Chapter 3
Extraction and Separation of Mycobacterial Proteins
Megan Lucas, Joan M. Ryan, Jackson Watkins, Kala Early,
Nicole A. Kruh-Garcia, Carolina Mehaffy, and Karen M. Dobos
Abstract
The extraction and separation of native mycobacterial proteins remain necessary for antigen discovery,
elucidation of enzymes to improve rational drug design, identification of physiologic mechanisms, use as
reagents for diagnostics, and defining host immune responses. In this chapter, methods for the manipulation of whole mycobacterial cells and culture exudates are described in detail as these methods are the
requisite first steps towards native protein isolation. Specifically, several methods for the inactivation of
viable Mycobacterium tuberculosis along with qualification assays are provided, as this is key to safe manipulation of cell pastes for downstream processes. Next, the concentration of spent culture filtrate media in
order to permit separation of soluble, secreted proteins is described followed by the separation of mycobacteria extracellular vesicles (MEV) from the remaining soluble proteins in spent media. We then describe
the generation of whole-cell lysate and facile separation of lysate into subcellular fractions to afford cell wall,
cell membrane, and cytosol-enriched proteins. Due to the hydrophobic nature of cell wall and cell
membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea
and SDS) are also provided. Finally, methods for separation of hydrophobic and hydrophilic proteins from
both whole-cell lysate and spent culture media are included. While these methods were optimized for the
manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate
Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins.
Key words French press, Probe sonication, Bead beating, Gamma irradiation, Subcellular fractionation, SDS, Guanidine HCL, Urea, Triton X-114, Ammonium sulfate, Culture filtrate, Extracellular
vesicles
1 Introduction
Two of the greatest hurdles a bacteriologist faces working with
Mycobacterium tuberculosis are the lag in culture (due to the slow
growth rate) and safe manipulation of cells for downstream uses.
For those in the research community, there is an acute need for
replicate samples, including bulk material for biochemical studies
and samples that can be used in animal models or eukaryotic cell
culture assays. Our laboratory is an established resource for the
generation of high-quality replicate samples and high quality and
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
77
Extraction and Separation of Mycobacterial Proteins
Megan Lucas, Joan M. Ryan, Jackson Watkins, Kala Early,
Nicole A. Kruh-Garcia, Carolina Mehaffy, and Karen M. Dobos
Abstract
The extraction and separation of native mycobacterial proteins remain necessary for antigen discovery,
elucidation of enzymes to improve rational drug design, identification of physiologic mechanisms, use as
reagents for diagnostics, and defining host immune responses. In this chapter, methods for the manipulation of whole mycobacterial cells and culture exudates are described in detail as these methods are the
requisite first steps towards native protein isolation. Specifically, several methods for the inactivation of
viable Mycobacterium tuberculosis along with qualification assays are provided, as this is key to safe manipulation of cell pastes for downstream processes. Next, the concentration of spent culture filtrate media in
order to permit separation of soluble, secreted proteins is described followed by the separation of mycobacteria extracellular vesicles (MEV) from the remaining soluble proteins in spent media. We then describe
the generation of whole-cell lysate and facile separation of lysate into subcellular fractions to afford cell wall,
cell membrane, and cytosol-enriched proteins. Due to the hydrophobic nature of cell wall and cell
membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea
and SDS) are also provided. Finally, methods for separation of hydrophobic and hydrophilic proteins from
both whole-cell lysate and spent culture media are included. While these methods were optimized for the
manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate
Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins.
Key words French press, Probe sonication, Bead beating, Gamma irradiation, Subcellular fractionation, SDS, Guanidine HCL, Urea, Triton X-114, Ammonium sulfate, Culture filtrate, Extracellular
vesicles
1 Introduction
Two of the greatest hurdles a bacteriologist faces working with
Mycobacterium tuberculosis are the lag in culture (due to the slow
growth rate) and safe manipulation of cells for downstream uses.
For those in the research community, there is an acute need for
replicate samples, including bulk material for biochemical studies
and samples that can be used in animal models or eukaryotic cell
culture assays. Our laboratory is an established resource for the
generation of high-quality replicate samples and high quality and
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
77