Structurally biased random peptide libraries based on different scaffolds
The invention relates to the use of scaffold proteins, particularly green fluorescent protein (GFP), in fusion constructs with random and defined peptides and peptide libraries, to increase the cellular expression levels, decrease the cellular catabolism, increase the conformational stability relative to linear peptides, and to increase the steady state concentrations of the library peptides and peptide library members expressed in cells for the purpose of detecting the presence of the peptides and screening peptide libraries. N-terminal, C-terminal, dual N- and C-terminal and one or more internal fusions are all contemplated. Novel fusions utilizing self-binding peptides to create a conformationally stabilized fusion domain are also contemplated.
1. A nucleic acid encoding a fusion protein comprising:
a) a fluorescent scaffold protein; and
b) a peptide;
wherein at least one amino acid of said fluorescent scaffold protein is replaced by at least one amino acid of said peptide, and wherein said peptide is inserted into a loop structure of said fluorescent scaffold protein.
2. The nucleic acid of claim 1 , wherein said peptide comprises a random sequence of amino acids.
3. The nucleic acid of claim 1 , wherein said peptide is encoded by a cDNA.
4. The nucleic acid of claim 1 , wherein said fluorescent scaffold protein is a green fluorescent protein (GFP).
5. The nucleic acid of claim 4 , wherein said GFP is from a Renilla species.
6. A cell comprising the nucleic acid of claim 1 .
7. A library of polynucleotides comprising the nucleic acid of claim 1 .
8. The nucleic acid of claim 1 , wherein said peptide further comprises at least one flexible linker linked to at least a portion of said fluorescent scaffold protein.
9. The nucleic acid of claim 8 , wherein said peptide comprises a second flexible linker, wherein each of said flexible linkers is linked to a portion of said fluorescent scaffold protein.
10. The nucleic acid of claim 8 , wherein said flexible linker comprises
-(gly) n -, wherein n≧2.
11. A method of screening for a bioactive peptide conferring a particular cellular phenotype comprising:
a) maintaining a cell comprising a nucleic acid encoding a fusion protein comprising:
i) a fluorescent scaffold protein; and
ii) a peptide;
wherein at least one amino acid of said luminescent or fluorescent scaffold protein is replaced by at least one amino acid of said peptide and wherein said peptide is inserted into a loop structure of said fluorescent scaffold protein,
under conditions suitable for expression of said fusion protein; and
b) assaying said cell for an identifiable phenotype, wherein said phenotype results from the expression of said fusion protein.
12. The method of claim 11 , wherein said peptide further comprises at least one flexible linker linked to at least a portion of said fluorescent scaffold protein.