Site‐Specific Protein Bioconjugation via a Pyridoxal 5′‐Phosphate‐Mediated N‐Terminal Transamination Reaction

Leah S. Witus1, Matthew Francis1

1 University of California, Berkeley, Department of Chemistry, Berkeley, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100018
Online Posting Date:  June, 2010
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Abstract

The covalent attachment of chemical groups to proteins is a critically important tool for the study of protein function and the creation of protein‐based materials. Methods of site‐specific protein modification are necessary for the generation of well defined bioconjugates possessing a new functional group in a single position in the amino acid sequence. This article describes a pyridoxal 5′‐phosphate (PLP)–mediated transamination reaction that is specific for the N‐terminus of a protein. The reaction oxidizes the N‐terminal amine to a ketone or an aldehyde, which can form a stable oxime linkage with an alkoxyamine reagent of choice. Screening studies have identified the most reactive N‐terminal residues, facilitating the use of site‐directed mutagenesis to achieve high levels of conversion. Additionally, this reaction has been shown to be effective for a number of targets that are not easily accessed through heterologous expression, such as monoclonal antibodies. Curr. Protoc. Chem. Biol. 2:125‐134 © 2010 by John Wiley & Sons, Inc.

Keywords: bioconjugation; N‐terminus; pyridoxal phosphate; PLP; oximation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Protein Labeling via a Site‐Specific N‐terminal Transamination Reaction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Protein Labeling via a Site‐Specific N‐terminal Transamination Reaction

  Materials
  • Target protein stock solution (see recipe)
  • PLP stock solution (see recipe)
  • Alkoxyamine solution (see recipe)
  • Additional reagents and equipment for protein separations and analysis of protein modification, including ESI‐MS or MALDI‐TOF MS methods (Coligan et al., )
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Figures

Videos

Literature Cited

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