Part 3 Slides 42-91 Flashcards
What is the biggest problem in affinity chromatography
Give example
Non specific binding to the beads
Get over this by using magnetic beads instead of sepharose beads
Also Need to make sure that the protien binding part on the protien is free to dock the site on the bead
Atp, when purifying need to know what part of it docks to make a affinity chromatography bead for it
What are the steps in affinity chromatography
Incubate the crude sample (or serum if purifying antigens) with the bead to let the target protien bind
Wash away unbound samples
Elite the target protien from the ligand in the bead by chasing buffer conditions (might be hard because if very high affinity it won’t come off easily)
Is affinity chromatography a very powerful purification method
Yes, gives more than 1000 fold purification
What are group specific ligands
Give examples of group specific ligands and their specificities
Ligand in beads that have affinity for a group of related substances instead of just a single type of molecule
Protien A: the Fc (constant) region of IgG
IgG: the epitope of igG
Benzamadine : serine proteases
What are non group specific ligands and their specificities
Bind specific recombinant protiens
Amylose: MBP tagged proteins
Glutathione: GST tagged protiens
Metal ion (like ni2+): protiens with poly his tags
A peptide or protien domain: protiens that dock a specific protien sequence or domain
How many enzymes are in cells that have bid ing pockets for atp
4
What are immobilized/directly coupled ligands
What are indirectly coupled ligands
Direct: you have your gst tagged protien in crude extract and it is bound to the 3 aa long glutathione on the matrix
Elute with excess glutathione and you get the gst tagged protien with the glutathione attached to the gst
Indirect:
a pull down where the binding partners get coupled to the immobilized tag
Have the tagged protein binding to the column
But now pass over a solution with binding partners inside it and your proteins binding partners also get attached to the column
How can nonspecific binding on affinity chromatography be minimized
What’s the downside
How do you get protien off off the matrix
Adding low levels of detergent to wash buffer
Increasing salt concentration in wash buffer
But this could disrupt the actual interaction of the target with the bead , this is why you need to also do controls experiments with this
Elute:
Extreme Low or high pH (glycine ph 2 to elute igG)
high salt
detergents (SDS)
chaotropic agents (naSCN for pp1)
competion with a higher concentration counter ligand (like glutathione for gst elution) : same ligand that you coupled to the matrix
Explain the phneomena of binding partners in yeast
Protien protien interactions are the rule not the exception
6200 protiens in S.cerevisiae
Experiment where they tagged every protien in yeast then did a pull down to see binding partners of every protien
Found Asocial protien group called DLR1, whcih has no binding partners because did pull down and nothing came out
So out of 6200 protiens, only 461 protiens didn’t have partners
Shows how Protien protien interactions are the rule not the exception.
Even Once you find some other condition of the affinity chromatography, some those 461 could still show binding partners
How do you elute igG
Low ph (ex. Glycine ph 2)
What percent of protiens pulled down in affinity chromatography are non specifically bound
What type of protiens usually get nonspecifically bound
70-90%
Very abundant protiens like ribosome and histones
What is the crapome or the beadome
Non-specifically bound protiens that bind directly to the agarose beads
What else can help remove non specifically bound protiens and get over the issue of using detergent and salt to remove non specific binders
The TAP-tag
Explain the IP with phosphorylates RVSF antibody experiment results
EXACTLY SAME AMOUNT OF 2 MICROGRAMS Compared 5 control preimmune serum igG immunopreciptations and 5 p-RVSF igG IP (so the antibody recognizes phospho RVSF)
When purifying the control igG or the experimental igG, checked the number of resulting unique peptides that came from the protien they were purifying
The protien that elutes would have the p-RVSF motif meaning it got phosphorylated at the entrance of mitosis
RICTR protien showed no unique peptides in the control so not a non specific binder
But grp 78 had the same number of unique peptides in control as in experimental, meaning grp 78 is a non specific binder
NUMA1 also shows in the control experiment but it is higher in the experimental, meaning there is some degree of non specific binding but it is still a real binder
If the RICTR protien in the IP with phosphorylates RVSF experiment didn’t have the p-RVSY motif whats a reason is could have been captured by the igG
It would have a binding partner with the P-RVSF
and get eluted with that protien
For the bacterial non specific binding, why would the untagged BL21 DE3 protiens bind to the ni-NTA matrix even without the his tag
Non specific binding to the agarose bead, then eluting with high charge imidazole bumps them off the matrix
Might naturally have string of his residues
For the bacterial non specific binding, why would the tagged SLP1 lane show an extra band but the tagged slp 2 doesn’t
Not a non specific binder because it would be with the non specific binding band
It’s a protien that interacts with slp1 and copurfies but not with slp2
What can be added to a protien when it is being tagged
What is the term for these
Extra Amino acids
A functional domain of a protien
A whole protien
Fusion tag
What are the fusion tags
6x his
GST
MBP
What are epitope tags
Examples
6-10 aA peptides that need a specific antibody to bind to them on the column and purify
HA, Myc, Flag
HA is from the human influenza virus called hemaglutinin
Why don’t we use epitope tags for large scale purification
Because antibody based resins are expensive
Use for small scale purification
Explain how to use the MBP tag
And the way to elute
In our protien gene sequence, We add a protease cleavage site after the MBP part but before the target part of the gene
The target protien with the MBP bind to amylose sugar
First way to elute:
We elute with maltose, cleave with protease, then dialysis to remove the excess maltose and another chromatography step to remove MBP from target protien
Second way to elute:
Add protease first while the protein on amylose resin
Collect flowthrough with cleaved protien and protease, remove protease from solution
What’s another way to bind fusion protien to a column
There are antibodies made that specifically recognize these tags so we don’t have to make our own thing for them to bind to
Like an anti his antibody for his tagged protiens
What metals do the his on his tagged protiens bind to
Nickel cobalt and copper
What signifies that a column is charge with nickle
Blue colour
How is a column for IMAC prepared
The bead has either NTA or IDA (nitrilotriacetic acid, iminodiacetic acid) as a chela for for metal ion
Then NTA is charged with the metal (ni) to make Ni-NTA
Then the nitrogen on the his tag interacts with the metal
What are the conditions needed to do IMAC
The tags bind best to the column at ph 7.5 and 150 mM ionic strength (physiological)
Wash buffer has low amount of imidazole to prevent non specific binding of protiens that naturally have his, by washing them out
Also salt and low concentration detergent to stop nonspecific binding
Elute with high imidazole
How can IMAC also work for insoluble protiens
Since high salt and chaotropic agents like 8M urea and 6M guandiunium are compatible with the column
You can use them to first denature the protien to get is soluble then do IMAC
What are inclusion bodies
The protiens that are insoluble and aggreagate
we use denaturants to solubilize them to purify in IMAC
After being purified, they refold and precipitate after the urea is removed
What is the difference in GST MBP and IMAC
Gst and MBP needs the protien to be properly folded and have the 3D binding site to be functional so it can bind to the matrix
But IMAC doesn’t need this since his tags aren’t 3D, which is why we can purify inclusion bodies using denaturants
What’s not comaptible with IMAC
Reducing agents and chelators (EDTA), so can’t use them to denature the inclusion bodies
Why do we purify inclusion bodies
To make antibody’s
To use in far western blots also called overlays (since western blots can use both soluble and insoluble protiens)
Why is western blot called that
The DNA binding to DNA on a membrane (southern blot) was done by a man named southern, they kept the theme
What is the difference in western vs far western blot (overlay)
Western blot: have a protien on the membrane with an epitope, primary antibody binds to this epitope, secondary antibody for labelling binds to primary
Far western: another protien bind to epitope instead of antibody. Now a protien protien interaction then the antibody binds the other protien
Explain how difference are seen in the far western and western blot of the Brest cancer samples
What is the reasons for the extra EGFR band showing up on the far western but no on western
In western blot have anti EGFR, see high expression of EGFR band in certain patients
In far western blot: had GST-SH2 PI3K, the SH2 domain in PI3K docks to the EGFR since the EGFR has phosphorylated tyrosine (protien protien interaction)
The extra band in far western could be:
The band in far western is actually a diff protien with phos tyrosines
the EGFR protien is cleaved, so epitope gone but still sh2 binding to phos tyrosine, causes band in far western and none in western
What are the types of affinity chromatography
Peptide AC: peptide bound to the matrix
Co IP: antibody bound to agarose bead, get protien plus binding partners
Small molecule AC: coupled microcystin to the matrix to purify PP1, couple ATP to the matrix
Pulldowns
Displacement AC
Tap-tag (tandem affinity chromatography)
Explain peptide AC with example
There is a peptide on a bead that is phos and dephos (has RVSF phos and no phos)
Get diff protiens bound to this peptide in phos and de phos form
Ex. PP1 binds and elutes with de phos version but not to phos version, phosphorylation prevent binding of pp1
What would the gels from immune precipitation look like
How do we adjust it
One band with 55 and one at 25 (heavy and light chain) and the antigen of the antibody that was bound to the protien A resin
To remove the chains in the SDs page , covalently couple the antibody to protien A to just get the antigen and no antibody
How was coimmunoprecpitation used to show that kif18a binds pp1 directly not through a binding partners
Used kif18a antibody on the matrix to bind kif18a and eluted
Did western blot with anti pp1 and anti kif18a, saw that pp1 was also in that eluted sample meaning it’s interacting with kif18a
Then did pull down assay with his tagged kif18a and saw that pp1 gets pulled down
What are pull down assays
Have a pure and tagged protien (bait) that pulls down its binding partner (prey)
For example, doing IMAC, his tagging kif18a and seeing if PP1 comes out with the kif18a
What was seen in each of the tubes of the pull down assay to show that kif18a binds pp1 directly not through a binding partners
Put each tube through ni-NTA column
Tube 1: no pp1, his kif18a or RARA kif18a , no bands
Tube 2: just pp1, control since could be binding non specifically, no bands
Tube 3: pp1 and his kif18a, saw pp1 band and kof18 band meaning pp1 pulled down with kif18a
Tube 4: pp1 and RARA kif18a, control, no pp1 band but kif18a band, show that if no RVSF motif PP1 doesn’t bind
So showed that pp1 binds to k18a directly through the RVSF motif
What is displacement affinity chromatography
Using a peptide to displace specific binding partners
For example want pp1 binding partners but not pp1
Use a peptide with RVRW motif to bind to pp1 and displace binding partners in elution
What is special about microcystin
VERY BAD TOXIN
A whole range of organisms create this small molecule/toxin that binds to enzymes to inhibit their function
Sweden releases raw sewage to the rivers, nitrogenous compounds in the sewage let’s the blue green algae to thrive and make Microcystin
Pollution make let’s microcystin in the water and kill us
What does the kif18a eluting from the mc matrix show
The RVRW peptide being able to elute kif18a from the matrix (displacement chromatography)
shows that kif18a binds to pp1 directly using a RVRW motif
What experiments showed the kif18a binds pp1 directly in the nucleus of hela cells
- The displacement chromatography with pp1 on the MC column, gave kif18a and K9 as binding partners
- The co-immunoprecipitation of kif18a, gave kif18 and pp1 in elution
- The pull down assay of pp1 with kif18a, showed pp1 binds kif18a through the motif RVRW since didn’t get pulled down with mutated RARA
What is kif18a as a protien
How was this found
It’s a Kinesin (motor protein) that binds pp1 directly in the nucleus to take it to the mitotic spindle kinetochore for a de phosphorylation event
Found by:
Cloned kif18a with RFP, and have the PP1 with GFP, Had a stain that recognizes the dna
Saw that kif18a and pp1 showed up at the end of the microtubules which were binding to the chromosomes
Why is yeast so good
Has easy genetics so we do large scale experiments with it
We can do homologous recombination to it where we have a tagged protien and expresses that tagged version in the yeast in place of the WT
What is the tap tag
How is it made
Tandem affinity purification (two affinity chromatography in a row)
You fuse the TAP tag genome to the target protien genome and use homologous recombination to replace the endogenous WT protien with the TAP tagged one
Do this because if the endogenous protien is being overexpressed, it accosiates with non-natural binding partners
So maintain the normal levels by just completely replacing the endogenous protien with the tagged one through homologous recombination
What are hubs
They are basically a map of different protien protien interactions
Ex. Can map that pp1 interacts with kif18a
What is the structure of the tap tag
The tag has two copties or the protien A igG binding domains and a calmodulin binding peptide (CBP)
Separated by a TEV protease cleavage site
Protien- CBP- protease site-A-A
Can be flipped so N-C term is protien then protons A
Or N-C is protien A then protien
When ca binds the CAM, the CAM has a conformational change with its binding site exposed and binds to the CBP
Why did the do two flipped versions of the tap tag
Because we’re looking for binding partners, the protien could bind at the n term or the c term
In each way either the n term or the c term of the protien is covered by the CAM binding protein
So we do this to make sure all binding sites can be exposed
Ex.
N-protein CAM- C (n term exposed with protien at the start)
N - CAM-protein -C (c term exposed with protien at the end)
What is the method of TAP tag
you’ve expressed the protien with the tag it, has its normal binding partners, expressed at normal levels
First affinity step:
Put the cell lysate onto an igG matrix, non working end binds the protien A with high affinity
While bound to matrix, use the TEV to cleave
Now binding partners-protien-CBP elutes and protease, leaving non specific binders on the bead
Second affinity step:
The eluate binds to CAM coated beads with calcium in the buffer
So now CAM on the bead has conf change, bind the CBP-protien-binding partners
Wash to remove contaminants
Use egta to chelate the ca ions to make CAM change conf to close
Elutes the pure CBP-protien-binding partners
All nonspecific binders gone because using egta to elute
Explain how the TAP tag gel shows the effectiveness of the tap tag
Ran 4 lanes with snu71p WT and TAP tagged
Just using cam bead to purify:
tap tagged showed band of the snu71p protein (with proteins A and CBP) and some contaminants, WT showed all contaminants and non specific binding
Using just igG with TEV protease:
tap tagged showed band of the cleaved snu71p protein (with just CBP), some contaminants, and the dirty TEV protease , WT showed all contaminants and non specific binding and tag contaminants
Just TEV protease:
Control , Showed that the protease is very dirty and contributes to some of the bands we see in the experiment
Tandem regular tap tag method: BEST RESULTS
tap tagged showed band of the snu71p protein and binding partners and barely any contaminants, WT no contaminants since all washed out
How can we see that all of the binding partners of snu71p are stochimetriaclly related
By seeing that each band gets fainter as it goes down, for every molecule of sun there one molecule of binding partner and one mocleule of another binding partner
Coosmasie binds the surface of the protiens, if longer protien more intense band
1:1 binding
What was the goal of the paper MODERN USE OF AFFINITY CHROMATOGRAPHY: TARGET LANDSCAPE OF CLINICAL KINASE DRUGS
Wanted to see if protien kinase inhibitors were specific or not by finding the targets of 243 kinase inhibitors
Did this by using kinobeads and using MS to see if the inhibitor were specific to what they were made for
What are kinobeads
They let us purify endogenous protein kinases (no non protien kinases like pyruvate kinase)
By using quantitative mass spec, let us do label free measurements (don’t have to buys expensive reagent) of the interaction of the kinase with a bunch of protiens in parallel
What are the two ways to do quantitative mass spec
TMT labelling (chemically label the peptides and quantify them using that label)
Label free
Why are small protien kinase inhibitors (KI) used as drugs/medicines
Since protien kinases are disregulated in diseases like cancer, we use these small protein kinase inhibitors to target and stop them
Kinases are control in many of the cellular processes so if diregulated they cause big effects
What are examples of drugs that inhibit kinases and what is special about them
Crizotinib and imatinib (gleevac)
They are tyrosine kinase inhibitors that aren’t as specific as we thought they were have off target/side effects
Need the drug to be a specific as possible to remove off target/side effects
What is the kinome tree
What does it show
It’s a phylogenetic tree showing the relationship of all kinases to other kinases based on their sequence
Branches show ones that are closer to each other in the tree and are more closely related to each other than the other kinases
The circles mark protien kinases that the drug (imatinib or crizotinib) bind to in the human kinome
The bigger the circle the tighter the binding of the drug to the kinase
Shows that the drugs bind to many different protien kinases
What does phophylation happen on
Give probability’s
Most protien get phosphorylated, 75%
Ser (86%) thr (12%) tyr (2%)
What do kinases do
Use atp to phosphorylate specific residues on specific protiens (not random process)
Add phosphryl group
What is the second biggest target of drug companies
The first
The 518 PK in the human genome
GPCR are first
What do the protein kinase inhibitor drugs actually target
What make this hard
The atp binding pocket of the protien kinase
The substrate and atp has to dock in the kinase
Drugs are trying to be specific to certain kinases to inhibit them, but each protien kinase binds atp in the same way,
hard to make the drugs specific if they all use the same way to bind atp
How does the kinome pull down assay work
On beads link 5 known protien kinase inhibitors that are non specific and bind all kinds of kinases
Mixed the beadS together
Run lysate of 4 different cell lines: want this because they express a bunch of diff kinases that can be extracted
Allow them to pull out all the protien kinases in the cell (missing some but may be because they weren’t expressed in the cell)
Do dose dependent competition with the small molecule inhibitors
Using LC-MS/MS Sequenced the peptides of the collected kinases to see which protein kinases were left on the beads after the dose dependent competition
How is dose dependent competition done
Have varied amount of small molecule drug competing for binding the kinase with the kino bead
Orange green yellow kinase
Have zero small molecule inhibitor drug, do label free lc-MS/MS on the bead, see that the protien kinases dock the kino bead and high amounts of each kinase are seen in ms/ms since drug didn’t displace them
When adding inhibitor, with add it to the cell extract to it binds to the kinases, then run across the beads. Any kinase the drug binds to will not bind to the kinobead (since active site occupied by the inhibitor) and barely show up in MS/MS
One that decreases most would mean drug bind to it with high affinity
Keep increasing the concentrations in the cell extracts, the MS/MS of the high concentration inhibitor would show less signal of the kinase the drug has high affinity for
same amount of yellow protien in zero as in high shows that the inhibitor doesn’t bind to that kinase
What is the concentration range they used in the kinobeads competitive pull down assay
Range of 3 nM to 30,000 nM
What did the paper show about the Fausidil inhibitor
Found that fausidil targets PRKX, not Rock 1 and rock 2
Hydroxyfausidil targets ROCK1 and ROCK2
How did the paper find that Fasudil binds PRKX and not Rock 1 and Rock 2
Did the kinobeads competitive pull-down assay
To test Fausidil:
The binding curve showed that as they increased the amount of Fasudil, the binding or PRKX to the column decreased more than ROCK1 and 2
The activity curve shows activity of PKRX decrease more than ROCK1 and ROCK2, meaning more of it is inactivated by Fasudil, more sensitive to the drug
Then did same but with hydroxyfasudil
Binding curve showed that PRKX not sensitive to drug at all , but ROCK1 and 2 decrease even more now
Same in the activity, ROCK1 and ROCK2 decrease
How many protiens kinases in the cell
518
How many kinases can the kinobeads pull down assay detect
250 out of the 518 in the cell
Why would we want to study the the range of targets for a KI drug?
Most KI drugs target more than one protien and can’t be extremely specific
So we study their actual targets to explain
undesired side effects and to see if we can accept this degree of side effects
To repurpose the drug (might find it binds to another type of kinase to stop a diff disease)
What was the conclusion of the TARGET LANDSCAPE OF CLINICAL KINASE DRUGS paper
Found that some drugs show exquisite selectivity but others bound more that 100 kinases simultaneously
Meaning it’s hard to make specific kinase inhibitor drugs using those inhibitors
Repurposing: Also found new targets for already made drugs , like SIK2 inhibitor targets TNF alpha and IL-10 production
What are pseudokinases
Kinases that are inactive but still expressed in cells
Explain the off target interactions of Protien kinase inhibitor
They found that many drugs target things other than their expected target
For example they target the kinase PDXK which phosphorylates vitamin B6 to make it, can lead to side effects
Explain the black circle map using examples
Each black circle is a kinase inhibitor drug
The size of the circle is how many kinases it binds to (bigger circle, bind more kinases)
For example the drug ponatinib which is a tyrosine kinase inhibitor, is supposed to target a kinase for chronic myeloid leukaemia
But it binds to 39 other PK and has side effects
So bigger black circle
