UNIT 10A.

4 Analyses of Mycobacterium tuberculosis

Proteins
Michelle H. Larsen,
1
Karolin Biermann,
1
and William R. Jacobs, Jr.
1
1
Albert Einstein College of Medicine, Bronx, New York
ABSTRACT
This unit includes protocols for the isolation of proteins from Mycobacterium
tuberculosis. Considerations for working with M. tuberculosis at Biosafety Level 3
containment are also discussed. Curr. Protoc. Microbiol. 6:10A.4.1-10A.4.5.
C 2007 by
John Wiley & Sons, Inc.
Keywords: tuberculosis
r
TB
r
protein
r
culture ltrate
r
Sauton
INTRODUCTION
Mycobacteriumtuberculosis, the causative agent of tuberculosis (TB), infects almost one
third of the worlds population. Yearly, there are an estimated 10 million new cases of TB
resulting in 2 to 3 million deaths. Over the last 15 years, the development of genetic tools
to manipulate M. tuberculosis has proliferated (UNIT 10A.2). Furthermore, availability of the
complete M. tuberculosis DNA genome sequence (as well as other related mycobacteria)
has yielded tremendous information towards a better understanding of the bacterium and
the disease it causes.
The protocols in this unit will detail bacterial protein extraction and culture ltrate
preparation. These proteins may then be analyzed using standard methods such as SDS-
PAGE or 2-D electrophoresis. See Braunstein et al., 2003, and Kriakov et al., 2003, for
examples of M. tuberculosis protein analyses.
CAUTION: Mycobacteriumtuberculosis is a Biosafety Level 3 (BSL-3) pathogen. Follow
all appropriate guidelines for the use and handling of pathogenic microorganisms. See
Strategic Planning, Safety Considerations for further discussion. See UNIT 1A.1 and other
pertinent resources (APPENDIX 1B) for additional information.
SAFETY CONSIDERATIONS
Standards of BSL-3 containment should be followed for work with M. tuberculosis.
Consult with the Safety Ofcer and/or Institutional Biosafety Committee for additional
guidance. See http://www.cdc.gov/OD/ohs/symp5/jyrtext.htm and UNIT 1A.1 for a brief de-
scription of general BSL-3 laboratory features. Complete inactivation of M. tuberculosis
cells is necessary to perform some studies outside the BSL-3 laboratory. All methods
for inactivation should be validated in individual laboratories, taking into consideration
the concentration of cells used. Schwebach et al. (2001) provides some guidance on
determining if particular conditions are adequate for inactivation of mycobacteria. For
many of the procedures, the authors have found the following conditions sufcient for
inactivation of M. tuberculosis: incubation for 12 hr at 65

C, 2 hr at 80

C, or 15 min at
95

C; or incubation for 1 hr in a nal concentration of one of the following solutions:

2.5%glutaraldehyde, 2% paraformaldehyde, 5%formalin, or Vesphene IIse. During heat
inactivation, care should be taken to ensure that the entire tube is immersed in a water
bath preheated to the appropriate temperature. Incubation of M. tuberculosis cultures
with gluteraldehyde, paraformaldehyde, formalin, or Vesphene IIse should be done in a
Current Protocols in Microbiology 10A.4.1-10A.4.5, August 2007
Published online August 2007 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/9780471729259.mc10a04s6
Copyright
C 2007 John Wiley & Sons, Inc.
Actinobacteria
(High G+C
Gram Positive)
10A.4.1
Supplement 6
Analysis of
M. tuberculosis
Proteins
10A.4.2
Supplement 6 Current Protocols in Microbiology
matter that ensures that the entire interior surface of the tube has been coated by gently
rolling and inverting the tube.
BASIC
PROTOCOL 1
EXTRACTION OF M. TUBERCULOSIS PROTEINS USING THE GLASS
BEAD METHOD
For preparation of protein extracts, the glass bead method is preferred. Some researchers
suggest more than one wash step (step 1) to remove any residual albumin from Mid-
dlebrook broth while other researchers prefer to avoid any albumin contamination by
growing cultures in Sautons medium (see UNIT 10A.1). Glass beads are necessary to lyse
the cells as the large amount of lipids retained by mycobacteria make boiling alone an
inefcient method for protein extraction. Protein extracts can then be used in SDS-PAGE
or 2-D protein gel electrophoresis for further analysis.
Materials
Mycobacteria (UNIT 10A.1)
Phosphate buffered saline (PBS; APPENDIX 2A)
Protein extraction buffer (see recipe), ice cold
2 SDS sample buffer (APPENDIX 2A)
15-ml screw-cap conical tubes
Refrigerated tabletop centrifuge
Vortex and tube adaptor
2-ml screw-cap tubes (Sarstedt)
Microcentrifuge
Glass beads (106-m, Sigma cat. no. G4649)
95

C water bath
1. Wash cells two times with an equal volume of PBS to remove BSA that might
interfere with resolution of mycobacterial proteins by SDS-PAGE.
2. Centrifuge culture 10 min at 2000 g, 4

C, in 15-ml screw-cap conical tubes.

3. Discard supernatant and add 10 ml PBS to cell pellet. Vortex to resuspend cell pellet
then centrifuge 10 min at 2000 g, 4

C.
4. Discard supernatant and resuspend cell pellet in 1.5 ml PBS. Vortex to resuspend
cell pellet.
5. Transfer cell suspension to a 2-ml Sarstedt screw-cap tube. Microcentrifuge 5 min
at 15,800 g, 4

C.
6. Discard supernatant and resuspend cell pellet in 300 to 400 l of ice-cold protein
extraction buffer.
7. Add 0.4 g of 106-m glass beads.
8. Vortex 5 min at top speed using an adaptor to hold the tubes.
9. Microcentrifuge 5 min at 15,800 g, 4

C, to pellet glass beads and debris at the

bottom of the tube.
10. Transfer supernatant to a fresh 2-ml Sarstedt screw-cap tube.
11. Add an equal volume of 2 SDS sample buffer.
12. Vortex briey to mix sample, then incubate 5 min in a 95

C water bath.
13. Store sample up to 2 weeks at 70

C; thaw at 2

C (10 min) just prior to use.

14. Perform SDS-PAGE (CPMB UNIT 10A.2).
Actinobacteria
(High G+C
Gram Positive)
10A.4.3
Current Protocols in Microbiology Supplement 6
BASIC
PROTOCOL 2
PREPARATION OF CULTURE FILTRATES
M. tuberculosis secretes a number of proteins of immunological interest. For preparation
of culture ltrates, it is essential that cultures be grown in Sauton medium, which does
not have the albumin enrichment that is found in Middlebrook medium (refer to UNIT
10A.1). Cultures will take longer to growin Sauton medium(both initial growth and overall
doubling time) compared to growth in Middlebrook 7H9 medium. Culture ltrates can
then be used in SDS-PAGE or 2-D protein gel electrophoresis for further analysis.
Materials
Bacterial culture in Sauton medium (UNIT 10A.1)
50-ml conical tubes
Sterile cell scraper
Refrigerated tabletop centrifuge
Filter units (0.45- and 0.2-m)
Cell concentrator (Amicon Ultraltration unit or equivalent)
1. Grow 100 to 200 ml bacterial culture in Sauton medium.
2. Transfer culture to 50-ml conical tubes.
Bacteria will be difcult to manipulate because of a lack of albumin and detergent in the
medium; the culture will grow as a pellicle on the surface of the medium and will stick
to pipets. For most analysis, it is useful to compare the supernatant and pellet; therefore,
it is important to collect as much of both components as possible. Decanting the culture
directly to 50-ml conical tubes and using a sterile cell scraper will help in increasing the
amount of material recovered.
3. Pellet bacteria by centrifuging 20 min at 2000 g, 4

C.
4. Transfer supernatant to a fresh tube and centrifuge again.
5. Filter supernatant through a 0.2-m lter unit, then through another 0.2-m lter
unit.
For BSL-3 organisms, dip closed tube in a disinfectant such as Vesphene II to disinfect
the outer surface of the tube. From this point on, the ltrate can be processed in a BSL2
laboratory.
6. In the BSL-2 laboratory, concentrate supernatant 100 in a cell concentrator with
appropriate cutoff lter.
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Protein extraction buffer
50 mM TrisCl, pH 7.5 (APPENDIX 2A)
5 mM EDTA
0.6% SDS
10 mM NaPO
4
1.1 mg/liter pepstatin (Roche, cat. no. 10253286)
Add 50proteinase inhibitor cocktail (PIC) to protein extraction buffer (see recipe)
for a nal 1 concentration (i.e., 2 l PIC to 98 l protein extraction buffer).
The protein extraction buffer without PIC can be stored for 1 year at room temper-
ature. The protein extraction buffer with PIC must be used on the same day and
cannot be stored for later use.
Analysis of
M. tuberculosis
Proteins
10A.4.4
Supplement 6 Current Protocols in Microbiology
Proteinase inhibitor cocktail (PIC), 50
Dissolve one Complete Proteinase Inhibitor Cocktail tablet (Roche cat. no.
11836145001) in 1 ml dH
2
O. Store up to 3 months at 20

C.
COMMENTARY
Background Information
Tuberculosis (TB) is a disease caused by M.
tuberculosis. M. tuberculosis is spread via the
aerosol route and predominately causes dis-
ease in lung tissue, although it can spread to
other organs. It is estimated that nearly one-
third of the worlds population is infected with
TB, many of those are cases of latent infection.
Upon waning of the immune system through
aging or infection with HIV, TB can reacti-
vate from its latent form, even after decades of
quiescence. Antibiotic therapy is available for
the treatment of TB, although due to the slow-
growth of the organism, long courses of treat-
ment (6 months to 1 year) are recommended.
Critical Parameters and
Troubleshooting
One of the most important aspects of work-
ing with M. tuberculosis is to minimize any
possible aerosol exposure. Second to impor-
tant safety considerations, scrupulous atten-
tion to detail in preparing mediumand reagents
is crucial. Given the long doubling time (18 to
20 hr, longer for some mutant strains) of the
organism, care should be taken to minimize
Figure 10A.4.1 SDS-PAGE gel. Typical distribution of M. tuberculosis proteins from culture
ltrates and whole-cell lysates. Cells were grown to an OD
600
of 0.4 in Sauton medium without
detergent. Proteins were extracted following Basic Protocols 1 and 2.
any possible sources of contamination of
medium.
Extraction of proteins
Contaminating albumin from the medium
is one of the major obstacles to recovering
protein samples. If albumin contamination is a
problem, increase the number of washes with
PBS. Furthermore, the time and speed that the
samples are processed in step 8 can be ad-
justed. If extended times are used, a 5-min rest
period with incubation on ice is recommended.
Preparation of culture ltrates
Care must be taken to use a non-albumin-
containing medium such as Sauton. The bulk
of the cells will grow as a sticky lawn on the
surface of the medium. For many applications,
it is important to compare the proteins in the
cell bulk to the proteins in the culture ltrate.
For this reason, as much of the cells should be
recovered and saved for potential analysis.
Anticipated Results
Extraction of proteins
Yield can be altered by increasing the
amount of starting culture and/or adjusting the
Actinobacteria
(High G+C
Gram Positive)
10A.4.5
Current Protocols in Microbiology Supplement 6
conditions for cell lysis. A Bradford assay is
useful for determining the protein concentra-
tion for each sample.
Figure 10A.4.1 shows a protein gel of cul-
ture ltrate proteins isolated from M. tubercu-
losis grown on either Middlebrook or Sauton
medium.
Time Considerations
Extraction of proteins
The protocol takes 2 hr.
Preparation of culture ltrates
Centrifugation and ltering of the culture
ltrate take <1 hr. Depending on the cutoff
lter used, application of the supernatant to
the cell concentrator can take 2 to 8 hr.
Acknowledgments
Special thanks to Kieran Bhatt, Bing Chen,
Zhiyan (Annie) Dai, Dee Dao, Robert Glover,
Morad Hassani, Tsugunda (Ko) Hsu, Jordan
Kriakov, Sunhee Lee, Catherine Vilcheze (also
see UNIT 10A.2), Brian Weinrick, and Torin
Weisbrod for contributions and helpful discus-
sions.
Literature Cited
Schwebach, J.R., Jacobs, W.R. Jr., and Casadevall,
A. 2001. Sterilization of Mycobacterium tuber-
culosis Erdman samples by antimicrobial xa-
tion in a Biosafety Level 3 laboratory. J. Clin.
Microbiol. 39:769-771.
Braunstein, M., Espinosa, B.J., Chan, J., Belisle,
J.T., and Jacobs, W.R. Jr. 2003. SecA2 functions
in the secretion of superoxide dismutase A and
in the virulence of Mycobacterium tuberculosis.
Mol. Microbiol. 48:453-464.
Kriakov, J., Lee, S., and Jacobs, W.R. Jr. 2003. Iden-
tication of a regulated alkaline phosphatase,
a cell surfaceassociated lipoprotein, in My-
cobacterium smegmatis. J. Bacteriol. 185:4983-
4991.