Determining the Localization of Carbohydrate Active Enzymes Within Gram-Negative Bacteria

  • Richard McLean
  • G. Douglas Inglis
  • Steven C. Mosimann
  • Richard R. E. Uwiera
  • D. Wade Abbott
Part of the Methods in Molecular Biology book series (MIMB, volume 1588)


Investigating the subcellular location of secreted proteins is valuable for illuminating their biological function. Although several bioinformatics programs currently exist to predict the destination of a trafficked protein using its signal peptide sequence, these programs have limited accuracy and often require experimental validation. Here, we present a systematic method to fractionate gram-negative cells and characterize the subcellular localization of secreted carbohydrate active enzymes (CAZymes). This method involves four parallel approaches that reveal the relative abundance of protein within the cytoplasm, periplasm, outer membrane, and extracellular environment. Cytoplasmic and periplasmic proteins are fractionated by lysis and osmotic shock, respectively. Outer membrane bound proteins are determined by comparing cells before and after exoproteolytic digestion. Extracellularly secreted proteins are collected from the media and concentrated. These four different fractionations can then be probed for the presence and quantity of target proteins using immunochemical methods such as Western blots and ELISAs, or enzyme activity assays.

Key words

Signal peptideSecretion Subcellular localization Cell fractionation Osmotic shock Whole cell dot blot Gram-negative bacteria 



This work was supported by an Alberta Innovates BioSolutions grant #BIOFS026 and Agriculture and Agri-Food Canada.


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Richard McLean
    • 1
    • 2
  • G. Douglas Inglis
    • 2
  • Steven C. Mosimann
    • 2
  • Richard R. E. Uwiera
    • 3
  • D. Wade Abbott
    • 1
    • 2
  1. 1.Functional Genomics of Complex Carbohydrate Utilization, Lethbridge Research and Development CentreAgriculture and Agri-Food CanadaLethbridgeCanada
  2. 2.Department of Chemistry and BiochemistryUniversity of LethbridgeLethbridgeCanada
  3. 3.Department of Agricultural, Food and Nutritional ScienceUniversity of AlbertaEdmontonCanada

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