Part 11 47-100 Flashcards
What are the three main sample sources
Recombinant sources
Biological (model) systems
Hybridoma cell lines used for production of monoclonal antibodies
What are recombinant sources
cells that are used to express a heterologous gene for overexpression of a specific protein (usually with an affinity tag)
Ex. In bacteria yeast insect mammal plant cells
What is codon bias
codon optimizing
Give an example
Codon bias is when the codon we want in the protien is not available in the recombinant host
So we make the gene that the host prefers (codon optimize) and then put that codon in the host
Ex. UCC is preferred by the host but we want UCA, keep it as UCC so that the host makes that protein
What is the first choice in recombinant sources
Bacterial cell line
Second it eukaryotic cell lines
How do we maximize bacterial protein expression into the soluble phase
Why would we want it in the soluble phase
You can change the
growth/induction temp
IPTG concentration
length of induction (overnight)
use specialized cell lines (getting rid of the proteases so that the protein of interest isn’t chopped up)
We want the protein in the solution after centrifuging it, not in the pellet, to do this the solution is the soluble faction
What are examples of specialized cell lines for protein expression
Arctic express (switch to these if the protein is not going into soluble phase in normal cell lines)
grow the cells at low temp and slow so that they become more soluble
What are two other examples of specialized cell lines for protein expression
Rosetta
BL21 DE3 codon plus cells
What is production of heterologous proteins limited by
What happens
In bacterial cells, there is a rarity of certain TRNA that are needed to make the protein
If we force high level expression of these heterologous proteins, the limited amount of rare TRNA gets used up and stalls further translation of the protein
What bacterial cell line can help get over the TRNA rarity dilemma
Why
BL21-CODON plus
They are engineered to have extra copies of genes that encode the rare TRNA that normally get used up
What are human cells that are used to express proteins
HEK293F cells
Human embryonic kidney cells
What is TEV
What is the tev sequence
A protease that cleaves the AA sequence [ENLYFQX]
It separates the linker and the protein of interest
Are large proteins expressed well in bacteria
No
That’s when we move on to express the protein in eukaryotic cells
What is an example of expressing a protein in a human cell culture
They wanted to purify TOP2 beta and alpha
Tagged them with YFP and expressed them in the HEK293F cells
They are able to see if the proteins being made in the cell by the amount of yellow showing from the YFP
They look at the same cells and stained the DNA, they saw that the dna is showing in the nucleus
What did they find when from the HEK293 experiment
Found that TOP2 is gets shuttled into the nucleus and is a topoisomerase
What is Sepharose
A modified agarose
How is the recombinant TOP2a isolated from the HEK293F CELLS
What do they have to keep adding during growth of the cells and why
The sepharose bead has a nano body attached to it
This bead+nanobody binds to the YFP on the TOP2A
While culturing the cells with YFP, they need to keep adding media because as the cells grow they use up the media
They lyse the cells and do chromatography on them
after the cell is lysed in the TOP2a purification what do they do
The lysate is poured into a chromatography matrix
They washed all the things that weren’t the protein out
The yellow looking protein (bc of YFP) stayed in the column
Then they added tev protease to the resin to cleave the protein
After tev cleavage of the top 2 a what happened
What does this mean
To see the progress of the tev cleavage, they did SDS page with the resin
This showed the YFP-TOP2A, TOP2A, and YFP
The sds cocktail separates YFP from the nanobody , which is why we see a band of that only
After tev cleavage of the top 2 a what happened
What does this mean
To see the progress of the tev cleavage, they did SDS page with the resin
This showed the YFP-TOP2A, TOP2A, and YFP
The sds cocktail separates YFP from the nanobody , which is why we see a band of that only
Why did it look like there was more TOP2 a than YFP in the TOP2a experiment SDS page
The two proteins get stained differently by coomassie blue
What is different in HEK293 and HEK293F
The F version is a fast growing variant of the original
They grow in a more massive scale and don’t need serum
What are biological model systems
Why do we use them
and give an example
When you purify the protein from an endogenous source
This is because Some activity or factor can only be found in a specific cell type under specific condition
Ex. CDK was a factor in frog Oocytes
Or purifying protein kinases in the insulin signal pathway
What factor drives frog oocytes into mitosis and how was this found
MPF
We extract the cytoplasm from a cell that’s already in m phase then inject that into another cell
That other cell goes into m phase meaning a factor was in the cytoplasm to make it into m phase
What type of organisms can be used as biological/model systems
Viruses, non recombinant bacteria, non recombinant yeast
Animal , human , plant
What is the primary difference in each cell type in humans
The metabolism of each cell and proteins they express
What is special about the cells of multicellular eukaryotes
What do the cells have in common with
They have many different cell types for diff functions
Despite being differentiated, these cells have lots of features in common (composed of the same organelles for example)
What is special about red blood cells
Fat/adipose cells
Highly differentiated (have no nucleus)
The store energy as fat so we can use it later when starving to death
What are the types of model cells
E. coli
Yeast
Arabidopsis (plant)
Human cells in a culture
Nematode
Drosophila
Mouse
A cell culture is mainly made of
Either mammalian or human cells
What do cells in a culture require
A media with: Hormones and growth factors
What is a primary culture
Secondary culture
Cell line
When cells are taken directly from the organsim
Derived from a previously made culture
Cells with genetic modifications that make them grow indefinitely (cancerous)
How do you get a primary culture
What is special about fat cells
For fat cells you digest the ECM (which holds them together) then centrifuge
They float at the top of a centrifuged solution
What is red media
This is blood serum which is very good in the media for cell cultures
Has amino acids, glucose, vitamins
Before protein extraction and purification what else’s should you consider
Your objectives for purity and quantity
The assays you’ll do to follow your target protein
The properties of the protein that can help you purify it
What are the requirements of purity needed to do the three types of analysis with the our protein
This is the objectives for purity and quantity part
For therapeutic use, purity need to be extremely high (>99%)
For x ray crystallography high 95-99
As an antigen for antibody production moderate < (or equal) 95
What assays could you develop to follow your target protein
SDS PAGE/western blot
enzyme or functional assay (like the injection of cytoplasm into oocytes)
Protein assay (Bradford)
Explain why the activity would increase in the nitrate reductase inhibitor assay to track the inhibitor
There was an increase in activity of the nitrate reductase
This meant that the inhibitor previously was bound to the reductase, keeping its activity low
but when in solution it dissociates from the reductase and increased its activity
Explain how they know that the inhibitor is a protein in the nitrate reductase inhibitor assay
If they boil the sample and the inhibitory factor disappears (precipitates out of solution)
This means that the inhibitory factor is a protein
Explain how they know which fraction the inhibitor is in in the nitrate reductase inhibitor assay
They put each fraction of the inhibitor in a vial of nitrate reductase
The activity of nitrate reductase with that fraction begins to decrease
This means the the inhibitor was in that fraction
Explain what the A280 solid line is in the nitrate reductase inhibitor assay
The general monitoring of the inhibitor protein in each fraction as it comes through the column
What is the first step in sample preparation
What is the purpose
Cell lysis to collect and extract the sample
This is to remove all the protein population we want into the soluble phase (after centifuging)
But we still keep the in vivo state of the proteins
What is the in vivo state of a protein
The natural state of it (ex. Keeps its PTM)
What things can you do after collecting the sample in the supernatent but before doing the chromatography column
Remove non protein contaminants (lipids, nucleic acids) that were in the intial biological source
Adjust the buffer composition of the sample so it’s compatible with the column (ex. Remove salt for IEC, or change pH)
Adjust the volume and total protein concentration for the next step (if it’s gel filtration you want to reduce the volume)
What are the challenges that need to be tackled for proper sample prep
The protein complexity and dynamic range
Protecting proteins from degredation
Global or complete protein extraction
What does the first challenge in sample prep (The protein complexity and dynamic range) mean
The samples are highly complex due to PTM
the abundance of different proteins in a sample can vary widely
Give two examples of the The protein complexity and dynamic range challenge
In s. Ceravisiae, the abundance of proteins ranges from less than 50 to more than 10^6 proteins per cell (wide range)
In blood serum there is 60/80mg/ml of protein but half of this is albumin and 1/4 is y-globulin (igG)
What is albumin
What is IgG
A carrier of hydrophobic molecules (fatty acids) in the blood
Involved in the immune response
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Blood plasma has a ______ dynamic range of protiens
What are some of these proteins
Very high
Cytokines, interleukins, interferons
Explain how MS has allowed for more proteins to be detected and extracted
Before, the amount low abundance/membrane associated proteins (not easily extractable) was high compared to the abundant and soluble proteins (easily detected/extractable)
But now, because of MS, the amount of detected proteins is much higher
Explain the challenge 2 of protecting proteins from degredation
How do we fix this challenge
Proteases in solution could potentially cleave your sample that you want to purify (especially after the cell has been broken open)
We’ve deleted a select few proteases from the genome of bacterial cell lines
Also we’ve can add protease inhibitors to the crude cell lysate and pooled fractions from the column
Why do we do protein purification in the cold room
This decreases the protease activity so it doesn’t cleave our protein
What are common protease inhibitors
PMSF
EDTA
BENZAMADINE
All cheap
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What is included in challenge 3 global protein extraction
Tissue vs cell homogenization
Lysis buffers
Is the protein soluble or membrane bound
Is the protein/factor in an organelle or chromatin bound
In Tissue vs cell homogenization what can be used to physically break the cell/tissue open
What type of lysis method is it
Waring blender/polytron (mechanical)
Dounce homogenizer/french press (liquid homogenization)
Sonicator (sonication)
Freezer or dry ice with ethanol (freeze thaw)
Mortar and pestle (Manual grinding)
How does a waring blender/polytron work
What is the setback
Blender bladed grinds and suck everything in the centre
Not good for suspension cells because they would just stay at the top and not blend
How does a french press work
How does a source homogenizer work
Liquid with the cells, the French press pushes the liquid through a small opening, the cells get busted open bc of pressure
Liquid with the cells, dounce homogenizer has a piston that pushes them down, small distance between the glass and position causes them to pop open when pushed
How does a sonicator work
Put the tip of machine in the solution
The vibration from high frequency sound waves bust the cells open
How does a sonicator work
Put the tip of machine in the solution
The vibration from high frequency sound waves bust the cells open
How does a freezer with dry ice or ethanol work
What’s the setback
Repeated freeze thaw cycles disrupts the cells due to ice crystals forming
Not as efficient
What buffers can be added to aid in cell lysis (for tissue/cell homogenization in challenge 3
Lysozyme: digests cell walls
Bead beater: glass beads move vigorously to crush cell walls
DNase/RNase : to get rid of released nucleic acids that cause viscosity
Precipitation with streptomycin sulfate (to precipitate out the dna)
Protease inhibitors
Which treatment to lyse cells is commonly used for yeast cells
Lysozyme/glass beads
Is DNASE/RNASE needed for cells that have been sonicated
No, this is because sonicetion also breaks the chromosomes
What needs to be in the lysis buffer for sample prep
Why
A buffer (tris or hepes) at pH 7.5 and 25mM
(Because cell ph is 7.4, buffering capacity of 25mM is enough to keep at ph 7.5)
PMSF, benzamadine, protease inhibitor tablets
(Used as protease inhibitors to get rid of proteases)
Glycerol
(To make viscous, same environment as cell, stabilizes proteins)
EGTA (ca), EDTA
(Chelates ca2+, and other metals that might be needed for proteases)
Detergent like DTT (Thiol)
(To stop disulfide bond formation)
What do proteases need to work
Metal ions in their active site
This is why we use EGTA/EDTA
Membrane bound proteins are _____
Very hard to purify
Where are ribosomes made
In the nucleolus
Timbit
What do you have to do if your protein is in an organelle
You have to isolate that organelle ex. Mitochondria or nucleolus
What is a method to separate mitochondria, lysosomes, and perixisomes
Place the resuspended cell pellet on top of a sucrose gradient
Centrifuge
The organelles stop at a point in the tube that’s equal to their own density and form bands at these points
Why do we use sucrose in the method to separate organelles
The sucrose maintains the osmotic potential so that the organelles don’t explode
What is the OMM and the IMS
Outer mitochondrial membrane
Inter membrane space
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How do you isolate nuclei
Filter the supernatent, then Centrifuge
The nuclei goes in the pellet
Resuspend the pellet in mgCl2 and sucrose
Then place that on a sucrose cushion (this time there’s no sucrose gradient)
Then centrifuge in a swing out rotor (only the intact nuclei are dense enough to go through the sucrose cushion
What g of centrifuge pellets the nuclei best
600
What are the things to consider from the point of lysing cells, things to do between columns, to the final product
Homogenization
Clarification/centrifugation
Buffer exchange/desalting
Concentration/volume reduction
Sample cleanup
What are the things to consider from the point of lysing cells, things to do between columns, to the final product
Homogenization
Clarification/centrifugation
Buffer exchange/desalting
Concentration/volume reduction
Sample cleanup
What does the clarification/centrifugation step mean for protein sample perp and handling
Removing cell debris/aggresgated protiens from the sample to avoid clogging the filters or columns in future steps
Done via centrifugation
Where is each thing after centrifugation
What is the best centrifugation speed and time for this
The soluble proteins are in the supernatent
The cell debris/aggregated proteins are in the pellet
Force of 15,000 g for 15 min
Why would you need to do buffer exchange/desalting
How can you do it
Before doing chromatography steps, the ph may need to be changed or the salt may need to be removed
This can be done by
Gel filtration (ph change)
Dialysis (if sample is large, the small salt molecules escape out, also the ph of the sample changes to the ph of the dialysis solution)
Concentrate the samples using vivaspin or stirred concentrators (can also use to change the pH)
Precipiatation: PEG and ammonium sulfate precipitation to precipitate the protein
How does a vivaspin concentrator work
The tube has a membrane separating the sample at the top from the lower compartment
The membrane has a MW cutoff (diff cutoff sizes for diff tubes 3,10,30, 100kda)
You centrifuge this and anything less than the sample cutoff goes through the membrane
sample at the top gets more concentrated sample at the bottom less
How does a vivaspin concentrator work
The tube has a membrane separating the sample at the top from the lower compartment
The membrane has a MW cutoff (diff cutoff sizes for diff tubes 3,10,30, 100kda)
You centrifuge this and anything less than the sample cutoff goes through the membrane
sample at the top gets more concentrated sample at the bottom less
How do you do do concentration/volume reduction of your sample
Similar to buffer exhange and desalting this uses
Vivaspin concentrators/ stirred concentrators (and concentrate a sample at 5ml down to 2ml)
Precipitation: ammonium sulfate and PEG
How do you do do concentration/volume reduction of your sample
Similar to buffer exhange and desalting this uses
Vivaspin concentrators/ stirred concentrators (and concentrate a sample at 5ml down to 2ml)
Precipitation: ammonium sulfate and PEG
Explain the sample cleanup part of sample prep and handling techniques
We want to remove contaminants like lipids, detergents, nucleic acids from our protein solution
We do this by precipitating the proteins with ammonium sulfate or PEG
What’s another way to remove nucleic acids during sample cleanup
Use nuceleases
OR streptomycin sulfate precipitation of the dna
What is special about PEG
peg can be covelently bounds to lipids
It binds water molecules and keeps the lipids soluble
Used to help constipation
What is special about PEG
peg can be covelently bounds to lipids
It binds water molecules and keeps the lipids soluble
Why would we do PEG or ammonium sulphate precipitation before doing a chromatography step
The precipitation helps remove other non protein molecules (clean up step)
Give some purification of the protein (through ammonium sulfate cuts)
Can reduce a large volume of sample (L) to smaller volume (mL)
What is the advantage of PEG
What happens if we do a long dialysis to remove ammonium sulfate?
It carries no charge, this means we don’t have to dialyze it, saves time
The proteases have a lot of time to cleave your protein during that dialysis, proteolysis happens
This is why PEG it better
What is the ionic strength inside cells
<0.15M
What is salting in
When you start with low salt (<0.15M) adding more salt makes the protein salinity go up
Because the salt ions shield the protein from other ions
What is salting out
When you get too high ionic strength (too much salt)
the salt competes with the protein for the water molecules
The hydrophobic patches of the proteins start to interact now and the proteins precipiates out
Why do we do ammonium sulfate “cuts”
During the salting out process, diff protein have diff amount of hydrophobic patches
This means the they aggregate a diff amount of salt
So we do cut to see at what percent salt the actually precipitate
Other than providing purification and precipitation, what else can ammonium sulfate cuts help wih
They can help reduce the large volume of a crude extract /lystae right after busting the cells open
After doing ammonium sulfate precipitation, what do you do to the precipitated sample
What is the exception to this
Can Resuspend it in the proper buffer, then dialyze the salt away
SLIDE 88
How do you do the calculation of amount of ammonium sulfate needed in g to get to a extraction percentage
First know which temp your doing it at
Then find the percent you’re starting at (ex. Zero)
Go to the percent you need (ex . 30)
That value is the g/L to add (same as mg/ml)
Times that by the ml sample you have to get mg of salt to add
Convert to g by dividing by 1000
If doing 30-60 precipitation what is the starting percent
30 then go to 60
If doing 30-60 precipitation what is the starting percent
30 then go to 60
Since leg comes in a range of masses, what mass do we use for PEG fractionation
Peg 6000
What does 50% w/v solution of PEG mean
50g of peg in a final volume of 100ml
50g/100ml
How do you do PEG fractionation
Start with 30-50% w/V of peg
Add slowly via gentle stirring to the sample
Let sit for 20min then centrifuge
What is chromatography
What is liquid chromatography
Separation of components using the different affinities of them for the mobile phase or stationary phase
The buffer (mobile phase) passes through the chromatography beads (stationary phase)
The diff properties of the bead and be used to purifying the proteins
The does the column volume/bed volume mean in chromatography
This is the volume of beads in the column
If we ran 10 column volumes what does this mean
Tan the entire column volume 10 times
If column was 1ml, ran 1ml of column volume 10times
What are the steps in chromatography
First equaillibrate the column but just flowing buffer through it (to keep proteins stable)
Flow the sample through it, if proteins in the sample have affinity for the matrix, it binds
If not it flows out as flowthrough
Wash the matrix (with same wash buffer) then the proteins that are still in the matrix get washed out
Then elute the protein of interest that bound to the column using a column specific method (ion is surgery change or affinity ligand)
What is important to remember for the column size during chromatography
The column size (column volume) needs to be greater than the amount of sample your adding
So that all your protein can bind to the matrix
All chromatography matrix have a ______
Maximum binding capacity
What properties of a protein do we exploit during chromatography
Charge (IEX)
Size (SEC, gel filtration)
Hydrophobicity (HIC, HPLC, REVERSE PHASE)
Ligand binding (Affinity chromatography)
For tagged proteins what is the general steps to purifying it
The capture step: AC
The intermediate: IEX
The polishing: SEC, or IEX
For endogenous proteins what is the general steps to purifying it
IF USING PEG
The capture step: IEX
The intermediate: HIC
The polishing: SEC
If using ammonium sulfate
The capture step: HIC (because we can start at high salt)
The intermediate: IEX
The polishing: SEC
IEX samples run at
Higher flow rates
Sec and gel filtration works best for
Very small sample
This is why we usually do it last
Sec and gel filtration works best for
Very small sample
This is why we usually do it last
What is the difference in manual and automated purification
Manual: don’t need much training or start up time
Easy to do parallel runs for increased throughput
Automated: convenient, high resolution, documentation
What is the difference in manual and automated purification
Manual: don’t need much training or start up time
Easy to do parallel runs for increased throughput
Automated: convenient, high resolution, documentation
WHAT are AKTA chromatography systems
This is where the analysis is automated
The two buffers go in the pump and mixer
Inject the sample into the machine
The sample goes through the column and the absorbance, conductive and ph it automatically calculated
Normally we would measure the things
What are cytvia and biorad
Companies that make these automated column machines
What are most chromatography beads made of
Cross linked Agarose
Cross linked turns it into the bead
It’s a carbohydrate polysaccharide
What is sepharose
The name for the cross linked agarose bead
What are the densities of the sepharose and what does this mean
4-6%
Means that a 1ml column volume is more than 0.9 (90%) water
Slide 99
Densities
