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Updated: Feb 26, 2026

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
Published on: June 6, 2022
Expression of Recombinant Phosphoproteins for Signal Transduction Studies
Karl W Barber1,2, Jesse Rinehart3,4
1Department of Cellular and Molecular Physiology, Yale University, ISTC 3rd Floor Rm 313, 850 West Campus Drive, New Haven, CT, 06516, USA.
Abstract:
Complex signaling cascades are difficult to study in vitro without phosphorylated proteins. Here, we describe a technique for the routine production of recombinant phosphoproteins by directly incorporating phosphoserine as a nonstandard amino acid. This protocol utilizes an optimized phosphoserine orthogonal translation system and an engineered strain of E. coli containing no genomic amber codons. This approach has been used to generate a variety of phosphorylated proteins to understand the role of protein phosphorylation in cell signaling.
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