A4.1.3.ProteinChromotography
Activity 4.1.3 Protein PurificationIntroductionThanks to you (and DNA derived from a jellyfish), tiny bacteria now glow a brilliant green under UV light. You transferred a gene from one organism to another and you now see, in living color, the relationship between DNA and a trait. But your work is not complete. You now have a source of GFP, but the protein is trapped inside of the cells. Once scientists have used a bacterial cell to produce a protein, such as insulin or GFP, how can they extract and isolate the protein so it can be used? Bacteria produce thousands of different proteins. Scientists must have a way of sifting through the mix and finding the “needle in the haystack.” As you continue Lesson 4.1, your job is to separate the GFP from the other bacterial proteins, verify its purity, and get it out to market. Before protein products can be released as pharmaceuticals, enzymes, or diagnostic tools, they must be isolated and purified in a series of laboratory procedures. Chromatography is one technique used to separate and purify components in a mixture of gases, liquids, or dissolved solids. In all chromatography methods, components are separated using a stationary phase and a mobile phase. The stationary phase is the medium that does not move; the mobile phase is the medium that does. A mixture is dissolved in the mobile phase, which may be gas or liquid, and the sample is then passed over a stationary phase, a filter such as paper, silica on a glass plate, or porous beads in a column. Just as a filter catches some materials and lets others slide through, the stationary phase either traps or releases items in the mixture based on their chemical and physical properties. This filter may separate out components in the mixture by size, by charge or by their affinity for the filter itself. Specific molecules, such as proteins, can be isolated as they are separated by chromatography. Once your bacteria have multiplied in liquid culture, you will perform a specific type of chromatography called hydrophobic interaction chromatography (HIC) to separate the GFP from the other proteins in the mixture. Proteins have a unique structure based on the sequence of their amino acids. GFP is a highly hydrophobic molecule because of its many water-fearing amino acids. Using a column filled with porous beads and a series of buffer solutions, the hydrophobic GFP proteins can be trapped in the beads of the hydrophobic column and then washed out and collected. Follow the glowing drops and isolate our protein of interest!Equipment Bio-Rad Green Fluorescent Protein (GFP) Purification KitDisposable pipetsInoculation loopsHIC chromatography columnsLB broth with ampicillin and arabinoseLysozymeTE bufferBinding bufferColumn equilibrium bufferCollection tubesMicrocentrifuge tubes – 2ml Computer with Internet accessLaboratory journal3D Molecular Designs – Amino Acid Starter KitTransformed bacteria on agar plates from Activity 4.1.2Activity 4.1.3 Student Response SheetGFP Purification – Quick Guide handoutMicrocentrifugeMicrocentrifuge tube rackHandheld UV lightFreezerRing stand (optional)Shaking incubator (optional)ProcedurePart I: Review of Protein StructureWith a partner, brainstorm what you remember about protein structure. Think back to the Designer Proteins activity you completed in PBS. Remember that a change in just one amino acid of a protein can lead to an illness such as sickle cell disease. Take notes in your laboratory journal. Research the structure of an amino acid. Draw a generic amino acid in your laboratory notebook and label each functional group. Obtain an Amino Acid Starter Kit from your teacher.Remove the magnetic chemical properties circle, the amino acid chart, and the magnetic amino acid molecules from the kit. Place each amino acid on the magnetic chemical properties circle according to its chemical structure. Note that the different amino acid sidechains have been colored to reflect their chemical properties. Describe the distinguishing features of each category of amino acid side chain in the space below the category. What do you notice about the molecules that make up this type of side chain?Hydrophobic (nonpolar) side chains are YELLOW.Hydrophilic (polar) side chains are WHITE.Acidic side chains are RED.Basic side chains are BLUE.Cysteine side chains are GREEN.Consult the amino acid chart to match each magnetic shape with the name of the amino acid and to position it correctly on the magnetic circle.Unroll a foam toober. This tube will represent the backbone of your protein.Place a blue end cap on the N-terminus (the beginning) of the protein, and a red cap on the C-terminus (the end) of the protein.Evenly space 15 magnetic clips along the toober. Randomly add the following mixture of amino acids by clipping the magnets on the amino acids to the magnets on the toober.6 hydrophobic amino acids2 acidic amino acids2 basic amino acids2 amino acids containing cysteine3 hydrophilic amino acids.As a class, discuss how each of these amino acids will interact with one another and come up with a list of “rules” for protein folding. Write these rules in your laboratory journal. Fold your protein according to the rules described in your list.Trade completed proteins with another group in the class. Analyze the new protein to make sure that the amino acids are oriented in a way that maintains the rules of protein folding. Provide feedback to the other group, but do not refold the protein. Allow the group to make changes (if necessary) based on your observations. Answer Conclusion questions 1-3. Part II: Protein Purification by Column ChromatographyGFP is composed of many hydrophobic amino acids. This property can be used to separate the protein from others in a mixture. In a high salt concentration, these hydrophobic amino acids move to the outside of the protein and push the hydrophilic amino acids to the inside. The more hydrophobic the compound, the more likely the compound will stick to a hydrophobic column. When the salt concentration is reversed, the 3-D structure of the protein changes as the hydrophobic amino acids move to the interior. Thus the protein is released from the beads in the column and can be collected by itself. Use the Internet to research the science of chromatography. Define the term chromatography in your laboratory journal. Under the definition, list at least two uses of chromatography in the real world. Obtain a copy of the Bio-Rad GFP Purification – Quick Guide handout and a Student Response Sheet from your teacher. Read the entire procedure as described on the Quick Guide. Notice that there are four lessons in the experiment. As Lesson 1 refers to the delivery of background information, you will begin the experiment at Lesson 2. Locate the name of each lesson of the experiment on the Student Response Sheet. Under the name, summarize the overall goal of this specific part of the experiment. Follow the steps of the Quick Guide and the instructions provided by your teacher to complete the experiment. Use the drawings located on the right side of the page as a visual reference for each step and make sure to follow the two bullets below. Your teacher will inform you of appropriate stopping points. Record any observations in your laboratory journal. The following additional instructions should also be followed. Note these changes on the Quick Guide.In Lesson 4, step 4, transfer an additional 50?l of the supernatant to a clean microcentrifuge tube labeled pre-purification supernatant. Label the tube with your initials or lab designation and the date and place this tube in the designated freezer. You will use this sample in the next experiment. In Lesson 5, you may be directed to use a ring stand to help stabilize your chromatography column. Note that in this lesson, the columns are designed to drip slowly. The entire process should take anywhere from 20-30 minutes. The column should only be disturbed when gently changing out the collection tube. You may need additional TE buffer to fully elute the glowing sample from the column. If so, label a new collection tube as Tube 4. In increments of 50?l, add TE buffer to the column until all of the GFP has been removed. Collect these drops in Tube 4. At the conclusion of each part of the experiment, answer the associated questions on the Student Response Sheet.Discuss the answers to the questions with your lab group and with the class.At the conclusion of the experiment, examine the three (or four) collection tubes and note any color differences. Describe the contents of each tube in your laboratory journal.Use a micropipettor to transfer the contents of each collection tube to a labeled microcentrifuge tube. The label should include the identity of the sample (ex. HIC fraction #1), your lab group initials or designation and the date. Make sure to use a fresh tip for each sample.Store your HIC fractions, along with the pre-purification supernatant sample, in the freezer. Further analysis will be completed on these samples in the following lab activity. Answer the remaining Conclusion questions. ConclusionDescribe how proteins fold, mentioning at least three rules followed by the amino acids. GFP contains a large number of hydrophobic amino acids. Describe how these amino acids would be oriented in the protein. How do you think the chemical properties of GFP can be used to isolate this protein from others in a mixture?By the end of the experiment, you should have a tube full of pure GFP. Describe one technique you could use to determine if this protein has been isolated from the other bacterial proteins or if additional proteins still remain in the mixture. (HINT: Think back to another molecular technique you have used to separate and visualize items in a mixture.) Explain how chromatography might be used in other career fields, such as forensic science. ................
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