1. with fluorescent proteins
Protein Purification
with fluorescent proteins

2. 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!

3. 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

4. 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)

5. 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

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

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

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

9. The “Central Dogma”
DNA
mRNA
Protein
Trait

10. 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).

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

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

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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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.

27. 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.

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

29. 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”

30. 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!

31. 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.

32. 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.

33. 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

34. 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.

35. 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.

36. Green Fluorescent Protein (GFP)

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

38. 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

39. 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.

40. 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

41. 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.

42. 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.

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

44. Roger Tsien and Rainbow Proteins

45. 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.