Humanized renilla reniformis green fluorescent protein as a scaffold
The present invention discloses green fluorescent protein (GFP) and GFP variants that are derived from Renilla reniformis . The Renilla reniformis GFP and variants there of, are optimized for expression in human cells and are further used as a scaffold for the in vivo display of peptides and peptide libraries.
1 . A recombinant polynucleotide comprising a first nucleic acid sequence encoding a humanized Renilla reniformis green fluorescent protein (hrGFP) and a second heterologous nucleic acid sequence inserted internally into said first nucleic acid sequence encoding humanized hrGFP, said recombinant polynucleotide encoding a scaffold GFP.
2 . The recombinant polynucleotide of claim 1 wherein said scaffold GFP is fluorescent.
3 . The recombinant polynucleotide of claim 1 wherein the said first nucleic acid sequence encoding a hrGFP is SEQ ID NO: 1.
4 . The recombinant polynucleotide of claim 2 wherein said second heterologous nucleic acid sequence is inserted between nucleotides 519 and 520 of said first nucleic acid sequence encoding hrGFP.
5 . The recombinant polynucleotide of claim 1 wherein said second heterologous nucleic acid sequence comprises a multiple cloning site sequence.
6 . The recombinant polynucleotide of claim 1 wherein said second heterologous nucleic acid sequence is the multiple cloning site sequence of SEQ ID NO: 2.
7 . The recombinant polynucleotide of claim 4 or 5 further comprising a third nucleic acid sequence inserted internally into said multiple cloning site, wherein said third nucleic acid sequence comprises a random nucleic acid sequence.
8 . The recombinant polynucleotide of claim 6 wherein said third nucleic acid sequence encodes a peptide in frame with said hrGFP coding sequences.
9 . The recombinant polynucleotide of claim 6 wherein said third nucleic acid sequence encodes a peptide of 2 to 50 amino acids.
10 . The recombinant polynucleotide of claim 6 wherein said third nucleic acid sequence encodes a polypeptide of 10 to 20 amino acids.
11 . A recombinant polypeptide comprising Renilla reniformis green fluorescent protein (GFP) and a heterologous peptide that is fused internally into said GFP.
12 . The recombinant polypeptide of claim 7 wherein said heterologous peptide is located between amino acid residues 173 and 174 of said GFP.
13 . The recombinant polypeptide of claim 7 wherein said second heterologous amino acid sequence is a random peptide sequence.
14 . A recombinant vector comprising the recombinant polynucleotide sequence of any of claims 1 - 6 .
15 . The recombinant vector of claim 11 wherein said vector is selected from the group consisting of a plasmid, a bacteriophage, a virus, and a retrovirus.
16 . A cell comprising the recombinant vector of claim 11 .
17 . A library of recombinant vectors comprising a plurality of recombinant polynucleotides wherein said recombinant polynucleotides comprise a first nucleic acid sequence encoding humanized Renilla reniformis green fluorescent protein (hrGFP) and a second heterologous nucleic acid sequence inserted internally into said first nucleic acid sequence encoding hrGFP, wherein the members of the library comprise a plurality of different said second heterologous nucleic acid sequences.
18 . The library of claim 17 wherein said plurality of different said second heterologous nucleic acid sequences comprise a pluriality of randomized nucleic acid sequences.
19 . A method for identifying a peptide conferring a phenotype of interest comprising the steps of:
a) providing a plurality of cells, each cell containing a recombinant vector comprising a recombinant polynucleotide that encodes a recombinant polypeptide comprising Renilla reniformis green fluorescent protein (hrGFP) and a heterologous random peptide wherein said heterologous random peptide is fused internally into said hrGFP, under conditions wherein said recombinant polypeptide is expressed; and
b) assaying said cells for said phenotype.
20 . A method for identifying a peptide that interacts with a protein of interest, the method comprising the steps of:
a) introducing a library of recombinant vectors comprising recombinant polynucleotides that encode recombinant polypeptides into a plurality of host cells and maintaining said cells under conditions wherein said recombinant polypeptides are expressed,
wherein said recombinant polypeptides comprise Renilla reniformis green fluorescent protein (hrGFP) fused to a transactivation domain and a heterologous randomized peptide fused internally into said hrGFP and,
wherein said host cells contain a gene that encodes a protein of interest fused to a DNA binding domain, and a reporter gene functionally linked to a DNA sequence that binds said DNA binding domain, wherein expression of said reporter gene is regulated by said transactivation domain and;
b) detecting expression of said reporter gene, wherein detection of reporter gene expression identifies said heterologous random peptide as a peptide that interacts with the protein of interest.