Work

One-Step Purification and N-Terminal Functionalization of Bioactive Proteins via Atypically Split Inteins

Year: 2024

Type: article

Abstract: Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our method─referred to as VidaL-tagged expression and protein ligation (VEPL)─enables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (β-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.

Source: Bioconjugate Chemistry

DOI: 10.1021/acs.bioconjchem.4c00223

Authors: Ryan Gharios, Annabella Li, Irina Kopyeva, Ryan M. Francis, Cole A. DeForest

Institution: University of Washington

Language: English

Topic: Biochemical and Structural Characterization

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green