: 22), pET17b-SD33-ELP4-60-IFNA2bSD (SEQ ID NO: 23), pET17b-SD34-ELP1-90-IFNA2bSD (SEQ ID NO: 24), pET17b-SD34-ELP4-60-IFNA2bSD (SEQ ID NO: 25), pET17b-SD22-ELP1-90-IFNA2bSD (SEQ ID NO: 26), pET17b-SD22-ELP4-60-IFNA2bSD (SEQ ID NO: 27), pET17b-SD35-IFNA2bSD-ELP1-90 (SEQ ID NO: 28), pET17b-SD35-IFNA2bSD-ELP4-60 (SEQ ID NO: 29), pET17b-SD37-IFNA2bSD-ELP1-90 (SEQ ID NO: 30), pET17b-SD37-IFNA2bSD-ELP4-60 (SEQ ID NO: 31), pET17b-SD31-IFNA2bSD-ELP1-90 (SEQ ID NO: 32) and pET17b-SD31-IFNA2bSD-ELP4-60 (SEQ ID NO: 33). 
     
     
         12 . The fusion protein of  claim 1 , selected from ELP4-60-S-S-Orexin B, ELP4-60-S-S-MMN, ELP4-60-S-S-NPY or ELP4-60-S-S-Gh. 
     
     
         13 . A polynucleotide comprising a nucleotide sequence encoding the fusion protein of  claim 1 . 
     
     
         14 . The polynucleotide of  claim 13 , selected from pET17b-SD33-ELP1-90-IFNA2bSD (SEQ ID NO: 34), pET17b-SD33-ELP4-60-IFNA2bSD (SEQ ID NO: 35), pET17b-SD34-ELP1-90-IFNA2bSD (SEQ ID NO: 36), pET17b-SD34-ELP4-60-IFNA2bSD (SEQ ID NO: 37), pET17b-SD22-ELP1-90-IFNA2bSD (SEQ ID NO: 38), pET17b-SD22-ELP4-60-IFNA2bSD (SEQ ID NO: 39), pET17b-SD35-IFNA2bSD-ELP1-90 (SEQ ID NO: 40), pET17b-SD35-IFNA2bSD-ELP4-60 (SEQ ID NO: 41), pET17b-SD37-IFNA2bSD-ELP1-90 (SEQ ID NO: 42), pET17b-SD37-IFNA2bSD-ELP4-60 (SEQ ID NO: 43), pET17b-SD31-IFNA2bSD-ELP1-90 (SEQ ID NO: 44) and pET17b-SD31-IFNA2bSD-ELP4-60 (SEQ ID NO: 45). 
     
     
         15 . An expression vector comprising the polynucleotide of  claim 13 . 
     
     
         16 . A host cell transformed by the vector of  claim 15 , wherein said host cell expresses the fusion protein. 
     
     
         17 . An ELP spider complex comprising two or more fusion proteins of  claim 1 , linked by at least one disulfide bond. 
     
     
         18 . An ELP spider complex comprising two or more fusion proteins exhibiting a phase transition, comprising:
 a) at least one target protein or peptide;   b) one or more proteins comprising oligomeric repeats of a polypeptide sequence selected from SEQ ID NOs: 1-17, wherein the one or more proteins exhibits a phase transition and are joined to the at least one target protein or peptide of a);   c) at least two residues capable of forming a disulfide bond; and   d) optionally, a spacer sequence separating any of the phase transition protein(s) of h) from any of the target protein(s) or peptide(s) of a).   wherein the two or more fusion proteins exhibiting a phase transition are linked by at least one disulfide bond.   
     
     
         19 . A method of providing a purified protein of interest, comprising:
 contacting a fusion protein comprising the protein of interest and an ELP tag, wherein the fusion protein contains at least one cleavage site that is cleavable to yield the protein of interest as a cleavage product with ELP-TEV1 that is effective to cleave said cleavage site, thereby yielding said protein of interest as a cleavage product in a cleavage product mixture comprising said ELP tag, any uncleaved fusion protein and said ELP-tagged cleavage agent; and   separating the protein of interest from the cleavage product mixture by inverse phase transition.   
     
     
         20 . The method of  claim 19 , wherein the fusion protein comprises a spider construct. 
     
     
         21 . A method of enhancing proteolytic resistance of a protein or peptide moiety in an ELP-based fusion peptide, comprising provision of the ELP-based fusion peptide in an ELP spider complex. 
     
     
         22 . An ELP fusion protein spider complex.

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