Fusion peptides isolatable by phase transition
OwnerAshutosh Chilkotiinventor
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Abstract
Genetically-encodable, environmentally-responsive fusion proteins comprising ELP peptides. Such fusion proteins exhibit unique physico-chemical and functional properties that can be modulated as a function of solution environment. The invention also provides methods for purifying the FPs, which take advantage of these unique properties, including high-throughput purification methods that produce high yields (e.g., milligram levels) of purified proteins, thereby yielding sufficient purified product for multiple assays and analyses. The high throughput purification technique is simpler and less expensive than current commercial high throughput purification methods, since it requires only one transfer of purification intermediates to a new multiwell plate.
Claims65
A fusion protein exhibiting a phase transition, the fusion protein comprising:
- (a) one or more biological molecules;
- (b) one or more proteins exhibiting a phase transition joined to the biologically active molecule;
- and
- (c) optionally, a spacer sequence separating any of the protein(s) of (b) from any of the biological molecule(s) of (a).
A fusion protein exhibiting a phase transition, the fusion protein comprising:
- (a) one or more protein(s) of interest;
- (b) one or more protein(s) exhibiting a phase transition joined at a C- and/or N-terminus of a protein of (a);
- and
- (c) optionally, a spacer sequence separating the any of the protein(s) of (a) and/or (b).
A fusion protein exhibiting a phase transition, said fusion protein comprising:
- (a) one or more proteins of interest;
- (b) one or more β-turn protein(s) joined at a C- and/or N-terminus of any of the proteins of (a);
- and
- (c) optionally, a spacer sequence separating any of the protein(s) of (a) and/or (b).
A fusion protein exhibiting a phase transition, the fusion protein comprising:
- (a) a protein of interest;
- (b) a protein exhibiting a phase transition joined at a C- and/or N-terminus of the protein of interest;
- and
- (c) optionally, a spacer sequence separating the protein of (a) from the protein of (c).
A fusion protein exhibiting a phase transition, said fusion protein comprising:
- (a) a protein of interest;
- (b) a protein exhibiting a β-turn joined at a C- and/or N-terminus of the protein of (a);
- and
- (c) optionally, a spacer sequence separating the protein of (a) from the protein of (c).
A polynucleotide comprising a nucleotide sequence encoding a fusion protein exhibiting a phase transition, said fusion protein comprising:
- (a) one or more protein(s) of interest;
- (b) one or more β-turn proteins exhibiting a phase transition joined at a C- and/or N-terminus of (a);
- and
- (c) optionally, a spacer sequence separating any of the protein(s) of (a) and/or (b).
A method of optimizing size of an ELP expression tag incorporated in a polynucleotide comprising a nucleotide sequence encoding a fusion protein exhibiting a phase transition, wherein the fusion protein comprises a protein of interest, said method comprising the steps of (i) forming a multiplicity of polynucleotides comprising a nucleotide sequence encoding a fusion protein exhibiting a phase transition, wherein each of said multiplicity of polynucleotides includes a different-sized ELP expression tag, (ii) expressing corresponding fusion proteins from said multiplicity of polynucleotides, (iii) determining a yield of the desired protein for each of said corresponding fusion proteins, (iv) determining size of particulates for each of said corresponding fusion proteins in solution as temperature is raised above Tt, and (v) selecting an optimized size ELP expression tag according to predetermined selection criteria for maximum recoverable protein of interest from among said multiplicity of polynucleotides.
A method of purification of fusion proteins to yield a protein of interest, comprising forming a polynucleotide comprising a nucleotide sequence encoding a fusion protein exhibiting a phase transition, expressing the fusion protein in culture, and subjecting a fusion protein-containing material from said culture to processing involving centrifugation and inverse transition cycling to recover said protein of interest.
Dependencies are inferred from the claim text.