Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

J Bacteriol. 2016 Jan 19;198(7):1035-43. doi: 10.1128/JB.00864-15.


The use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryote Escherichia coli. We show that functional protein fusions of the HaloTag can be detected both in vivo and in vitro when expressed within the cytoplasmic or periplasmic compartments of E. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.

Importance: Visualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryote Escherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Cytoplasm / metabolism*
  • Escherichia coli / metabolism*
  • Gene Expression Regulation, Bacterial / physiology
  • Models, Molecular
  • Mutation
  • Periplasm / chemistry
  • Periplasm / metabolism*
  • Plasmids
  • Protein Conformation
  • Protein Engineering
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Rhodococcus / enzymology*
  • Rhodococcus / metabolism
  • Staining and Labeling


  • Bacterial Proteins
  • Recombinant Fusion Proteins