Molecular indexing of proteins by self assembly (mipsa) for efficient proteomic investigations
AssigneeThe Johns Hopkins University
InventorsHarry B. Larman, Joel Credle, Jonathan Gunn, Puwanat Sankapreecha
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Abstract
The present disclosure relates to the field of proteomics. More specifically, the present disclosure provides compositions and methods for molecular indexing of proteins by self-assembly. In one aspect, the present disclosure provides a library of self-assembled protein-DNA conjugates. In particular embodiments, each protein-DNA conjugate comprises (a) a cDNA comprising a barcode, wherein the cDNA is conjugated with a ligand that specifically binds a polypeptide tag; and (b) a fusion protein comprising the polypeptide tag and a protein of interest, wherein the ligand is covalently bound to the polypeptide tag.
Claims31
1-87. (canceled)
A library comprising a plurality of protein-cDNA conjugates, the protein-cDNA conjugates in the plurality comprising:
- (a) a fusion protein comprising:
- (i) a protein of interest;
- and
- (ii) a polypeptide tag,
- wherein the protein of interest and the polypeptide tag are present in a single polypeptide;
- and
- (b) a single-stranded cDNA comprising:
- (i) a barcode, and
- (ii) a ligand that specifically binds the polypeptide tag.
A method for studying protein interactions with a molecule of interest, the method comprising the steps of:
- (a) contacting the library of claim 1 with a sample comprising the molecule of interest under conditions sufficient for one or more of the protein-cDNA conjugates in the plurality to interact with the molecule of interest;
- (b) performing a pull-down of the molecule of interest;
- and
- (c) determining the identity of the barcodes present in the one or more of the protein-cDNA conjugates that interact with the molecule of interest, optionally correlating the identified barcodes with the identity of the one or more proteins of interest in the one or more of the protein-cDNA conjugates.
A protein-cDNA conjugate, comprising:
- (a) a fusion protein comprising:
- (i) a protein of interest;
- and
- (ii) a polypeptide tag,
- wherein the protein of interest and the polypeptide tag are present in a single polypeptide;
- and
- (b) a single-stranded cDNA comprising:
- (i) a barcode, and
- (ii) a ligand that specifically binds the polypeptide tag.
A method comprising the steps of:
- (a) providing a vector library, wherein the vector library encodes a plurality of proteins, and wherein each vector of the vector library comprises in the 5′ to 3′ direction:
- (i) a polymerase transcriptional start site;
- (ii) a barcode;
- (iii) a reverse transcription primer binding site;
- (iv) a ribosome binding site (RBS);
- and
- (v) a nucleotide sequence encoding a fusion protein, the fusion protein comprising:
- (A) a protein of interest;
- and
- (B) a polypeptide tag,
- wherein the protein of interest and the polypeptide tag are a single polypeptide;
- (b) transcribing or having transcribed the vector library to produce mRNA;
- (c) contacting or having contacted the transcribed mRNA with (i) a primer that binds the reverse transcription primer binding site, wherein the primer is conjugated to a ligand that specifically binds the polypeptide tag;
- and (ii) reverse transcriptase;
- (d) reverse transcribing or having reverse transcribed the 5′ end of the transcribed mRNA, to produce a complementary deoxyribonucleic acid (cDNA) comprising the ligand and the barcode;
- and
- (e) translating or having translated the transcribed mRNA under conditions wherein the ligand of the cDNA specifically binds the polypeptide tag of the fusion protein in cis during translation.
A vector library, wherein the vector library encodes a plurality of proteins, and wherein each vector of the vector library comprises in the 5′ to 3′ direction:
- (i) a polymerase transcriptional start site;
- (iii) a barcode;
- (iii) a reverse transcription primer binding site;
- (iv) a ribosome binding site (RBS);
- and
- (v) a nucleotide sequence encoding a fusion protein, the fusion protein comprising:
- (A) a protein of interest;
- and
- (B) a polypeptide tag,
- wherein the protein of interest and the polypeptide tag are a single polypeptide.
Dependencies are inferred from the claim text.