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Biotech (Recomb. Protein Production) > Exam 2 > Flashcards

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)
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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
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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
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4
Q

Antibody (ligand)

A

Antigen, virus, cell (counterligand)

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5
Q

Inhibitor (ligand)

A

Enzyme (counterligand)

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6
Q

Lectin (ligand)

A

Polysaccharide, glycoprotein, cell surface receptor, membrane protein, cell (counterligand)

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7
Q

Nucleic Acid (ligand)

A

Nucleic acid-binding protein (enzyme or histone) (counterligand)

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8
Q

Hormone, vitamin (ligand)

A

receptor, carrier protein (counterligand)

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9
Q

Sugar (ligand)

A

Lectin, enzyme, or other sugar binding protein (counterligand)

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10
Q

Broadly Specific Dye Ligand

A

-used to purify different proteins
- wide varitey

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11
Q

Protein Binding by
Polysaccharide Heparin

A
  • herparin is a sugar
  • used to purify different enzymes and proteins
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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
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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

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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
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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

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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
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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
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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
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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)
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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
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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
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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)
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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)
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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
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25
Q

thiol group coupling

A
  • 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
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26
Q

Coupling Procedures (NHS-activated matrix)

A
  • 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
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27
Q

Purification Steps

A

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)

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28
Q

Binding

A

type of buffer that makes proteins bind

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29
Q

Elution

A

type of buffer that weakens binding

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30
Q

Wash

A

type of buffer that seperates bound and unbound proteins

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31
Q

Ligand Coupling

A

provides affinity

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32
Q

Pre-activated Matrices

A

chemically modified matrices that facilitate the coupling of specific types of ligand

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33
Q

Elution Methods

A

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

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34
Q

Gradient vs. Step Elution

A

Gradient: must be run first to find desired conditions, slowly increase salt [ ] or pH

Step: used after gradient, can jump to desired conditions immediately

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35
Q

Condition Optimization

A

scan for the optimal binding/elution condition
- [ ] gradient or pH gradient
optimization
- flow rate
* not same for each protein

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36
Q

Affinity Purification Procedures

A

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

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37
Q

MBP fusion protein

A

-monospecific affinity tag purification
- maltose binding protein (MBP)
- ligand is dextrin
- not expensive

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38
Q

Immobilized Metal Ion Affinity Chromatography

A
  • 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
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39
Q

Histidine-tagged proteins

A
  • most common process
  • can denature and purify proteins
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40
Q

Imidazole

A
  • 5 membered ring w/ 2 nitrogens and two double bonds
  • kind of competing agent
  • will compete w/ ligand
  • use low [ ] to start
41
Q

Procedure (IMAC)

A

1) Equilibrate
2) Load Sample
3) Wash
4) Elute
5) Re-equilibrate
approx. 20 mins total

42
Q

General Considerations (for IMAC)

A

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
Q

Competing Substance

A

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
Q

Commercially Available Chromagraphic Supports for IMAC

A

Chelating Sepharose Fast Flow
Chelating Sepharose 6B
Chelating Superose
Ni Sepharose High Performance
HiTrap Chelating

45
Q

Characteristics of Ni Sepharose

A
  • only differs in particle size
  • if particle size is smaller, flow rate is slower bec. small particle size indicates that its packed tightly
46
Q

Compatibility of Ni Sepharose

A
  • stable when adding additives
  • compatible w/ detergents
  • if too high get micelle formation
  • be careful w/ EDTA
  • not really used w/ IMAC
47
Q

Procedure (IMAC)

A

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
Q

Magnetic Bead based Purification

A
  • collect beads using a magnetic device
49
Q

Condition Optimization (IMAC)

A

1) Imidazole
2) Different metal ions
3) Using multistep purifications

50
Q

Detection Methods (IMAC)

A

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
Q

Gel Filtration (GF): Size Exclusion Chromatography (SEC)

A
  • steric exclusion
  • no direct binding
    -separate by size
  • low volumes used
  • reserved for very last step
52
Q

Vs

A

matrix volume
inaccessible to solvent

53
Q

Vo

A

void volume

54
Q

Vi

A

volume in pore
distance between Vt and Vo
Vi = Vt - Vo

55
Q

Vt

A

total liquid volume
Vt = Vi + Vo

56
Q

Vc

A

total geometric volume
Vc = Vo + Vi + Vs

57
Q

Fractionation Range

A
  • 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
Q

Group Separation: Desalting

A
  • 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
Q

Factors Affect Rs (GF)

A

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
Q

Viscosity (Gf)

A
  • low is better than high
  • need slow flow rate
  • can downgrade resolution
  • avoid additives bec. that changes
    viscosity
61
Q

Matrix Porosity (Vi) and Fractionation Range

A

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
Q

Sephacryl Matrix

A
  • 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
Q

Superdex Matrix

A
  • made out of composite gels
  • ratio of agarose and dextrin
64
Q

16/60

A

first # is length (cm)
second # is diameter (mm)

65
Q

Procedure (GE)

A

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
Q

Distribution Coefficient KD

A

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
Q

The Partition Coefficient Kav

A

Kav = Ve - Vo / Vt - Vo
interchangeable w/ KD

68
Q

Selectivity Curves

A

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
Q

Molecular Weight Estimation

A
  • 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
Q

Procedure for MW Estimation

A

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
Q

Troubleshooting (GF)

A

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
Q

FPLC Injection Valve

A

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
Q

Hydrophobic Interaction Chromatography (HIC)

A

-similar to IEX
- hydrophobic ligand
- high salt buffer
- target protein is also hydrophobic
- more hydrophobic = more binding

74
Q

HIC vs IEX

A

HIC: starts w/ high salt [ ], then decreases
IEX: starts w/ low salt [ ] , then increases

75
Q

Effect of Water (HIC)

A

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
Q

Effect of Salt (HIC)

A

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
Q

Typical Steps (HIC)

A

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
Q

Matrix (HIC)

A
  • stationary
  • agarose beads
  • hydrophillic environment
79
Q

Ligand (HIC)

A
  • 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
Q

Procedure (HIC)

A

1) Equilibrate
2) Apply
3) Wash
4) Elution
5) Wash
6) Re-equilibrate

81
Q

Gradient vs Step Elution (HIC)

A
  • 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
Q

Additives (HIC)

A

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
Q

Effect of Ions

A

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
Q

Other Factors Affect Rs

A

Salt Concentration

Temperature

Gradient Slope
- if slope decreases then separation could be better

Flow Rate
- too fast: loose resolution
- too slow: more resolution

85
Q

Troubleshooting (HIC)

A

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
Q

Reversed-Phase (RPC)

A
  • 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
Q

Inclusion Bodies

A
  • can be 30% of mass
  • large metabolic load for host so can’t fold correctly
  • large aggregation of proteins
88
Q

Advantages & Disadvantages
of Refolding Inclusion Bodies

A

Advantages
- high expression levels
- high purity
- protection from protease
- allows expression of toxic proteins

Disadvantages
- refolding is cumbersome
- not predictable
- low success rate

89
Q

Try Soluble Expression First (Inclusion Bodies)

A
  • reduced growth rate usually leads to more soluble expression
  • lower growth temp.
  • better to spend time purifying than refolding
90
Q

Typical Steps (Refolding of Inclusion Body)

A

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
Q

Isolation of Inclusion Body

A

1) cell lysate in wash buffer
2) centrifuge
3) save pellet, suspend, and wash then centrifuge
4) collect pellet (where inclusion bodies are)

92
Q

Solubilization (Inclusion Bodies)

A
  • need this step to stop aggregation
  • may have to optimize pH
  • need denaturant (high [ ])
  • can optimize temp + time
93
Q

Solubilization buffer vs Refolding buffer

A

Solubilization buffer: has high [ ] of urea (6-8M)

Refolding buffer: has low [ ] of urea (1-2M)

94
Q

Factors that Affect Refolding

A

protein [ ]
temp
rxn. time
disulfide exchange reagants
buffer additives

95
Q

Refolding of Inclusion Body

A
  • 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
Q

Refolding Methods

A

1) Dialysis
2) Dilution
3) Gel filtration (SEC)
4) IMAC
5) IEX (or IEC)

97
Q

Dialysis

A
  • its simple
  • classic
  • slow
  • uses large volume of buffers
  • majority of protein gets aggregated
98
Q

Dilution

A
  • it’s simple
  • classic
  • slow
  • when protein is diluted the [ ] of protein is low
99
Q

Refolding and Purification: Gel Filtration (SEC)

A
  • 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

Biotech (Recomb. Protein Production) (2 decks)

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