with fluorescent proteins Protein Purification with fluorescent proteins

Laboratory Introduction What is a protocol? What is a protein? Why do scientists use protocols? Why would we need to purify proteins? Now let’s practice pipetting!

Organisms produce thousands of different proteins, each having a different function… Hair and Nails Hormones Muscle Contraction Structural Support Have students give examples of human proteins within these categories Receptors, membrane channels Enzymes Nutrient Storage Antibodies

Proteins… …are created by living organisms (DNA → RNA → PROTEIN → trait) …have unique structures that determine function (insulin, cobratoxin, fluorescence) …can be isolated from living things (humans, cobras, jellies) …can be studied and modified by humans (fluorescent proteins)

Protein Structure Not only do proteins act different, they look different. Their function is determined by their structure, and vice versa. Bottom center-similar to hemoglobin with four “lobes” each with an internal heme group for oxygen binding

Protein Structure 1° = amino acids 2° = basic structure (hydrogen bonds) 3° = 3D structure 4° = interaction of subunits

Structure determines function. Protein Structure Structure determines function. Now you be the protein!

DNA RNA Protein Trait The “Central Dogma” Transcription Translation Nucleic Acids Nucleic Acids Amino Acids Trait

The “Central Dogma” DNA mRNA Protein Trait

Making a Protein in the Lab “Transformation” Bacterial genome Plasmid E. coli bacterial cell We use E. coli bacteria “transformed” with a plasmid (a loop of DNA).

Making a Protein in the Lab DNA RNA Protein E. coli bacterial cell The bacteria now express (make) the fluorescent protein.

Engineered Fluorescent Proteins From organism… Why are we here? What is a protein? What is purification? …to purified protein product

Engineered Fluorescent Proteins From GFP: From RFP: 1. Green 4. Cherry 2. Blue 5. Tangerine 3. Grape 6. Yellow We have six different plasmids!

Our Plasmids Plasmid Mix 1 Plasmid Mix 2 GFP FP gene Cherry BFP Tangerine Grape YFP “Ampicillin resistance” means the bacteria will survive even when exposed to antibiotics! AmpR Ampicillin resistance gene

to understand structure Why Purify Proteins? Research Medicine Recombinant vaccines expressed in bacteria or yeast to produce large quantities of a single viral or bacterial protein. -purified protein injected into the patient -patient's immune system makes antibodies to the disease agent's protein, -patient protected from natural disease. Advantages of the recombinant vaccine technology are that there is virtually no chance of the host becoming ill from the agent, since it is just a single protein, not the organism itself. to understand structure to make vaccines to treat disorders

Why Purify Proteins? Insulin for diabetics Pancreas → Identify Cells → Isolate Gene → Insert Gene How many of you know a diabetic? Before we were able to synthesize insulin, we needed to find a way to make large quantities of insulin for diabetic patients. Through genetic engineering and protein purification methods, we were able to produce insulin protein for humans through bacterial transformation. Step !-Normal Insulin Producing Human Liver Step 2-Identify and collect insulin-producing cells Step 3-Identify and remove insulin-producing gene Step 4-Insert insulin-producing gene into bacterial DNA (plasmid) Step 5-Have living bacterial cell take in plasmid Step 6-Bacterial cell produces insulin protein product Step 7-Open cell, isolate insuline protein, purify (remove all other material) Step 8-Provide to Human Patient → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein → Human Use Insulin for diabetics

Why Purify Proteins? Insulin for diabetics Pancreas → Identify Cells → Isolate Gene → Insert Gene How many of you know a diabetic? Before we were able to synthesize insulin, we needed to find a way to make large quantities of insulin for diabetic patients. Through genetic engineering and protein purification methods, we were able to produce insulin protein for humans through bacterial transformation. Step !-Normal Insulin Producing Human Liver Step 2-Identify and collect insulin-producing cells Step 3-Identify and remove insulin-producing gene Step 4-Insert insulin-producing gene into bacterial DNA (plasmid) Step 5-Have living bacterial cell take in plasmid Step 6-Bacterial cell produces insulin protein product Step 7-Open cell, isolate insuline protein, purify (remove all other material) Step 8-Provide to Human Patient → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein → Human Use Insulin for diabetics

Why Purify Proteins? Insulin for diabetics Pancreas → Identify Cells → Isolate Gene → Insert Gene How many of you know a diabetic? Before we were able to synthesize insulin, we needed to find a way to make large quantities of insulin for diabetic patients. Through genetic engineering and protein purification methods, we were able to produce insulin protein for humans through bacterial transformation. Step !-Normal Insulin Producing Human Liver Step 2-Identify and collect insulin-producing cells Step 3-Identify and remove insulin-producing gene Step 4-Insert insulin-producing gene into bacterial DNA (plasmid) Step 5-Have living bacterial cell take in plasmid Step 6-Bacterial cell produces insulin protein product Step 7-Open cell, isolate insuline protein, purify (remove all other material) Step 8-Provide to Human Patient → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein → Human Use Insulin for diabetics

Why Purify Proteins? Insulin for diabetics Pancreas → Identify Cells → Isolate Gene → Insert Gene How many of you know a diabetic? Before we were able to synthesize insulin, we needed to find a way to make large quantities of insulin for diabetic patients. Through genetic engineering and protein purification methods, we were able to produce insulin protein for humans through bacterial transformation. Step !-Normal Insulin Producing Human Liver Step 2-Identify and collect insulin-producing cells Step 3-Identify and remove insulin-producing gene Step 4-Insert insulin-producing gene into bacterial DNA (plasmid) Step 5-Have living bacterial cell take in plasmid Step 6-Bacterial cell produces insulin protein product Step 7-Open cell, isolate insuline protein, purify (remove all other material) Step 8-Provide to Human Patient → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein → Human Use Insulin for diabetics

Why Purify Proteins? Insulin for diabetics Pancreas → Identify Cells → Isolate Gene → Insert Gene How many of you know a diabetic? Before we were able to synthesize insulin, we needed to find a way to make large quantities of insulin for diabetic patients. Through genetic engineering and protein purification methods, we were able to produce insulin protein for humans through bacterial transformation. Step !-Normal Insulin Producing Human Liver Step 2-Identify and collect insulin-producing cells Step 3-Identify and remove insulin-producing gene Step 4-Insert insulin-producing gene into bacterial DNA (plasmid) Step 5-Have living bacterial cell take in plasmid Step 6-Bacterial cell produces insulin protein product Step 7-Open cell, isolate insuline protein, purify (remove all other material) Step 8-Provide to Human Patient → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein → Human Use Insulin for diabetics

Why Purify Proteins? Insulin for diabetics Pancreas → Identify Cells → Isolate Gene → Insert Gene How many of you know a diabetic? Before we were able to synthesize insulin, we needed to find a way to make large quantities of insulin for diabetic patients. Through genetic engineering and protein purification methods, we were able to produce insulin protein for humans through bacterial transformation. Step !-Normal Insulin Producing Human Liver Step 2-Identify and collect insulin-producing cells Step 3-Identify and remove insulin-producing gene Step 4-Insert insulin-producing gene into bacterial DNA (plasmid) Step 5-Have living bacterial cell take in plasmid Step 6-Bacterial cell produces insulin protein product Step 7-Open cell, isolate insuline protein, purify (remove all other material) Step 8-Provide to Human Patient → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein → Human Use Insulin for diabetics

Why Purify Proteins? Insulin for diabetics Pancreas → Identify Cells → Isolate Gene → Insert Gene How many of you know a diabetic? Before we were able to synthesize insulin, we needed to find a way to make large quantities of insulin for diabetic patients. Through genetic engineering and protein purification methods, we were able to produce insulin protein for humans through bacterial transformation. Step !-Normal Insulin Producing Human Liver Step 2-Identify and collect insulin-producing cells Step 3-Identify and remove insulin-producing gene Step 4-Insert insulin-producing gene into bacterial DNA (plasmid) Step 5-Have living bacterial cell take in plasmid Step 6-Bacterial cell produces insulin protein product Step 7-Open cell, isolate insuline protein, purify (remove all other material) Step 8-Provide to Human Patient → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein → Human Use Insulin for diabetics

Why Purify Proteins? Insulin for diabetics Pancreas → Identify Cells → Isolate Gene → Insert Gene How many of you know a diabetic? Before we were able to synthesize insulin, we needed to find a way to make large quantities of insulin for diabetic patients. Through genetic engineering and protein purification methods, we were able to produce insulin protein for humans through bacterial transformation. Step !-Normal Insulin Producing Human Liver Step 2-Identify and collect insulin-producing cells Step 3-Identify and remove insulin-producing gene Step 4-Insert insulin-producing gene into bacterial DNA (plasmid) Step 5-Have living bacterial cell take in plasmid Step 6-Bacterial cell produces insulin protein product Step 7-Open cell, isolate insuline protein, purify (remove all other material) Step 8-Provide to Human Patient → Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein → Human Use Insulin for diabetics

How do you purify proteins? Fluorescent proteins are just one of thousands of proteins in the cell! A bacterial cell contains several thousand other proteins along with DNA, RNA polysaccharides, lipids, and small molecules. Brainstorm ideas. Know your protein, have bacteria make lots of protein, open the bacterial cell, wash through a column, grab protein of interest, let everything else go through. Fluorescent Protein

How do you purify proteins? 1. Open the cells 2. Separate cell components 3. Distinguish the protein of interest Ni2+ We are going to be using a method called column chromatography, to separate the fluorescent proteins away from everything else that is in the bacterial cell. First, we are going to lyse (or break) open the cells so that everything the cell is released into solution. Second, we are going to spin the cell contents down in a centrifuge to separate the large cellular contents from the smaller cellular contents. Third, we are going to add the liquid (supernatant) to the nickel bead mixture. (This will give the flourescent proteins a chance to bind to the nickel beads) 4. Separate the protein of interest “Column Chromatography” 5. Retrieve the protein of interest

How do you purify proteins? Ni2+ Supernatant We are going to be using a method called column chromatography, to separate the fluorescent proteins away from everything else that is in the bacterial cell. First, we are going to lyse (or break) open the cells so that everything the cell is released into solution. Second, we are going to spin the cell contents down in a centrifuge to separate the large cellular contents from the smaller cellular contents. Third, we are going to add the liquid (supernatant) to the nickel bead mixture. (This will give the flourescent proteins a chance to bind to the nickel beads) Snap freeze on dry ice Pellet Lyse (cut) open the cells. 2. Centrifuge to create pellet. 3.Mix supernatant with nickel beads.

How do you purify proteins? “Snap Freeze” Cell Lysis Freezing and then thawing… …causes ice crystals to break the cell open. By cooling the bacterial cells very quickly, we will make ice crystals form in the cell membrane. This will cause the cell membrane to break and open up the cell.

Lysozyme Lysozyme is a naturally occurring enzyme that is used to break open cells.

The “his tag” is how the protein attaches to the “nickel bead”! Purpose of the Nickel Beads The “his tag” is how the protein attaches to the “nickel bead”! Now that we have separated the mix of proteins from everything else in the cell, we have to separate our fluorescent proteins from the rest of the other proteins. The fluorescent protein being expressed in the bacteria has been engineered to carry a his tag. This tag is just a long chain of histidine amino acids that are attached to the rest of the fluorescent protein. his-his-his-his-his-his Fluorescent Protein with “his tag” The “his tag”

Purpose of the Nickel Beads The nickel bead binds to the “his tag” of the fluorescent protein. Ni2+ The his tag that is on the fluorescent protein, is attracted to something called a nickel bead. Much like a magnet to metal. You will be taking your mix of proteins and adding it to a tube that contains nickel beads. You will want to mix the proteins and nickel beads together well so that every fluorescent protein has a chance to interact with, and bind to a nickel bead. The nickel beads are large, so once they are bound to the fluorescent proteins, they fluorescent proteins that are attached to the nickel beads will lay on the cotton with the nickel beads. Joined together, the FP and nickel bead are too BIG to pass through the cotton!

How do you purify proteins? Once the flourescent proteins have bound to the nickel beads, we are going to pass the entire mixture through the glass “column” or pipette. The FP’s that have attached to the nickel beads will lay on top of the cotton while any other proteins or cell debris will flow through the column into your waste tube. Next we get the FP’s to release using the elution buffer. This buffer will “knock” the proteins off the nickel beads allowing them to flow through the column and into your clean tube. You then have a pure sample of only flourescent proteins. 4. Pass the supernatant through the column. 5. Add elution buffer. 6. End with a pure sample containing only the fluorescent protein.

How do you purify proteins? Fluorescent Proteins The nickel beads are too BIG to pass through the column, so the FPs that are stuck to nickel beads stay on top of the cotton. When you pass your mixture of proteins and nickel beads through the column, all of the proteins except your fluorescent proteins attached to the nickel beads, will be small enough to flow through the cotton ball. The fluorescent proteins that are bound to the nickel beads will be stuck above the cotton ball because they will be too big to flow through. All of the other proteins that were not attracted to the nickel beads because they did not have the his tag, will flow through the cotton ball into a waste tube and be discarded. All other proteins will flow through the cotton ball into the waste tube.

How do you purify proteins? Elution Imidazole FP are separated from nickel beads by the imidazole (elution buffer). Now FP is small enough to pass through the column. Ni2+ That that we have the FPs stuck to the nickel beads, and everything else has been discarded, we need to separate the FPs from the nickel beads and get them to flow through the cotton ball on their own. Do to this, we will add the elution buffer. The elution buffer contains a compound called imidazole. Imidazole competes and with the FP and cuts the bond between the FPs and the nickel beads. The FP then flows through the column and is collected in a clean tube – now a purified protein. Histidine

How do you purify proteins? Like your body, the E. coli bacterial cells that we will be using have many different proteins. From the over 4,000 proteins on an E. coli cell, we will be separating out just the specific protein that we are interested in looking at. The protein we will be purifying is the fluorescent protein. A bacterial cell is full of stuff. This includes DNA, RNA, lipids, and many different types of proteins. If we only want to separate out the fluorescent protein, how are we going to do that? Purify a specific protein from over 4,000 naturally occurring E. coli gene products.

How do you purify proteins? 1. Lyse (cut) open the cells. 2. Centrifuge to create pellet. 3. Mix supernatant with nickel beads. Summary of lesson or lab steps? 4. Pass the supernatant through the column. 5. Add elution buffer. 6. End with a pure sample containing only the fluorescent protein.

Green Fluorescent Protein (GFP)

Discovery of GFP (1960’s) Osamu Shimomura Co-winner of Nobel Prize Aequorea victoria Osamu Shimomura Co-winner of Nobel Prize

Fluorescent Organisms Corals There are lots and lots of organisms found in nature that can fluoresce all on their own. Here are some examples: Amphipods Coral Jellyfish Some animals, like this spider in the lower right hand corner, even use fluorescence to attract their mates. Jellyfish Amphipod Spider’s palps

How does fluorescence work? There are lots and lots of organisms found in nature that can fluoresce all on their own. Here are some examples: Amphipods Coral Jellyfish Some animals, like this spider in the lower right hand corner, even use fluorescence to attract their mates.

Blue light (High energy) Green light (Lower energy) How does fluorescence work? Excited state There are lots and lots of organisms found in nature that can fluoresce all on their own. Here are some examples: Amphipods Coral Jellyfish Some animals, like this spider in the lower right hand corner, even use fluorescence to attract their mates. Blue light (High energy) Green light (Lower energy) Ground state

Fluorescence vs. Bioluminescence Natural Light Scorpion- Natural Light In the Dark Scorpion- UV Light Fluorescent organism: Absorbs light at one wave- length (UV) and re-emits light at a visible wavelength (color) Bioluminescent organism: Produces its own light.

Fluorescence vs. Bioluminescence Fluorescent organism: Absorbs light at one wave- length (UV) and re-emits light at a visible wavelength (color) Bioluminescent organism: Produces its own light.

Roger Tsien and Rainbow Proteins GFP Why are we here? What is a protein? What is purification? RFP

Roger Tsien and Rainbow Proteins

Protocol Summary 1. Lyse (cut) open the cells. 2. Centrifuge to create pellet. 3. Mix supernatant with nickel beads. Ni2+ Summary of lesson or lab steps? 4. Pass the supernatant through the column. 5. Add elution buffer. 6. End with a pure sample containing only the fluorescent protein.