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

Expression of Recombinant Phosphoproteins for Signal Transduction Studies.

Author information

1
Department of Cellular and Molecular Physiology, Yale University, ISTC 3rd Floor Rm 313, 850 West Campus Drive, New Haven, CT, 06516, USA.
2
Systems Biology Institute, Yale University, New Haven, CT, 06520-8114, USA.
3
Department of Cellular and Molecular Physiology, Yale University, ISTC 3rd Floor Rm 313, 850 West Campus Drive, New Haven, CT, 06516, USA. jesse.rinehart@yale.edu.
4
Systems Biology Institute, Yale University, New Haven, CT, 06520-8114, USA. jesse.rinehart@yale.edu.

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

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