The University of North Carolina at Chapel Hill

Other

Johns Hopkins University

Cytogen Corporation

Utrecht University

Andrew B Sparks

Brian K Kay

Dana M Fowlkes

Channing J Der

Judith M Thorn

Lawrence A Quilliam

Stephen J McConnell

Noah Hoffman

Bert Vogelstein

Kenneth W Kinzler

Nils B Adey

James E Rider

Victor E Velculescu

Patrice J Morin

Hans Clevers

Nick Barker

Vladimer Korinek

Novel polypeptides having functional domains of interest are described, along with DNA sequences that encode the same. A method of identifying these polypeptides by means of a sequence-independent (that is, independent of the primary sequence of the polypeptide sought), recognition unit-based functional screen is also disclosed. Various applications of the method and of the polypeptides identified are described, including their use in assay kits for drug discovery, modification, and refinement.

Polypeptides having a functional domain of interest and methods of identifying and using same

Peptides having general and specific binding affinities for the Src homology region (SH3) domains of proteins are disclosed in the present invention. In particular, SH3 binding peptides have been isolated from three phage-displayed random peptide libraries which had been screened for isolates that bind to bacterial fusion proteins of SH3 domains and glutathione S-transferase (GST). Preferred peptides are disclosed having a core 7-mer sequence (preferably, a consensus motif) and two or more, preferably at least six, additional amino acid residues flanking the core sequence, for a total length of 9, preferably at least 13, amino acid residues and no more than about 45 amino acid residues. Such peptides manifest preferential binding affinities for certain SH3 domains. The preferred peptides exhibit specific binding affinities for the Src-family of proteins. In vitro and in vivo results are presented which demonstrate the biochemical activity of such peptides.

Src SH3 binding peptides and methods of isolating and using same

Serial analysis of gene expression, SAGE, a method for the rapid quantitative and qualitative analysis of transcripts, has been improved to provide more genetic information about each analyzed transcript by use of MmeI restriction endonuclease. In SAGE, defined sequence tags corresponding to expressed genes are isolated and analyzed. Sequencing of over 1,000 defined tags in a short period of time (e.g., hours) reveals a gene expression pattern characteristic of the function of a cell or tissue. Moreover, SAGE is useful as a gene discovery tool for the identification and isolation of novel sequence tags corresponding to novel transcripts and genes.

Serial analysis of transcript expression using MmeI and long tags

Peptides having general and specific binding affinities for the Src homology region 3 (SH3) domains of proteins are disclosed in the present invention. In particular, SH3 binding peptides have been isolated from phage-displayed random peptide libraries which had been screened for isolates that bind to bacterial fusion proteins having an SH3 domain and glutathione S-transferase (GST). Preferred peptides are disclosed which comprise a core 7-mer sequence (preferably, a consensus motif) and two or more, preferably at least six, additional amino acid residues flanking the core sequence, for a total length of 9, preferably at least 13, amino acid residues and no more than about 45 amino acid residues. Such peptides manifest preferential binding affinities for certain SH3 domains. The preferred peptides exhibit specific binding affinities for the Src-family of proteins. In vitro and in vivo results are presented which demonstrate the biochemical activity of such peptides.

Nck SH3 binding peptides

Peptides having general and specific binding affinities for the Src homology region 3 (SH3) domains of proteins are disclosed in the present invention. In particular, SH3 binding peptides have been isolated from three phage-displayed random peptide libraries which had been screened for isolates that bind to bacterial fusion proteins of SH3 domains and glutathione S-transferase (GST). Preferred peptides are disclosed having a core 7-mer sequence (preferably, a consensus motif) and two or more, preferably at least six, additional amino acid residues flanking the core sequence, for a total length of 9, preferably at least 13, amino acid residues and no more than about 45 amino acid residues. Such peptides manifest preferential binding affinities for certain SH3 domains. The preferred peptides exhibit specific binding affinities for the Src-family of proteins. In vitro and in vivo results are presented which demonstrate the biochemical activity of such peptides.

Scr SH3 binding peptides and methods of isolating and using same

GRB2 SH3 binding peptides and methods of isolating and using same

A novel method for producing novel and/or improved heterofunctional binding fusion proteins termed Totally Synthetic Affinity Reagents (TSARs) is disclosed. TSARs are concatenated heterofunctional proteins, polypeptides or peptides comprising at least two functional regions: a binding domain with affinity for a ligand and a second effector peptide portion that is chemically or biologically active. In one embodiment, the heterofunctional proteins, polypeptides or peptides further comprise a linker peptide portion between the binding domain and the second active peptide portion. The linker peptide can be either susceptible or not susceptible to cleavage by enzymatic or chemical means. Novel and/or improved heterofunctional binding reagents as well as methods for using the reagents for a variety of in vitro and in vivo applications are also disclosed.

Totally Synthetic Affinity Reagents

The APC tumor suppressor protein binds to .beta.-catenin, a protein recently shown to interact with Tcf/Lef transcription factors. Here, the gene encoding a Tcf family member that is expressed in colonic epithelium (hTcf-4) was cloned and characterized. hTcf-4 transactivates transcription only when associated with .beta.-catenin. Nuclei of APC.sup.-/- colon carcinoma cells were found to contain a stable .beta.-catenin-hTCF-4 complex that was constitutively active, as measured by transcription of a Tcf reporter gene. Reintroduction of APC removed .beta.-catenin from hTcf4 and abrogated the transcriptional transactivation. Constitutive transcription of TCF target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium. It is also shown here that the products of mutant APC genes found in colorectal tumors are defective in regulating .beta.-catenin/Tcf-4 transcrpitional activation. Furthermore, colorectal tumors with intact APC genes were shown to contain subtle activating mutations of .beta.-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of .beta.-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or .beta.-catenin.

.beta.-catenin, Tcf-4, and APC interact to prevent cancer

Random peptide library

Growing community of inventors

Pikesville, MD, United States of America

Andrew B Sparks