Expression of Recombinant Phosphoproteins for Signal Transduction Studies

Methods Mol Biol. 2017:1636:71-78. doi: 10.1007/978-1-4939-7154-1_5.

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.

Keywords: Biochemistry; Bioengineering; Kinase signaling; Molecular biology; Protein phosphorylation; Synthetic biology.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, N.I.H., Extramural

MeSH terms

  • Gene Expression*
  • Genes, Reporter
  • Humans
  • Phosphoproteins / genetics*
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphoserine
  • Recombinant Proteins*
  • Signal Transduction*

Substances

  • Phosphoproteins
  • Recombinant Proteins
  • Phosphoserine