Method for the identification of suitable fragmentation sites in a reporter protein
AssigneeEcole Polytechnique Federale de Lausanne
InventorsPetra Tafelmeyer, Kai Johnsson
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Abstract
The invention concerns a combinatorial method for the generation of new split-protein sensors, and its application towards the (β/αa)8-barrel enzyme N-(5′-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae is demonstrated. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. This powerful selection thus complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening.
Claims31
A method for the identification of suitable fragmentation sites in a reporter protein, wherein the reporter protein is detectable when active, the method comprising the steps of:
- (a) providing a DNA sequence encoding for said reporter protein;
- (b) creating a library based on the DNA sequence as defined in (a),
- wherein each individual of said library comprises a randomly created first subsequence of the DNA sequence as defined in (a), encoding for a first subdomain of said reporter protein, and
- wherein each individual of said library comprises a randomly created complementary second subsequence of the DNA sequence as defined in (a), encoding for a complementary second subdomain of said reporter protein;
- (c) screening and/or selection for restoration of detectable activity of said reporter protein, when said first subdomain and said complementary second subdomain are brought into close proximity;
- (d) identifying said first subdomain and/or said first subsequence, and said complementary second subdomain and/or said complementary second subsequence, that lead to restoration of detectable activity of said reporter protein.
A recombinant DNA sequence for use in securing expression in a prokaryotic or eukaryotic host cell of a polypeptide product having the primary structural conformation of a first subdomain of a reporter protein or a complementary second subdomain of a reporter protein, wherein detectable activity of said reporter protein is restored, when said first subdomain and said complementary second subdomain are brought into close proximity, and wherein said first and said complementary second subdomain are not subdomains of one of the group of proteins consisting of transcriptional activators, ubiquitin, dihydrofolate reductase, β-lactamase, green fluorescent protein, β-galactosidase, inteins, cAMP cyclase, glycinamide ribonuc leotide transformylase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, luciferase.
A first subdomain of a reporter protein and/or a complementary second subdomain of a reporter protein, wherein detectable activity of said reporter protein is restored, when said first subdomain and said complementary second subdomain are brought into close proximity, and wherein said first and said complementary second subdomain are not subdomains of one of the group of proteins consisting of transcriptional activators, ubiquitin, dihydrofolate reductase, β-lactamase, green fluorescent protein, β-galactosidase, inteins, cAMP cyclase, glycinamide ribonucleotide transformylase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, luciferase.
A kit of parts, comprising a first and a second DNA-based expression vector, wherein
- said first expression vector contains an expression cassette encoding for a polypeptide product having at least a substantial part of the primary structural confirmation of a first subdomain of a reporter protein;
- and
- said second expression vector contains an expression cassette encoding for a polypeptide product having at least a substantial part of the primary structural confirmation of a complementary second subdomain of a reporter protein;
- and
- wherein detectable activity of said reporter protein is restored, when said first subdomain and said complementary second subdomain are brought into close proximity, and wherein said first and said complementary second subdomain are not subdomains of one the group of proteins consisting of transcriptional activators, ubiquitin, dihydrofolate reductase, β-lactamase, green fluorescent protein, β-galactosidase, inteins, cAMP cyclase, glycinamide ribonucleotide transformylase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, luciferase.
A process for the identification of fragmentation sites in a reporter protein for use in a two-hybrid system, comprising a step of random circular permutation of a gene and/or the expressed polypeptide derived thereof.
A process for the generation of a recombinant DNA molecule that secures for expression of both a polypeptide product comprising a first subdomain of a reporter protein and a complementary second subdomain of a reporter protein from said recombinant DNA molecule, comprising a step of transforming a host cell line that allows for homologous recombination of DNA.
Dependencies are inferred from the claim text.