Fusions of scaffold proteins with random peptide libraries

Details Fusions of scaffold proteins with random peptide libraries Fusions of scaffold proteins with random peptide libraries

1999-10-08

2003-04-15

Brusca, John S. (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S350000, C435S006120, C435S320100, C536S023400, C536S023500

Reexamination Certificate

active

06548632

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the use of scaffold proteins, particularly detectable genes such as green fluorescent protein (GFP), luciferase, &bgr;-lactamase, etc., 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 random peptides and random peptide library members expressed in cells for the purpose of detecting the presence of the peptides and screening random 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.
BACKGROUND OF THE INVENTION
The field of biomolecule screening for biologically and therapeutically relevant compounds is rapidly growing. Relevant biomolecules that have been the focus of such screening include chemical libraries, nucleic acid libraries and peptide libraries, in search of molecules that either inhibit or augment the biological activity of identified target molecules. With particular regard to peptide libraries, the isolation of peptide inhibitors of targets and the identification of formal binding partners of targets has been a key focus. However, one particular problem with peptide libraries is the difficulty assessing whether any particular peptide has been expressed, and at what level, prior to determining whether the peptide has a biological effect.
Green fluorescent protein (GFP) is a 238 amino acid protein. The crystal structure of the protein and of several point mutants has been solved (Ormo et al., Science 273, 1392-5, 1996; Yang et al., Nature Biotechnol. 14, 1246-51, 1996). The fluorophore, consisting of a modified tripeptide, is buried inside a relatively rigid beta-can structure, where it is almost completely protected from solvent access. The fluorescence of this protein is sensitive to a number of point mutations (Phillips, G. N., Curr. Opin. Struct. Biol. 7, 821-27, 1997). The fluorescence appears to be a sensitive indication of the preservation of the native structure of the protein, since any disruption of the structure allowing solvent access to the fluorophoric tripeptide will quench the fluorescence.
Abedi et al (Nucleic Acids Res. 26, 623-30, 1998) have inserted peptides between residues contained in several GFP loops. Inserts of the short sequence LEEFGS (SEQ ID NO:1) between adjacent residues at 10 internal insertion sites were tried. Of these, inserts at three sites, between residues 157-158, 172-173 and 194-195 gave fluorescence of at least 1% of that of wild type GFP. Only inserts between residues 157-158 and 172-173 had fluorescence of at least 10% of wild type GFP. When -SAG-random 20 mer-GAS-peptide sequences were inserted at different sites internal to GFP, only two sites gave mean fluorescence intensities of 2% or more of the GFP-random peptide sequences 10-fold above background fluorescence. These sites were insertions between residues 157-158 and 172-173.
It is an object of the invention to provide compositions of fusion constructs of peptides with scaffold proteins, comprising for example detectable proteins such as GFP, and methods of using such constructs in screening of peptide libraries.
SUMMARY OF THE INVENTION
In accordance with the objects outlined above, the present invention provides fusion proteins comprising a scaffold protein and a random peptide, fused to said scaffold protein, and nucleic acids which encode such fusion proteins. In an additional aspect, the present invention provides libraries of: a) fusion proteins; b) fusion nucleic acids; c) expression vectors comprising the fusion nucleic acids; and d) host cells comprising the fusion nucleic acids. The present invention further comprises methods for screening for a bioactive peptide capable of confering a particular phenotype.
In one aspect, a library of fusion proteins comprises a scaffold protein, a random peptide fused to the N-terminus of the scaffold protein and a representation structure that will present the random peptide in a conformationally restricted form. In a preferred embodiment, each of the random peptide in the library is different.
In one aspect, a library of fusion proteins comprises a scaffold protein, a random peptide fused to the C-terminus of the scaffold protein and a representation structure that will present the random peptide in a conformationally restricted form. In a preferred embodiment, each of the random peptide in the library is different.
In one aspect, a library of fusion proteins comprises a scaffold protein, a random peptide inserted into the scaffold protein and at least one fusion partner. In a preferred embodiment, each of the random peptide in the library is different. In another preferred embodiment, the random peptide is inserted into a loop structure of said scaffold protein.
In one aspect of the invention, the scaffold protein is a green fluorescent protein (GFP).
In one aspect of the invention, the GFP is from Aequrea and the random peptide is inserted into the loop comprising amino acids 130 to 135 of said GFP.
In another aspect of the invention, the GFP is from Aequrea and the random peptide is inserted into the loop comprising amino acids 154 to 159 of said GFP.
In another aspect of the invention, the GFP is from Aequrea and the random peptide is inserted into the loop comprising amino acids 172 to 175 of said GFP.
In another aspect of the invention, the GFP is from Aequrea and the random peptide is inserted into the loop comprising amino acids 188 to 193 of said GFP.
In another aspect of the invention, the GFP is from Aequrea and the random peptide is inserted into the loop comprising amino acids 208 to 216 of said GFP.
In one aspect of the invention, the GFP is from a Renilla species.
In another aspect of the invention, the scaffold protein is &bgr;-lactamase.
In another aspect of the invention, the scaffold protein is DHFR.
In another aspect of the invention, the scaffold protein is &bgr;-galactosidase.
In another aspect of the invention, the scaffold protein is luciferase.
In another aspect of the invention, a library of fusion proteins is provided, comprising a linker between the random peptide and the scaffold protein.
In another aspect of the invention, a library of fusion proteins is provided, comprising a second linker between the other end of the random peptide and the scaffold protein.
In another aspect of the invention, a library of fusion proteins is provided, comprising a -(gly)
n
-linke, wherein n≧2.
In another aspect of the invention, a library of fusion proteins is provided, comprising a scaffold protein and a random peptide, wherein the random peptide replaces at least one amino acid of said scaffold protein. In a preferred embodiment, the amino acid of said scaffold protein which is replaced by the random peptide is located within a loop structure of said scaffold protein.
In one aspect of the invention, the library of fusion proteins and the library of nucleic acids comprise at least 10
5
different members.
The invention further provides fusion nucleic acids encoding the fusion proteins. In a preferred embodiment, the nucleic acid encoding the fusion protein comprises a nucleic acid encoding a random peptide, a nucleic acid encoding a scaffold protein and a nucleic acid encoding a fusion partner. In another preferred embodiment, the nucleic acid encoding the random peptide is inserted internally into the nucleic acid encoding the scaffold protein.
In another aspect of the invention, expression vectors are provided. The expression vectors comprise one or more of the nucleic acids encoding the fusion proteins operably linked to regulatory sequences recognized by a host cell transformed with the nnucleic acids. In a preferred embodiment the expression vectors are retroviral vectors. Further provided herein are host cells comprising the vectors and t

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