Exercise #2 -
Induction of Stress Protein Synthesis in E. coli.
Author: Lee
Weber, Biology Dept, University of Nevada, Reno
Minor modifications by Grant Mastick
[Return
to BIO 395 Schedule]
I. Overview
The purpose of this laboratory is to examine the effects of physiological stress on the pattern of proteins synthesized by cells. In this exercise, you will look at the effect of heat shock and other forms of physiological stress on protein accumulation in the bacterium E. coli. Cellular proteins will be fractionated according to molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spectrum of proteins contained in the cells before and after the period of stress will be visualized by staining the gel with Commassie blue dye. The molecular weight ratio (MWr) of each polypeptide induced by heat or other type of stress will then be determined by comparing the electrophoretic mobilities of the induced polypeptides with those of marker proteins of known MWr. Western immunoblotting, a method that allows detection and quantification of a know protein antigen, will then be used to determine if the stress treatment causes induction of a specific E. coli protein know as DNAK.
The experiments that you will be performing can potentially generate new information about the induction of stress proteins. Although heat shock is known to induce a specific set of stress proteins known as heat shock proteins, the effect of the type of stress that you decide to impose on the cells will not be known until we see your results.
II. Background
A. Stress Proteins:
Most cell types from all known organisms respond to thermal stress by synthesizing
a small number of evolutionarily conserved polypeptides that have come to be
known as heat shock proteins. It has recently been shown that many other kinds
of physiological stress besides heat also induce synthesis of these same
polypeptides. Thus, heat shock proteins belong to a larger class of proteins
called stress proteins that are induced by a wide variety of physiological
insults. Some conditions that are known to induce heat shock protein synthesis
at normal temperatures include drugs, amino acid analogs, ethanol, heavy
metals, ionizing radiation, viral infection and neoplastic transformation. The
metabolic signal that coordinately regulates expression of the same family of
genes in response to different type of stress is not completely understood at
this time. However, most treatments that induce stress protein synthesis cause
an increase in protein denaturation. At least two of the stress proteins (HSP60
and HSP70) function as molecular chaperons that facilitate correct folding of
newly synthesized polypeptides during normal cell growth. Thus, protein
denaturation is thought to be part of the signaling mechanism that up-regulates
the genes encoding the heat shock proteins. Although the precise function of
all of the stress proteins is not known, cells subjected to a mild heat shock
become increasingly resistant to thermal killing as they accumulate the heat
shock proteins. Interestingly, the heat shocked cells also become resistant to
killing by the other stresses mentioned above. The heat shock proteins are
therefore believed to be part of a general adaptation mechanism that functions
at the cellular level. The heat shock proteins are now often called stress
proteins (SP) or stress regulated proteins (SRP) in the literature.
B. Growth of E. coli and Stress Protein Induction:
The bacterium Escherichia coli is the best studied genetic organism to date. E. coli is a mammalian gut symbiont which is unique in its ability to ferment lactose, the mammalian milk sugar. Early workers selected this microorganism for genetic studies because it is very easy to grow in the laboratory and because of an assumption (now known to be incorrect) that its genetic mechanisms co-evolved along with those of mammals. E. coli are grown in the laboratory either as colonies on agar plates containing nutrient medium or in suspension culture in liquid media. In this laboratory exercise, you will work with liquid suspension cultures that have been inoculated with a few cells from an individual colony. E. coli grow very rapidly in rich media, doubling in number about every 20-30 min at 37oC. In order to enhance stress protein induction, you will work with cells growing at 30oC, where the doubling time should be closer to 45 min. At any temperature, bacteria will increase in number until they either deplete the nutrients in the medium or accumulate sufficient waste products to inhibit growth. Under these conditions of saturation, the cells undergo a physiological change that allows survival in the absence of growth. This state is called "stationary phase", where the number of cells in a culture increases very slowly if at all. When stationary phase cells are diluted into fresh medium, they undergo another physiological change and begin to divide at an exponential rate. This is known as the logarithmic phase or log phase. Most physiological experiments are conducted using rapidly dividing log phase cultures. Cell growth can be measured by following the increase in absorption of a culture at 600 nm using a spectrophotometer. A culture with an absorbance of 1.0 at 600 nm contains approximately 2 X 108 cells/ml.
In your lab exercise, you will start with cell cultures growing
exponentially at 30oC. When the cultures reach mid log phase, you
will stress the cells by either shifting the temperature to 42oC or
by adding a stressing agent. You will then harvest samples of each culture,
collect the cells, and then analyze the proteins by SDS gel electrophoresis.
C. Protein Molecular Mass Estimation by SDS-PAGE:
The unit of molecular mass is the dalton, which is the mass of hydrogen atom. The molecular mass of a protein that does not contain covalent modifications is the sum of the atomic weights of all the constituent atoms. SDS polyacrylamide gel electrophoresis (SDS-PAGE) is the most commonly used method of estimating the molecular mass of protein subunits. SDS is a powerful detergent and protein denaturant. It is an amphipathic molecule composed of hydrophobic alkyl chain attached to a negatively charged sulfate group. SDS disrupts membranes by binding to lipids and membrane proteins via the hydrophobic region and the highly polar sulfate groups makes the complexes soluble in aqueous solutions. SDS binds tightly to nearly all proteins and as long as excess SDS is present, produces rod shaped protein-SDS complexes with consistent linear charge densities. The longer the amino acid chain, the more SDS that will bind. The sulfate groups of the SDS molecules impart a negative charge to the complex and thus effectively eliminate charge differences between different kinds of protein molecules. Thus, protein-SDS complexes migrate toward the anode (+ pole) during electrophoresis. In a sieving matrix such as a polyacrylamide gel at a constant current flux, the rate of movement of an individual protein toward the anode is inversely proportional to the logarithm of its molecular mass. This is because the degree of retardation by the gel matrix is inversely related to the radius of the migrating molecule, which is a log function of the length of the molecule. Larger molecules fictionally interact with the gel matrix to a greater extent than smaller molecules and thus the larger molecule migrate slower.
In practice, the sieving effect of polyacrylamide gels can be varied by changing the concentration of acrylamide monomer and bisacrylamide crosslinker. Gels can be constructed with acrylamide concentrations between 2.5 and 20%. A gel with a given concentration of acrylamide is capable of resolving (separating) proteins only over a range of molecular masses. Proteins too large to enter the gel pores do not enter the gel. Proteins below a size determined by the gel concentration all migrate at the same rate as the ion front (usually determined by using a tracking dye) and are not resolved. By selecting appropriate gel concentrations, the molecular mass ratio (Mr) of polypeptides with molecular masses of 1000 to 500,000 can be determined using marker proteins with know molecular masses. Mr is usually a fairly accurate estimate of the molecular mass of most proteins (within a range of about 10% error). Notable exceptions include glycoproteins, which migrate much slower during SDS-PAGE that predicted by their molecular mass, and highly basic proteins such as histone, which also migrate more slowly than would be predicted from molecular mass.
Following SDS-PAGE, the distribution of the major proteins can be detected by staining the gel for protein. Several different stains are used to detect proteins in gels. The method you will employ uses Coomassie Blue, the same dye used the Bradford protein assay. Staining proteins in a gel gives information about the mass of proteins that are present in the cells but tell nothing about the types of proteins that are actually being synthesized during the labeling period. Most of the most abundant cellular proteins are very stable and the overall protein composition of the cell changes very little during a 1 hr period even if dramatic changes in protein synthesis have taken place.
D. Immunoblotting:
Immunoblotting, also known as Western blotting, combines the resolution of polyacrylamide gel electrophoresis with the specificity of immunological detection. Immunoblotting can be used to determine the presence and quantity of a specific protein antigen. In addition, the relative molecular weight of the protein can also be measured. A major advantage of Western blotting over other immunological detection systems is that interactions between an antibody preparation and antigens other than the one of interest can be detected and this nonspecific background can be discounted if necessary.
Specific antibodies are raised in animals such as rabbits, mice, and goats by injecting large doses of the purified molecule of interest. The animals respond by producing antibodies that bind very tightly to short regions of the antigen known as epitopes. In the case of proteins, antibodies recognized short regions of the polypeptide backbone, typically about 7 amino acids in length. Thus, an animal injected with a large protein can produce multiple different antibodies that recognize many different regions of that protein. Such antibody preparations are called polyclonal since each antibody producing cell can produce only a single antibody molecule specific for 1 epitope. It is possible to isolate or clone a single antibody producing cell and to develop a stable cell line that produces a single type of antibody molecule. Such antibodies are called monoclonal since they are produced by a single clone of cells. Polyclonal antibodies have the advantage of increased sensitivity, since many different antibody molecules can bind to a single protein antigen molecule. Monoclonal antibodies usually have the advantage of increased specificity, since they are less likely to react with other proteins than are polyclonal antibodies. Antibodies have become essential tools for identifying and quantifying specific proteins in research and diagnostic laboratories.
The immunoblotting procedure can be divided into several steps. First, an unlabeled protein solution, often a whole cell or tissue lysate, is prepared in electrophoresis sample buffer. This buffer can contain protein denaturing reagents such as SDS or urea. Thus, even proteins that are insoluble in standard aqueous solutions can be studied. Next, the proteins are separated by gel electrophoresis and transferred to a membrane that binds proteins nonspecifically. The remaining protein binding sites on the membrane are then blocked by reaction with excess inexpensive protein such as casein or bovine serum albumin. Finally, the location of the target antigen on the membrane is determined using either a labeled primary antibody or an unlabeled primary antibody followed by a labeled secondary antibody.
In this exercise you will use an unlabeled mouse
anti-DNAK antibody to bind a specific heat shock protein on the Western blot
membrane. Binding of this primary antibody will then be detected by reacting
with goat anti-mouse immunoglobulin that has been conjugated with alkaline
phosphatase. The location and amount of DNAK-primary antibody-secondary
antibody complex on the membrane filter will be measured by allowing the
alkaline phosphatase to react with bromochloroindolyl phosphate (BCIP) and
nitro blue tetrazolium (NBI). Alkaline phosphatase cleaves the phosphate group
from BCIP and the resulting indol product is rapidly oxidized by NBT. The
reduced NBT precipitates, producing a dark blue-grey stain. The intensity of this
stain at a protein band of approximately 70,000 MW indicates the amount of
total DNAK in the preparation. If
successful, the Western blot analysis will allow you to determine if the stress
treatment that you are studying will specifically induce accumulation of the
DNAK stress protein.
III. Procedure:
Week #1
A. Set Up of E. coli Cultures:
1. Obtain 2 cultures of E. coli from your instructor. A stationary culture of cells was diluted 1:50 in broth and allowed to grow at 30oC in the shaker incubator.
2. Measure the absorbance of one of the cultures until A600=0.45 (mid log phase) is reached.
3. Take a 1 ml aliquot of each culture as the unstressed control sample and hold on ice.
4. Transfer one culture to a shaker waterbath at 42oC. Take 3-1 ml aliquots after 5, 10, and 20 min of incubation and hold on ice in 1.5 ml microcentrifuge tubes.
5. Add a stressing agent of your choice to the second culture. Be creative. Chose any liquid food item that you might think could contain a toxic compound. The instructor will provide you with additional choices. Take 3-1 ml samples after 10, 20, and 30 min of incubation and hold on ice.
6. Collect the cells by centrifugation for 30 sec at full speed in the microcentrifuge. Discard the supernatants as directed by your instructor. Suspend each cell pellet in 100 ul of 1X sample buffer (Laemmeli) containing 0.5 mM PMSF (a proteinase inhibitor).
7. Boil your samples for 7 min. They are now ready for electrophoresis.
B. Analysis of Proteins Accumulated During Stress Using SDS-PAGE:
The most widely used system for analysis of proteins is the method
originally described by Laemmli in 1970. It is a discontinuous buffer system
for resolving proteins denatured with SDS. You can read about this technique in
the reference material provided in the lab. Your lab instructor will explain
how leading and trailing ion fronts are used to "stack" protein bands
in this system. Your instructor will prepare 10% polyacrylamide Laemmli gels in
advance of the lab period.
1. Underlay your samples in the wells as directed by your instructor. Be careful not to contaminate adjacent wells with each sample.
2. Load at least one well of each gel with a mixture of proteins of known MW to serve as markers.
3. Connect the electrodes to the power supply and carry out electrophoresis at 100V until the tracking dye reaches the bottom of the gel.
4. Turn off the power supply. Disconnect the leads and remove the lid of the electrophoresis cell. Pour out the buffer into a waste beaker by inverting the entire unit over the sink.
5. Disassemble the unit as directed by your instructor. Remove the side clamps and lift the gel sandwich out of the buffer chamber pod.
6. Use an extra spacer to pry the gel sandwich from the bottom to avoid breaking glass plates.
7. Remove the spacers and peel the gel off the plate and place it into a tray of fixative/stain solution containing 0.125% Coomassie blue, 50% methanol, 10% acetic acid. Allow the gel to stain for at least one hour or overnight.
8. Replace the fixative/stain with destain solution I (50% methanol, 10% acetic acid). Destain for 1 hr or overnight.
9. Your instructor will dry your gels onto a sheet of 3MM filter paper. [End Week 1]
C. Analysis of Gel Data:
Estimate the MWr of the stress-induced polypeptides by using a standard curve prepared by plotting the distance that each marker protein has migrated in the gel vs the log of each MW (the MW information will be provided by the instructor). Use semilog paper for this, or the Excel program. Plot the distance that each stress induced polypeptide has migrated on the same graph. Calculate the MWr of the induced proteins relative to the standards. Are any stress proteins induced that are different from the heat shock proteins? How could you demonstrate that a stress induced protein is or is not a heat shock polypeptide?
Week #2
A. Gel Electrophoresis for Immunoblotting:
1 . Run 20 ul of each cell sample on a 10% SDS-polyacrylamide gel as in the earlier lab exercises (~45 min @ 50 mA pers gel). Use prestained molecular weight standards.
2. Presoak 16 sheets of precut 3MM filter paper and 1 nitrocellulose filter per gel in transfer buffer (39 mM glycine, 98 mM Tris, pH 8.8, 0.0375% SDS, and 20% methanol) for at least 2 min.
3. Wet the electrodes of the semi-dry gel transfer apparatus under tap water for at least 15'.
4. Put lower electrode in place and stack 8 sheets of 3MM paper and the nitrocellulose filter. Add transfer buffer to the nitrocellulose filter until there is a pool of buffer on top of the filter.
5. Remove the gel from the electrophoresis apparatus, discard the stacking gel, and soak in transfer buffer for at least 1 min.
6. Orient the gel on top of the nitrocellulose filter. Layer 8 pieces of 3MM paper on top of the gel.
7. Put upper electrode into place, attach the lid, and electroblot for 30 min @ 70 mA per gel.
8. Terminate electrophoresis and discard the gel. The molecular weight standards should be visible on the nitrocellulose filter. If necessary, rinse the filter with transfer buffer to remove fragments of gel. Place filter in a ziplock bag, seal, label with the names of your lab group, and store in fridge. [END WEEK 2]
Week #3
A. Gel Electrophoresis for Immunoblotting:
[Steps 1, 2, and 3 may be done by the instructor the night before class].
1. Place filter (protein side toward center) into a 50 ml tube. Fill tube with blocking buffer (TBS + 0.5% nonfat dry milk) and incubate for 1 hr on the rock&roller apparatus. TBS = Tris buffered saline (0.1 M NaCl, 0.05 M Tris, pH 7.5).
2. Dilute antiserum 1000-fold in TBS containing 0.2% powdered milk and 0.01% sodium azide.
3. Incubate the filter overnight with 2 ml of diluted antiserum in the rock&roller apparatus.
4. The following day, pour off the antiserum and rinse briefly with TBS + 0.5% milk. Rinse 2 X 15 min with 45 ml of TBS + 0.5% milk.
5. Add the second antibody diluted into 2 ml of TBS + 0.5% milk. Incubate in the rock&roller for 1 hr. The second antibody is goat anti-mouset immunoglobulin that is conjugated with alkaline phosphatase.
6. Pour off the antiserum and rinse briefly with TBS + 0.5% milk. Rinse 2 X 15 min with 45 ml of TBS + 0.5% milk.
7. Rinse 2 additional times with TBS without milk.
8. Transfer the filter to a pipet tip box top containing the following solution freshly prepared:
10 ml developing buffer (0.1 M Tris, pH 9, 0.1 M NaCl, 0.005 M MgCl2) 33 ul BCIP
66 ul NBT
9. Allow color to develop until the darkest band is dark grey, not black. Stop the reaction by diluting in tap water. Store the filter in the dark.
D. Data Analysis
Using semilog paper, determine the molecular weight of the major protein band stained by the anti-DNAK antibody in the Western blot. Use the values for the molecular weight standards provided for you by the instructor. Did your stress treatment induce DNAK? Did your stress treatment induce DNAK as strongly as did the heat treatment?
REFERENCES
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D. (1989) The Molecular Biology of the Cell. pp. 169-172. Garland, New York.
Harlow, E. and Lane, D., Antibodies, A laboratory manual, Cold Spring Harbor Laboratory, 1988. pp. 361-411.
Welch, W.J. (1993) How cells respond to stress. Sci. Am. 268:56-64.