Exam 2 Flashcards
1
Q
Chromatofocusing
A
- similar to IEX but runs different
-runs from high to low pH (pH gradient) - no fixed pH
- as pH changes so does the proteins
- charges change
- salt [ ] is the same throughout
- separate by pI
- high pH will fall out first (elute out first)
2
Q
Affinity Chromatography
A
- selectivity is determined by affinity
- has stationary & mobile phase
- need ligand, target protein, spacer (sometimes)
-can be tagged or untagged
3
Q
Tagged vs Untagged Proteins
A
- Tagged: something added to protein to be able to mark it throughout the process
- Untagged: nothing added to the protein to mark it
4
Q
Antibody (ligand)
A
Antigen, virus, cell (counterligand)
5
Q
Inhibitor (ligand)
A
Enzyme (counterligand)
6
Q
Lectin (ligand)
A
Polysaccharide, glycoprotein, cell surface receptor, membrane protein, cell (counterligand)
7
Q
Nucleic Acid (ligand)
A
Nucleic acid-binding protein (enzyme or histone) (counterligand)
8
Q
Hormone, vitamin (ligand)
A
receptor, carrier protein (counterligand)
9
Q
Sugar (ligand)
A
Lectin, enzyme, or other sugar binding protein (counterligand)
10
Q
Broadly Specific Dye Ligand
A
-used to purify different proteins
- wide varitey
11
Q
Protein Binding by
Polysaccharide Heparin
A
- herparin is a sugar
- used to purify different enzymes and proteins
12
Q
Sugar Binding by Lectins
A
- lectin acts as ligand
- good amount of lectins come from plants
- binds to galactosamines
-have to see which one binds best to your protein - same lectin might not work as well as others
13
Q
Components of an Affinity Medium
A
1) Matrix: for ligand attachment, should be chemically + physically inert
2) Spacer Arm: used to improve binding between ligand + target by overcoming any effects of steric hindrance
3) Ligand: molecule that binds reversibly to a specific target molecule or group of target molecules
14
Q
Matrix
A
- similar to IEX
- should be:
- macroporous (more surface area)
- hydrophilic + neutral (prevent protein from nonspecific interactions)
- functional groups (allow derivatization by wide variety of chemicals) (key difference form IEX)
- physically + chemically stable (to withstand harsh conditions)
- readily available
15
Q
Ligand
A
Orientation is important but is difficult to control unless you know structure of protein
Must be:
- selective (not bind too weak or strong)
- reversible
- compatible w/ anticipated binding + elution conditions
- carry chemically modifiable functional groups which can be attached to matrix w/o loss of activity
16
Q
Spacer
A
- length is critical
- if too short = ineffective
- if too long = reduce selectivity
- rule of thumb about spacer: if ligand is small (Mr < 5000) need spacer if greater than 5000 then no spacer
- helps avoid steric hindrance
17
Q
Reversibility
A
- the higher the KD = weaker
- KD > 10^-4 = too weak
- the lower the KD = stronger
- KD < 10^-8 = too strong
- range typically is 10^-4 to 10^-8 M
18
Q
Effect of KD on Antigen Binding
A
- low to high binding affinity
- low KD needs less of target protein so when KD is strong you lose some of your target protein
19
Q
Coupling Methods
A
- one is one matrix and other is on ligand or spacer
- amino (NH2-R) w/ carboxyl (COOH on matrix)
- carboxyl (COOH) w/ amino (NH2-R on matrix)
20
Q
Ligand Coupling
A
- NHS-activated sepharose
- CNBr-activated sepharose
- EAH and ECH sepharose
- epoxy-activated sepharose with 12-C spacer
- thiol group coupling
21
Q
NHS-activated sepharose
A
- NHS: N-hydroxysuccinimide
- CO-O-N(in 5 membered ring w/ 2 double bonded oxygens)
- ring helps activate ester bonds
- NHS makes it more active
-10 atom spacer (# of carbons) - mix w/ amino (NH2-R) ligand
- spontaneous rxn
22
Q
CNBr-activated sepharose
A
- its a matrix
- mix w/ ligand (CHBr + NH2-R)
- isourea: circle sepharose OH group and O-C-NHR (C=NH is on top)
- rxns. of CNBr (cyanide bromide)
- 95% is wasted (oxidized)
- only little amount acts (less than
5%) - active cyanate ester (OCN)
- activation & coupling
- start w/ cyanate
- either will attach to amino group to
form isourea
-more chemically stable when
doesn’t attach to ligand (matrix)
23
Q
EAH and ECH sepharose
A
- EAH: think A for amino group, for
carboxyl group (available) - ECH: think C for carboxyl, for
amino group (available)
24
Q
epoxy-activated sepharose with 12-C spacer
A
- has epoxy ring
- more universal application but not
widely used bec. it attaches to so
many things - use as last resort
25
thiol group coupling
- if ligand is heavy metal, thiol will
react
- alkyl or aryl halide as ligand give
thioether derivatives
- ligands can contain C=O, N=N, and sometimes C=C
26
Coupling Procedures (NHS-activated matrix)
- need 2 solutions: buffer (high pH)
and HCl solution
- coupling in range of pH 6.5 to 9
- need good candidate for ligand
Steps
1) dissolve ligand in buffer
2) remove cap + add additional
solution (no bubbles) - prep step
3) wash column w/ low pH solution
4) inject ligand into column w/
syringe or pump
5) wait 15-30 mins. then start rxn.
* when rxn. done need to clean up
6) need 2 solutions buffers A
(ethanolamine) and B (acetate,
low pH)
* use to block unreactive sites
* switch between buffers
* helps matrix become inert
(neutral)
* don't use tris
27
Purification Steps
1) Equilibration (no protein, only
buffer)
2) Loading (add sample to certain
volume, huge broad peak)
* Washing: doesn't always happen,
gets rid of unbound materials*
3) Elution (change to elution buffer,
protein will get eluted)
4) Re-equilibration (switch to loading
buffer)
28
Binding
type of buffer that makes proteins bind
29
Elution
type of buffer that weakens binding
30
Wash
type of buffer that seperates bound and unbound proteins
31
Ligand Coupling
provides affinity
32
Pre-activated Matrices
chemically modified matrices that facilitate the coupling of specific types of ligand
33
Elution Methods
1) simplest, change buffer composition w/o harming either it or the ligand
ex.) pH elution, ionic strength, reduced polarity
2) extreme change of pH or high [ ] of chaotropic agents, may cause permanent or temporary damage, not used for purification
ex.) chaotropic elutents
3 and 4) competitive elution: additives will either compete w/ target protein to bind w/ ligand or bind to target protein to make sure it doesn't bind to ligand
34
Gradient vs. Step Elution
Gradient: must be run first to find desired conditions, slowly increase salt [ ] or pH
Step: used after gradient, can jump to desired conditions immediately
35
Condition Optimization
scan for the optimal binding/elution condition
- [ ] gradient or pH gradient
optimization
- flow rate
* not same for each protein
36
Affinity Purification Procedures
Prep column
- wash w/ binding buffer
- wash w/ elution buffer
- equilibrate w/ binding buffer
Purification
1) apply sample
2) wash w/ binding buffer
3) elute w/ elution buffer
4) re-equilibrate by washing w/ binding buffer
37
MBP fusion protein
-monospecific affinity tag purification
- maltose binding protein (MBP)
- ligand is dextrin
- not expensive
38
Immobilized Metal Ion Affinity Chromatography
- special type of affinity chromatography
- need ligand (metal ion), spacer, chealting (IDA) functional group that binds metal ion
- can purify native and denaturing proteins
- imidazole is key component
39
Histidine-tagged proteins
- most common process
- can denature and purify proteins
40
Imidazole
- 5 membered ring w/ 2 nitrogens and two double bonds
- kind of competing agent
- will compete w/ ligand
- use low [ ] to start
41
Procedure (IMAC)
1) Equilibrate
2) Load Sample
3) Wash
4) Elute
5) Re-equilibrate
approx. 20 mins total
42
General Considerations (for IMAC)
1) Types of media + formats
- can use magnetic bead based or
charged or uncharged
2) Metal ions
- Ni2+ (most popular), Co2+, Zn2+
- depends on nature of histine-
tagged protein
43
Competing Substance
Imidazole
- [ ] must be optimized at each step
- high purity: reducing nonspecific
binding of host cell proteins
- high yield: strong binding of
histine-tagged proteins
- is protein dependent
- if [ ] of imidazole is high in
beginning the yield will be low in
end
44
Commercially Available Chromagraphic Supports for IMAC
Chelating Sepharose Fast Flow
Chelating Sepharose 6B
Chelating Superose
Ni Sepharose High Performance
HiTrap Chelating
45
Characteristics of Ni Sepharose
- only differs in particle size
- if particle size is smaller, flow rate is slower bec. small particle size indicates that its packed tightly
46
Compatibility of Ni Sepharose
- stable when adding additives
- compatible w/ detergents
* if too high get micelle formation
- be careful w/ EDTA
* not really used w/ IMAC
47
Procedure (IMAC)
Buff prep
- 2 buffers (binding has low [ ] imidazole, elution has high [ ] )
Medium Prep
- use centrifuge water + binding buffer
Purification
1) Add sample
2) Let gravity pull it through column
3) Wash
4) Elute
48
Magnetic Bead based Purification
- collect beads using a magnetic device
49
Condition Optimization (IMAC)
1) Imidazole
2) Different metal ions
3) Using multistep purifications
50
Detection Methods (IMAC)
Amersham ECL is a chemiluminescent detection reagent
- use antibody
Westernblot
- very sensitive, can find tiny amount, doesn't detect purity, use ECH
Coomassie stain
- shows purity
Silver stain
- similar to coomassie stain, shows purity, more sensitive
Functional assay
- monitor product or substrate to see [ ] of enzyme
51
Gel Filtration (GF): Size Exclusion Chromatography (SEC)
- steric exclusion
- no direct binding
-separate by size
- low volumes used
- reserved for very last step
52
Vs
matrix volume
inaccessible to solvent
53
Vo
void volume
54
Vi
volume in pore
distance between Vt and Vo
Vi = Vt - Vo
55
Vt
total liquid volume
Vt = Vi + Vo
56
Vc
total geometric volume
Vc = Vo + Vi + Vs
57
Fractionation Range
- depends on pore size
- each column has a unique range
- Molecules smaller than the fractionation range can enter the pores
- molecules larger than the fractionation range are excluded from entering the pores
58
Group Separation: Desalting
- separate protein from salt
- salt remains in column
- very common, quick
- salt comes out close to Vt
- protein comes out earlier
- size of sample matters
59
Factors Affect Rs (GF)
Medium related
- particle size
- particle uniformity
- match between pore size + analyte
size
Column related
- bed height
- column packing quality
Experimental
- flow rate
- sample volume
- viscosity
60
Viscosity (Gf)
- low is better than high
- need slow flow rate
- can downgrade resolution
- avoid additives bec. that changes
viscosity
61
Matrix Porosity (Vi) and Fractionation Range
Traditional and High Resolution SEC Media
- watch for range of each column
- larger pore = larger + small
proteins through
- small pores = small proteins
- larger column # = larger range
- particle size range increases,
resolution decreases
- Sephacyl usually gives low
resolution
- composite gels: microporous polymers in macroporous pores
62
Sephacryl Matrix
- composite medium prepped by covalently cross-linking ally dextrin
- form hydrophilic matrix of high mechanical stress
- Dextran Linked to Bisacrylamide
- loses resolution
- fractionation range is high
-wide range of particles
63
Superdex Matrix
- made out of composite gels
- ratio of agarose and dextrin
64
16/60
first # is length (cm)
second # is diameter (mm)
65
Procedure (GE)
1) Equilibrate w/ buffer
2) Apply sample
3) Add buffer and run it
4) Elute
5) Re-equilibrate
- need one buffer, degassed
- need low salt [ ]
66
Distribution Coefficient KD
normalized retention factor, k
k = Vr/Vo -1 or Vr = Vo(1+k)
KD = Vr - Vo / Vi = Vr - Vo / Vt - Vo
Vr = retention volume
# is always a fraction (0 to 1)
- 0 protien is too big
- 1 is max #
to lower non-specfic binding increase [ ] of NaCl
67
The Partition Coefficient Kav
Kav = Ve - Vo / Vt - Vo
interchangeable w/ KD
68
Selectivity Curves
are almost linear in the range Kav = 0.1 to Kav = 0.7 and can be used for determining the fractionation range of a gel filtration medium
- also called a callibration curve
- can estimate molecular weight for each peak
- affected by protein shape
- larger # = larger fractionation range
69
Molecular Weight Estimation
- need MW size standard to estimate
- blue dextrin has huge MW, so won't get into pores
- need to choose right column w/ fractionation range
- run the MW standard through column first, can graph this
- then run sample through
70
Procedure for MW Estimation
1) determine void volume (Vo) and check column packing
2) Dissolve standard in buffer
3) Apply standard to column
4) determine elution volume (Ve)
5) calculate Kav for standard and prep standard curve of Kav vs logarithm of MW
6) apply sample to column
7) calculate Kav of sample and determine it MW from standard curve
71
Troubleshooting (GF)
1) Poor resolution
- media selection, particle size, sample volume, column volume
2) Leading peaks
- asymmetric: sample elutes before void volume
- overpacked column
3) Tailing peaks
- asymmetric: sample application is uneven
- underpacked column, low flow rate
4) Late elution
- peaks seen after buffer has passed through
- aggreagtion occurs
72
FPLC Injection Valve
3 positions
Position 1: Load
- loading sample into loading loop
Position 2: Inject
- sample is injected into column
Position 3: Waste
- washing pump, column is bypassed
73
Hydrophobic Interaction Chromatography (HIC)
-similar to IEX
- hydrophobic ligand
- high salt buffer
- target protein is also hydrophobic
- more hydrophobic = more binding
74
HIC vs IEX
HIC: starts w/ high salt [ ], then decreases
IEX: starts w/ low salt [ ] , then increases
75
Effect of Water (HIC)
surface tension phenomenon
- water molecules form highly ordered shell around hydrophobic substance, due to an inability to form h-bonds in all directions
- minimizing this shell leads to decrease in the # of ordered molecules
- more thermodynamically favored (entropy increases)
hydrophobic interaction depends on behavior of water molecules rather than on direct attraction
in pure water hydrophobic effect is too weak to cause an interaction between ligand + protein
76
Effect of Salt (HIC)
don't need salt theoretically but salt enhances the binding affinity (increasing entropy)
some salts are better than others
(NH4)2SO4 is most commonly used
77
Typical Steps (HIC)
1) Equilibration (high salt [ ] + hydrophobic ligand)
2) Sample Application (load sample, non-hyrophobic will fall out)
3) Elution 1 (decrease salt [ ] to weaken binding, makes sure weaker bonds fall out first)
4) Elution 2 (further decrease in salt [ ] )
5) Elution 3
6) Wash (no salt wash removes any hydrophobically bound proteins)
78
Matrix (HIC)
- stationary
- agarose beads
- hydrophillic environment
79
Ligand (HIC)
- intermediate or short chain of hydrophobic functional groups which increase or decrease binding affinty
- choice of ligand makes a difference
- density of ligand also makes a difference
80
Procedure (HIC)
1) Equilibrate
2) Apply
3) Wash
4) Elution
5) Wash
6) Re-equilibrate
81
Gradient vs Step Elution (HIC)
- step 4 of procedure can be done either by gradient or step
Gradient
- allows you to figure out exactly which peaks come out at certain salt [ ]
Step
- start w/ high salt [ ]
- then decrease to certain salt [ ]
- saves time
- makes selectivity further apart (more selective)
82
Additives (HIC)
3 types
1) Alcohols
- different types
- weaken interaction to make come out earlier
2) Detergents
- remember critical micelle threshold
- same effect as alcohol
3) Chaotropic Salts
- decrease hydrophobic effect, weakening interactions and causing dissociation
- reduce binding
Can optimize additives
83
Effect of Ions
To the left is increasing precipitation (salting-out) effect
- for anions as - charge increases so does this effect
- for cation as + charge decreases this effect increases
To the right is increasing chaotropic (salting-in) effect
- for anions as - charge decreases this effect increases
-for cations as + charge increases so does this effect
Most commonly used salts are:
(NH4)2SO4, Na2SO4, NaCl, KCl, and CH3COONH4
amount of protein that will bind to HIC media increase almost linearly up to a specific salt [ ]
once limit is hit protein will either precipitate or denature
ions make a difference they can reduce # of peaks
84
Other Factors Affect Rs
Salt Concentration
Temperature
Gradient Slope
- if slope decreases then separation could be better
Flow Rate
- too fast: loose resolution
- too slow: more resolution
85
Troubleshooting (HIC)
1) Target protein eluted early
- binding is weak
- adjust salt [ ]
- try different ligand
2) Binding too strong
- reduce salt [ ]
- change ligand
3) Eluted in middle of gradient
- adjust gradient
- add additives
- may have to change chromatography
86
Reversed-Phase (RPC)
- its reversed HIC
RPC
- more hydrophobic environment (matrix, stationary)
- water/organic, nonpolar solvent (mobile, solute)
ex.) methanol, acetonitrile
- stronger interactions
- no salt used
-not recommended for purification
- rarely used bec. can denature proteins
HIC (normal)
- more hydrophilic environment (matrix, stationary)
- salt/organic solvent
ex.) hexane, ethylene chloride
87
Inclusion Bodies
- can be 30% of mass
- large metabolic load for host so can't fold correctly
- large aggregation of proteins
88
Advantages & Disadvantages
of Refolding Inclusion Bodies
Advantages
- high expression levels
- high purity
- protection from protease
- allows expression of toxic proteins
Disadvantages
- refolding is cumbersome
- not predictable
- low success rate
89
Try Soluble Expression First (Inclusion Bodies)
- reduced growth rate usually leads to more soluble expression
- lower growth temp.
- better to spend time purifying than refolding
90
Typical Steps (Refolding of Inclusion Body)
1) Protein expression
2) Cell harvest + disruption
3) Isolation of Inclusion Bodies
4) Solubilization
5) Refolding
6) Purification
* Steps 3 to 5 are unique to inclusion body prep
91
Isolation of Inclusion Body
1) cell lysate in wash buffer
2) centrifuge
3) save pellet, suspend, and wash then centrifuge
4) collect pellet (where inclusion bodies are)
92
Solubilization (Inclusion Bodies)
- need this step to stop aggregation
- may have to optimize pH
- need denaturant (high [ ])
- can optimize temp + time
93
Solubilization buffer vs Refolding buffer
Solubilization buffer: has high [ ] of urea (6-8M)
Refolding buffer: has low [ ] of urea (1-2M)
94
Factors that Affect Refolding
protein [ ]
temp
rxn. time
disulfide exchange reagants
buffer additives
95
Refolding of Inclusion Body
- no known general rules
- suitable refolding conditions thus have to be found by trial + error
- intermediates can quickly collapse into aggregates
- for successful refolding: promoting the main pathway + suppressing competing pathways that lead to aggregation
for disulfide bridges
- reducing agent is replaced by disulfide exchange reagents for refolding
- the protein disulfide bonds are broken or formed until correct formation is found (oxidative folding)
96
Refolding Methods
1) Dialysis
2) Dilution
3) Gel filtration (SEC)
4) IMAC
5) IEX (or IEC)
97
Dialysis
- its simple
- classic
- slow
- uses large volume of buffers
- majority of protein gets aggregated
98
Dilution
- it's simple
- classic
- slow
- when protein is diluted the [ ] of protein is low
99
Refolding and Purification: Gel Filtration (SEC)
- stretched peptide could not enter pore of the microsphere, while refolded could get inside due to its compact structure
- cage like effect
- prescence of aggregates, even small amounts, is believed to induce accelerated aggregation
2 Methods
1) Refolding
- buffer + low/reduced concentration of urea (or GdnHCl)
- faster
- lower yield
- no gradient
2) Solubilization
- buffer + high concentration of urea (or GdnHCl)
- slower
- higher yield
- gradient
