DEVICES AND METHODS FOR DISPLAY OF ENCODED PEPTIDES, POLYPEPTIDES, AND PROTEINS ON DNA

摘要

A novel method for displaying proteins and peptides is disclosed in which individual proteins or peptides remain associated with the DNA encoding them. Proteins or peptides can be generated by in vitro translation of DNA templates, either free in solution or arrayed on a solid support, such that the proteins or peptides remain immobilized on their DNA templates. In particular, high throughput sequencing can be combined with high throughput functional characterization of encoded proteins and peptides, wherein the identity of each protein or peptide is determined by DNA sequencing, and functional studies are carried out directly on each protein or peptide while immobilized on the DNA template encoding it. The methods of the invention should find numerous applications, for example, in high throughput genetic or pharmacological screening, epitope mapping, and protein engineering and directed evolution.

主权项

1. A method for displaying a polypeptide on a DNA template encoding it, the method comprising:
a) providing a DNA template that encodes the polypeptide, wherein the DNA template comprises:
i) a promoter operably linked to an open reading frame (ORF); andii) a molecular roadblock that blocks progress of an RNA polymerase during transcription of the DNA template; b) adding an RNA polymerase that can be blocked by the molecular roadblock, wherein the RNA polymerase binds to the promoter of the DNA template and carries out transcription of the DNA template until the RNA polymerase stalls at the molecular roadblock where the RNA polymerase is blocked from further progress, such that the DNA template and transcribed mRNA remain associated; and c) adding a ribosome that binds to a ribosomal binding site on the mRNA, wherein the ribosome carries out protein translation until the ribosome reaches the stalled RNA polymerase at the molecular roadblock where the ribosome is blocked from further progress, such that the ribosome displays the nascent polypeptide while remaining associated with the mRNA.