1.14: Column Chromatography

Learning Objectives

Goals:

• Explain the conditions for bacterial growth and relate it to the goal of collecting protein.
• Explain what is meant by protein folding.
• Describe the relationship between a protein’s conformation (three-dimensional shape) and function of the protein.
• Use column chromatography to separate proteins.

Student Learning Outcomes:

Upon completion of this lab, students will be able to:

• Lyse bacterial cells to release protein.
• Purify GFP via hydrophobic interaction column.
• Identify the location of GFP throughout the purification process.

Introduction

Proteins are biological molecules made of chains of amino acids that take on a three-dimensional shape called a conformation. A specific conformation is achieved by folding of the amino acid chain in a way that the protein can perform a specific job or function for the cell. If the protein does not fold in the specific way, it cannot do the job it otherwise would. Proteins can be purified and used as therapeutic agents (drugs) to treat patients with specific conditions. Because purifying specific human proteins can be difficult or impossible from human or animal tissues, we can employ cells such as bacteria like E. coli to do the job. These cells are genetically engineered to make the proteins of interest in large amounts that are more easily purified.

An example of a therapeutic protein is insulin, used to treat diabetes. This was the first genetically engineered protein to be introduced as a therapeutic agent. The gene for human insulin was engineered into a plasmid and this recombinant plasmid was introduced into E.coli. The bacterial cells made insulin which was purified to be used for treating diabetes.

Similarly, green fluorescent protein (gfp) can be produced and purified. Prior to this lab, the gfp gene was introduced by the process of genetic engineering into a recombinant plasmid capable of allowing E. coli to produce protein from the genetic instructions. Producing protein from a gene is termed expression. This recombinant plasmid was introduced into E. coli using the process of transformation. This laboratory uses the process of obtaining purified green fluorescent protein to model how therapeutic proteins are purified for human treatments.

You will be provided a culture of bacteria that have been grown and induced (stimulated) to produce gfp. You will treat the bacteria with a solution that will lyse (break open) the cells to release the protein. Then you will employ the process of column chromatography, the focus of this laboratory. Column chromatography is a method of allowing a solution to flow over a substance that will selectively bind and separate the components of the solution. In this case, tiny beads are packed into a tube-like column and the solution obtained through bacterial lysis is allowed to flow over the beads. As you pass other solutions over the column, you will be able to collect some of the solution flowing through the beads which will contain a much more concentrated and pure gfp.

Growing Bacterial Cells

The pattern of growth for bacterial cells is well understood. In the laboratory setting, the goal is to grow cells and have them produce the protein we are interested in purifying. When using optimal conditions to grow E. coli, four phases of growth will occur (see Figure 1)

Figure 1. Bacterial growth curve outlining change in growth over time

1. During lag phase no cell division occurs. Cells are preparing to divide by making new enzymes and proteins as well as copies of their DNA. Cells enlarge.
2. In the log phase, a doubling of the population is occurring. This is termed logarithmic growth. Cells undergo binary fission (cells divide in half) approximately every 20 minutes for E. coli under optimal conditions. Other types of bacteria, and E. coli under less than ideal conditions, will divide at different rates.
3. The stationary phase occurs after the log phase when there is no overall change in the population size. During this period, cell division is equivalent to cell death and happens as resources such as nutrients and oxygen are depleted, and waste products are building up in the environment.
4. During the decline or death phase of a bacterial culture, cells are dying faster than they replicate. The cell population is decreasing. This occurs as waste builds further and the food supply is exhausted.

CONSIDER: If the gene of interest is controlled by an operon, such as the lac operon, when is the best time to turn on the gene? Keep in mind:

• Production of the protein takes energy away from the processes of cell growth and cell division.
• A greater number of cells will produce more protein
• Proteins can degrade over time

Pre-lab Activity: Think Pair Share - Discuss What You Already Know

Prior to the lab period you should read the material, take some notes on your thoughts on the following questions (Think). In lab, you will be asked to discuss with your partner your thoughts and hear theirs (Pair). Take additional notes on new insights during this small discussion. Finally, you may be asked to discuss your ideas with the class (Share). It is not expected that you know all the answers before the class discussion but by the end of the activity, you should have the questions answered completely and correctly.

1. What is the term for bacterial reproduction? Describe this process.
2. What roles do proteins play within cells?
3. What happens to protein function if a protein loses, or never correctly achieves, the prescribed conformation?
4. How does the order of amino acids relate to protein conformation and thus protein function?
5. Since we can control when to “turn on” or express our gene, when is the best time to do so? To help you decide ask yourself:
   1. Would producing protein take energy?
   2. Would a greater number of cells produce more of your protein of interest than fewer cells?
   3. Can proteins degrade if left too long?
6. Compare your flow charts with each other prior to beginning lab. Note any differences and attempt to resolve which flow chart(s) has/have more accurate information. Adjust your own flow chart as necessary.

Protein Purification

Transformed bacteria can multiply in culture and produce the protein of interest. If this protein is to be used therapeutically, it will need to be purified. This means that other cellular components, including other proteins, must be separated from your protein. Column chromatography is a common method to separate proteins.

Proteins are made of amino acids. Individual amino acids have different properties such as hydrophobicity (water-hating) or hydrophilicity (liking water), ionic charge, or the ability to form weak or strong bonds with other amino acids. When a protein is first made in a cell, it is a long chain of amino acids in an order determined by the gene. The order of the amino acids in a protein determines how the chain will fold to produce a three dimensional protein conformation (See Figure 2). This specific conformation will have different amino acids interacting with each other in specific ways. Amino acids facing the environment in a folded protein can interact with other molecules. Specific groups of amino acids near each other can form binding sites to interact with other specific molecules. Overall, these relationships determine protein structure and thus protein function. Imagine proteins involved in enzymatic reactions, as channels in membranes, in transporting other molecules, or for binding DNA. These proteins all have very specific binding interactions determined by amino acids in specific locations in a folded protein.

Figure 2. The folding of a protein

QUESTION: If individual amino acids are swapped or deleted in an amino acid chain, do you imagine this would affect the function of a protein?

The rules for protein folding are not perfectly understood and is an area of active scientific investigation. However, a few basic rules have been discovered. Factors that cause proteins to fold in specific ways include:

1. Weak bonds will form between amino acids with a negative and a positive charge.
2. Strong (covalent) bonds will form between sulfur-containing amino acids. These are called disulfide bridges.
3. Hydrophilic amino acids locate to the outer surfaces of proteins because they interact with the cell environment, which is mostly water. Hydrophobic amino acids hide on the inside of proteins or embed within cell membranes to avoid contact with the water in the environment.

Figure 3. Liquid column chromatography

Depending on the content of amino acids in a specific protein, overall it will take on a hydrophobic or hydrophilic character. Column chromatography can separate hydrophilic and hydrophobic proteins from the rest of the cell contents. Small beads coated with a material called a resin are packed into a column. The resin attracts proteins which will bind to the resin as other cell contents flow past. For hydrophobic proteins to stick, they must be treated to expose the typically buried hydrophobic amino acids. Buffer solutions can be used to cause proteins to denature (unfold) and expose the amino acids that will be attracted to the resin.

Different buffers are passed over the column in an order determined to best separate the proteins of interest from the rest of the cell contents. Figure 14.4 shows three solutions used to separate green fluorescent protein from the rest of the cell. The binding buffer denatures proteins so that the hydrophobic amino acids stick to the resin. The wash buffer removes loosely adherent proteins and material from the column leaving the more strongly attached protein of interest. Finally, the elution buffer, which has a low buffer concentration, causes the protein to begin to refold to hide the hydrophobic amino acids which releases the protein from the resin coated beads in the column. The portion or fraction of fluid exiting the column that contains your protein can be captured in a container and saved.

Figure 4. Separation of green fluorescent protein by hydrophobicity using column chromatography

QUESTION: Do you believe that all types of protein would use the same types of resin-coated beads and the same types of buffers to become purified? Explain your answer.

Part I: Lysis of Bacterial Cells

Previously, bacteria were transformed with a recombinant plasmid capable of expressing gfp when cells were induced. The reason the cells can be induced to produce protein is that within the plasmid, in front of the gene for gfp, there is a special sequence of DNA that will respond if a chemical is placed in the media. This chemical is called an inducer (ind) and signals that the gene should be “turned on” and messenger RNA should be transcribed from the DNA instructions and the protein should be produced. The plasmid also contains the selectable ampicillin (amp) resistance gene to ensure that the cells growing in your culture are cells that contain your plasmid.

Prior to this lab period, cells were grown in the presence of ampicillin until late in log phase. The cells in culture divide and each cell contains many copies of the plasmid. At late log phase, the chemical inducer was added to the medium to turn on the gfp gene and the cells were allowed to continue to grow and produce gfp.

First, you will collect your cells and break them open. This process is called cell lysis. After the cells are lysed, you will use column chromatography to purify gfp.

MATERIALS

Reagents

• Microfuge tube rack with the following tubes:
  • LB/amp/ind culture of E. coli cells (EC)
  • Elution buffer (EB)
  • Lysis buffer (LyB)
• Extra 1mL of the EC culture – from the instructor

Equipment and Supplies

• P-200 micropipette
• Pipette tip box
• Permanent marker
• Microcentrifuge (shared with class)
• Vortex mixer (shared with class)
• Long wave UV light
• Liquid waste container
• Sharps container
• Biohazard bag for materials that come into contact with E. coli cells (shared with class)

Safety Reminders

Appropriate safety precautions should be used at all times. These will be reviewed by your instructor and can be found in the beginning of your laboratory manual which you should refer to before you begin this procedure. Aseptic technique is required when handling E.coli and materials that have come in contact with the bacterial culture. Remember that aseptic technique are the procedures used to protect your culture and samples from contamination but also protect you.

1. Disinfect your work area and wash your hands before beginning an experiment.
2. Never touch anything that has come in contact with the E.coli. This includes pipettes, spreaders, and the interior of tubes. Pipet tips should never touch anything except the material to be transferred. Spreaders and pipettes should only be handled from the end that will not touch bacteria.
3. When handling petri dishes, only open the lid enough to work with the agar surface and then close the lid immediately. This will avoid contamination, such as fungal spores from the air, landing on your agar plate.
4. If something becomes accidentally contaminated, speak to your instructor to inquire if a replacement is appropriate and available.
5. Avoid spills. If one occurs, notify your instructor immediately for help in cleaning it appropriately.
6. Contaminated waste such as used microfuge tubes and cell spreaders will be placed in the biohazard bag. Pipet tips will be placed in a sharps container.
7. Only when directed to do so will you dispose of your used petri dishes in the biohazardous waste.
8. Be sure to clean your work area and wash your hands before exiting the lab.

Procedures

1. Take a long wave UV light and look at the EC tube, record your observations.
2. Weigh your EC tube. Look for another tube with a similar weight; +/- 0.1g or create a balance tube for the microcentrifuge.
3. Spin the EC tube for 5 minutes at 13,000 rpm (or as high speed as possible) in a microcentrifuge. Make sure to balance the tubes correctly.
4. Very carefully take out the EC tube from the microcentrifuge. Avoid disturbing the cell pellet at the bottom of the tube.
5. Take the P-200 micropipette, set it to 200.0 µL and get a tip. Press to the first stop before going into the supernatant (liquid layer) and gently pull out the old liquid growth media. Do not disturb the cell pellet when doing so.
6. Discard the liquid into the liquid waste container, and the tip in sharps container.
7. Bring your cell pellet (the EC tube) to your instructor to dispense 1 ml of the same culture into your tube.
8. Repeat steps 2-6, so spin down the cells for 5 min again and remove the supernatant. Record the color of the supernatant and pellet at this step.
9. Take the P-200 micropipette and a new tip and carefully try to fully remove all the liquid from the pellet without taking up the cells. Discard the tip in sharps.
10. Set the P-200 to 150.0 µL and get a new tip. Add 150 µL of elution buffer (EB) to the EC tube. Discard the tip.
11. Firmly close the EC tube and resuspend the cell pellet with a vortexer. If one is unavailable, drag the tube quickly across an empty microfuge tube rack. This should cause the cell pellet to dislodge from the bottom of the tube and the buffer should become turbid. Repeat this movement until the entire pellet is gone.
12. Take the P-200 and get a new tip. Add 150 µL of lysis buffer (LyB) to the EC tube. Mix the tube contents with a vortexer or the microfuge tube rack method like previously.
13. The EC tube will incubate in the lysis buffer overnight at room temperature. Label your tube with class period and group number and give to your instructor to do this step.
14. Clean your work area and discard all contaminated tubes and tips into the appropriate biohazardous waste.

Part II: Using Column Chromatography to Separate the Green Fluorescent Protein

Materials

Reagents

• Microfuge tube rack with the EC tube that contains cells, elution buffer, and lysis buffer
• Bottles of:
• Binding buffer (BB) = 4.0 M Ammonium sulfate solution
• Equilibration buffer (EQ) = 2.0 M Ammonium sulfate solution
• Wash buffer (WB) = 1.3 M Ammonium sulfate solution
• Elution buffer (EB) = 10 mM Tris, 1 mM EDTA, pH 8.0 solution
• 20% Ethanol solution (For cleaning and storing resin in columns at the end of lab)

Equipment and Supplies

• P-1000 micropipette
• Pipette tip box
• 2-3, 1.5 mL microfuge tubes
• Chromatography column
• Microcentrifuge (shared)
• Liquid waste container
• Biohazardous waste container
• Sharps container

Procedure

1. Divide the work by assigning one person to do step 2-3, another 4-5, and a third do 6-7.
2. Verify that you have all the necessary materials.
3. Label one microfuge tube as “SUPER” and another as “GFP”.
4. Set up your column as directed, always maintain it in an upright position. Do not ever allow the column resin to go completely dry.
5. To set up the column:

• Take off the caps at the top and bottom of the column. Do not confuse the bottom cap with the stopcock.
• Place the column liquid waste container at the base of the column.
• Turn the stopcock valve to a vertical position and drain the column until 1-2mm of liquid remains above the column resin. Turn the stopcock to a horizontal position to close the valve.
• Take the P-1000 micropipette, attach a tip, and set to 1000 µL. Add 1000 µL of equilibration buffer (EQ) gently down the side of the column, trying to disturb the resin bed as little as possible.
• Drain the column until 1-2mm of liquid remains above the column resin then close the valve.
• Using the same tip, add another 1000 µL of equilibration buffer (EQ) gently down the side of the column, trying to disturb the resin bed as little as possible. Discard the tip afterwards.
• Drain the column until 1-2mm of liquid remains above the column resin then close the valve.
• Double check that the liquid stopped draining when the valve is closed.

6. Weigh the EC tube, find a balance tube, and spin the tube down for 5 min at 13,000 rpm (or the highest speed) in a microcentrifuge. Be sure to balance the microcentrifuge.
7. Carefully remove the tube and bring it back to your workspace. Record your observations when using a long wave UV light to examine the tube. What do you observe regarding the pellet and supernatant?
8. Take the P-1000 micropipette, set to 250 µL and get a tip. Very carefully, try to remove as much of the supernatant and transfer into the labeled “SUPER” tube. If the pellet is disturbed during this step, spin down the tube again and repeat this step. Discard the empty tip in sharps.
9. Get a new tip and add 250 µL of binding buffer (BB) to the SUPER tube. Gently pipette up and down two times to mix the solution. There should be approximately 500 µL of liquid total now.
10. With the same tip, add 200 µL of the SUPER tube solution to the column two times. The entire contents of the SUPER tube should be added to the column. Slowly drip the solution down the sides of the column, so the resin bed is disturbed as little as possible. Discard the tip in sharps.
11. Turn the stopcock valve and drain the liquid from the column until there is 1-2 mm of liquid above the resin.
12. Use the long wave UV light to examine the column and record your results. Where is the green fluorescent protein located in this step?
13. Take the P-1000 micropipette and set it to 1000 µL and get a new tip. Add 1000 µL of wash buffer (WB) gently down the side of the column, again trying to disturb the resin bed as little as possible. Discard the tip in sharps.
14. Drain the wash buffer until there is 1-2 mm of liquid above the resin.
15. Use the long wave UV light to examine the column and record your results. Where is the green fluorescent protein located in this step?
16. With a new tip for the P-1000 pipette, add 1000 µL of elution buffer two times to the column (a total of 2 mL of elution buffer). Slowly add the buffer down the sides of the column. Discard the tip in sharps.
17. Hold your “GFP” tube under the column stopcock. Another person should shine the UV light on the column so GFP can be located. Open the stopcock and collect the GFP into the GFP tube. OPTIONAL: For this step, if you have an extra microfuge tube, you can collect the fainter fluorescent liquid in one and the stronger glow in another to concentrate the GFP.
18. Once the GFP is mostly collected, drain the column into the waste container until 1-2 mm of liquid is above the resin.
19. With a new tip, add 1000 µL of storage solution- 20% ethanol three times down the side of the column.
20. Drain the column until there is 1 cm of liquid above the resin bed.
21. Put the caps back onto the top and bottom of the column for storage.
22. Dispose of the column flow through waste container by pouring the contents down the drain in the sink.

Data Analysis

While under the UV light, compare your GFP tube with GFP tubes from other groups. Record any observed differences in the color intensity in your lab book.

Study Questions

1. What characteristics of amino acids are important for protein conformation?
2. How is protein conformation related to protein function?
3. Did you notice if your lysed cell solution looked more or less bright than the fraction you collected from the column? If you observed a difference, why might that be the case?
4. Why are you able to use the column to separate your green fluorescent protein from the other cellular components?
5. For this procedure, what could be adjusted to increase purity of your protein in the sample?