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AP0609 - Advanced Analytical Techniques > Protein Purification > Flashcards

Protein Purification Flashcards

1
Q

What must you consider before protein purification?

A
  • yield of enzyme
  • enzyme’s functional activity (ensure that purification process doesn’t impact function)
  • ease of subsequent purification
  • cost (could be perfect technique but costs thousands, look for alternative approaches)
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2
Q

Where could you get your protein from?

A
  • Recombinant protein produced in bacteria
  • Recombinant protein produced in other organisms (e.g. mammalian cells, insect cells)
  • Endogenous protein from tissue – e.g. is protein highly expressed in a particular tissue?
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3
Q

Recombinant proteins

A
  • Gene of interest is cloned into an expression plasmid (vector)
  • Plasmid (manipulated circular DNA) is transferred into host cells (e.g. bacteria or human cells)
  • High levels of protein can be produced in host cells
  • Protein can be purified for functional studies
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4
Q

Bacterial expression in BL21 (DE3) cells

A
  • start codon (ATG)
  • affinity tag (6-His)
  • T7 promoter; needs IPTG which removes a transcriptional repressor
  • restriction enzyme sites (Xhol and HindIII)
  • coding sequence
  • protein expression (His-Protein)
  • antibiotic resistance gene (only bacteria that contain this plasmid will grow)
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5
Q

Advantages of protein expression in E.coli

A
  • Fast growth rate (20min doubling time) – can generate lots of protein-expressing bacteria very quickly
  • Can transform bacteria with plasmid DNA rapidly (less than 5 minutes)
  • Relatively cheap
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6
Q

Disadvantages of protein expression in E.coli

A
  • Proteins may not fold correctly, impact on protein function
  • High concentration of protein can be insoluble (inclusion bodies)
  • Lack some post-translational modifications (e.g. phosphorylation)
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7
Q

General protocol for bacterial expression

A
  • Transform BL21 E.coli with expression plasmid
  • Pick a single colony and grow in 5ml of medium (37C 6-8h)
  • 200ml medium (37C 16h)
  • 1-litre medium (37C 1.5h)
  • 0.1 - 1mM IPTG (37C 2-4h)
  • Centrifugation to pellet cells
  • Lysis of bacterial cells and protein purification
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8
Q

General protocol for bacterial expression

A
  • Transform BL21 E.coli with expression plasmid
  • Pick a single colony and grow in 5ml of medium (37C 6-8h)
  • 200ml medium (37C 16h)
  • 1-litre medium (37C 1.5h)
  • 0.1 - 1mM IPTG (37C 2-4h)
  • Centrifugation to pellet cells
  • Lysis of bacterial cells and protein purification
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9
Q

Step 1 of Purification of protein from bacteria

A
  • lyse (break open) bacterial cells without degrading or denaturing your protein of interest (want functional protein)
  • Centrifuge again after cell lysis – supernatant contains soluble cellular material, including proteins
  • Same methods can be applied if using other sources of protein (e.g.human tissue)
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10
Q

What ways could you break open bacterial cells?

A
  • freeze thawing using liquid N2 (liquid nitrogen)
  • non-ionic detergent(e.g. Triton X-100)
  • sonication (ultra high frequency sound)
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11
Q

Step 2 of protein purification from bacteria

A
  • Purify your protein from the crude cell extract/lysate (contains all cellular components)
  • could take multiple rounds of purification
  • Need to track your protein throughout the purification process
  • Commonly use Western blotting (immunoblotting) but could also use an assay to measure biological activity (e.g. an enzyme)
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12
Q

Ways to purify protein

A
  • differential solubility
  • ion exchange chromatography
  • affinity chromatography
  • size exclusion chromatography
  • hydrophobic interaction chromatography
  • isoelectric focusing
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13
Q

Western blotting

A
  • can be used to track your target protein and estimate how successful your purification strategy has been
  • If protein has a known activity (e.g. enzyme), then can perform a functional assay – e.g. kinase assay
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14
Q

Differential solubility

A
  • Often used as an initial purification step
  • Techniques include salting out (high salt conc), polyethylene glycol (PEG),heat denaturation and altering pH
  • Precipitated protein (solid) can then be re-dissolved and subject to additional purification
  • Polar water molecules interact with hydrophilic regions of protein (polar/charged), increasing protein solubility
  • Anything that affects protein charge, protein structure or protein-water interactions will affect protein solubility
  • (NH4)2SO4 dissociates into [NH4]+ and [SO4]2-
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15
Q

Ammonium Sulphate Precipitation

A
  • Can also be used to concentrate/enrich a protein
  • Proteins fold such that charged/polar aa’s (e.g. acidic/basic/polar) are on the surface of the protein (hydrophilic) and hydrophobic aa’s (uncharged) hidden inside structure
  • Proteins are solubilised by hydrogen bonding with polar water molecules
  • Addition of high salt concentration; e.g. ammonium sulphate (ionic) leads to displacement of the water molecules and precipitation of the protein – ‘salting out’
  • Water bonds with salt ions instead of proteins
  • Proteins bind each other and precipitate
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16
Q

Why Ammonium Sulphate?

A
  • Highly water-soluble
  • Relatively cheap
  • Available at high purity
  • No permanent denaturation of proteins (e.g. enzymes will remain active)
  • Salt may need to be removed prior to next purification step – e.g. can’t immediately perform ion exchange chromatography
  • may not need to be removed prior to next step – gel filtration chromatography and hydrophobic interaction chromatography (carried out in the presence of high salt concentration)
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17
Q

Three common methods of salt removal/buffer exchange

A
  • dialysis
  • gel filtration chromatography
  • diafilration
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18
Q

Dialysis

A
  • Sample is placed in a bag with semi-permeable membrane (‘pores’)
  • Choose permeability based on target protein
  • Pores too small to allow passage of your protein but big enough to allow passage of salt ions (salt reaches equilibrium)
  • Several changes of buffer eventually remove the salt from your sample
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19
Q

Gel filtration

A
  • Separates sample components based on size
  • Resin has pores/holes that some components can enter
  • Load dissolved protein (and salt) onto column – flush sample through with buffer
  • Small salt ions enter the pores of resin, whilst large proteins pass straight through (carried in the buffer)
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20
Q

Diafiltration

A
  • Pressure-driven filtration membrane
  • Salt passes through membrane (permeate)
  • Protein is retained in sample (retentate)
  • New buffer can be added and protein can also be concentrated
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21
Q

What is meant by the isoelectric point (pI)?

A
  • the pH at which a protein has no net charge
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22
Q

pH and protein solubility

A
  • Proteins have an overall charge, dictated by the presence of amino acid side chains that can gain or lose H+
  • Charged amino acids are hydrophilic – form hydrogen bonds with water, increasing protein solubility
  • The overall protein charge changes with pH (i.e. change in [H+])
  • isoelectric point (pI) – least solubility due to lack of interaction with water molecules - precipitation
  • pH often used to precipitate contaminating proteins
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23
Q

Heat denaturation

A
  • Heating normally causes denaturing (unfolding of proteins)
  • Unfolding of proteins exposes the normally hidden hydrophobic areas that tend to bind each other, causing protein aggregation/precipitation
  • Some proteins don’t unfold after heating (thermostable – e.g. above 45C)
  • If purifying a thermostable protein, heat can therefore be used to remove(precipitate) other proteins
  • precipitation via pH and heat occurs when making cheese and milk going lumpy
24
Q

What is the affinity resin/matrix composed of?

A
  • an affinity molecule bound to a solid support e.g. Sepharose beads
25
Q

Affinity chromatography

A
  • Affinity matrix specifically recognises protein of interest
  • Protein may be engineered to have specific tag
  • When mixed with cell extract, target protein should bind to affinity resin
  • Beads can then be centrifuged and washed, removing unbound extract components (batch purification)
  • Purified target protein can then be eluted from beads
  • Affinity resin can also be packed into a column (‘column purification) for larger scale purifications
  • Add cell extract, then several wash steps and then elute target protein
  • Wash steps with increasing NaCl concentrations
26
Q

What are epitope tags and affinity tags used for?

A
  • epitope tags used for protein detection
  • afffinity tags used for purification
27
Q

Principle of Gel Filtration Chromatography (GFC)

A
  • AKA size exclusion chromatography
  • separates proteins (and protein complexes) based on size
  • Column packed with porous resin (beads)
  • Add cell extract and allow to pass through column
  • Large proteins pass through more quickly than small proteins
  • Can load sample in high salt buffer, therefore can perform straight after protein precipitation
28
Q

How does GFC work?

A
  • load cell extract (mix of small and large proteins) into column contain with beads
  • larger proteins will avoid the beads and elute very quickly from column
  • smaller proteins move into holes in the beads so they move through much slower
  • fractions collected at end of column in usually 1mL tubes
  • multiple fractions are collected until all proteins have passed through column
  • collecting fractions over time - increasing volumes of buffer
29
Q

What are elution volumes?

A
  • the volume of buffer at which a particular protein exits the column e.g. larger protein may have 1ml, smaller proteins may have 8ml
  • monitor protein elution with UV absorbance
30
Q

How would you look for protein of interest after GFC?

A
  • take a sample from each fraction and perform a Western blot (immunoblotting)
31
Q

Column calibration

A
  • A range of protein standards are used to calibrate a column
  • Protein elution monitored by UV Abs and elution vol matched to mass
  • Larger proteins elute first (lower elution volume)
  • elution volume is measured against UV absorbance on calibration
32
Q

Protein complexes

A
  • Proteins can exist by themselves as a monomer, dimer or trimer as well as multi-protein complex
  • Proteins can form large protein complexes with themselves and other proteins
  • Complexes generally intact when proteins in native state
33
Q

Key factors affecting separation in GFC

A
  • Size/mass of protein - in effect, the molecular radius, which is generally proportional to mass
  • Shape of protein (e.g. globular vs fibrous)
  • Length of column (some columns >1m long!) – longer columns give better separation
  • Amount of protein – too much protein can cause broad elution peaks, causes overlap and proteins aren’t purified efficiently
  • Resin material (e.g. pore size)
34
Q

Gel filtration resins

A
  • Resins designed to have pores that allow separation of proteins within a particular mass range
  • Can check mass range of sample using SDS-PAGE, then choose resin
35
Q

Ion Exchange Chromatography (IEC) - Protein Charge

A
  • separates proteins based on charge
  • Protein charge comes from ionisation of amino acid side chains (form ions via loss or gain of H+)
  • At physiological pH, Glu and Asp lose H+ (acidic side chains), Lys and Arg gain H+ (basic side chains)
  • seven amino acids have side chains that can be ionised, each has a pKa value – acid dissociation constant - pH at which 50% ionisation occurs
  • At pH below pKa, side chain accepts H+ (protonated), at pH above pKa, side chain loses H+ (deprotonated)
36
Q

What is overall/net protein charge determined by and how can it be changed?

A
  • determined by the proportion of acidic and basic amino acids
  • changed by increasing or decreasing pH
  • this can be exploited during protein purification
37
Q

IEC - Isoelectric Point

A
  • Isoelectric point (pI) = pH at which protein has no net charge
  • pH below pI = net positive – decreasing pH = increased H+ ions – protonation of protein
  • pH above isoelectric point = net negative – increasing pH = decreased H+ - deprotonation of proteins
  • If you know pI of protein, then you can adjust the pH to alter net protein charge
38
Q

Cation exchange resin

A
  • Resin has a –’ve charge (e.g. CM-cellulose, S-Sepharose)
  • Binds to positively charged proteins (‘cations’)
  • 0.5-1.5 pH units BELOW pI of molecule of interest
39
Q

Anion exchange resin

A
  • Resin has a +’ve charge (e.g. DEAE-Sepharose, Q-Sepharose)
  • Binds to negatively charged proteins (anions)
  • 0.5-1.5 pH units ABOVE pI of molecule of interest
40
Q

How does Ion Exchange Chromatography work?

A
  • To purify target protein, need to use appropriate buffer pH and the correct resin, want target protein to bind to resin
  • +ve proteins bind to –ve resin, -ve proteins pass straight through
  • Bound proteins are then eluted with buffer containing increasing salt concentration
  • Salt gradient used to elute proteins (typically 0 - 0.5M)
  • Salt ions compete for ionic interactions and displace proteins from resin
  • Different proteins elute at different NaCl concentrations
41
Q

Hydrophobic Interaction Chromatography (HIC)

A
  • Requires interaction between hydrophobic patches on protein and resin coated with hydrophobic material
  • In aqueous solution, proteins have hydrophilic surface with hydrophobic patches
  • In aqueous solution, water forms a ‘shield’ around the protein surface – hinders hydrophobic interactions
  • sample is prepared and loaded onto column in high salt buffer (e.g. ammonium sulphate)
  • This displaces water and exposes hydrophobic patches
  • For protein binding to the resin, salt concentration is inversely proportional to protein hydrophobicity
  • For protein elution, a decreasing salt gradient is used
42
Q

HIC – Other Factors that Impact Elution

A
  • Choice of salt in buffer (see Hofmeister series)
  • Include non-ionic detergents (reduce hydrophobic interactions)
  • Reduce temperature
  • Change pH – remember proteins are least soluble (most hydrophobic) at their isoelectric point
43
Q

Isoelectric Focusing (IEF)

A
  • Protein is loaded onto a gel with stable pH gradient
  • An electric field is then applied – proteins migrate based on their charge
  • Proteins will migrate along the pH gradient until they reach their isoelectric point – the pH at which they have no net charge
  • Proteins with different pIs can be purified/separated using IEF
  • phosphorylation adds a negative charge to proteins and alters migration in IEF
44
Q

2-Dimenisonal Electrophoresis

A
  • Proteins can be separated in two dimensions
  • First separation based on charge – IEF
  • Then place IEF gel strip on top of regular SDS-PAGE gel and perform electrophoresis
  • Can also perform Western blotting after 2D electrophoresis to detect protein of interest
  • Interpretation of gel and Western blot can help inform purification strategy
45
Q

Checking Expression and Purity of your Protein

A
  • Protein assay to measure protein concentration, then analyse purity using SDS-PAGE
  • Lysis buffer and wash steps can be modified to improve yield and purity of your target protein (e.g. salt and buffer concentration)
  • Important to reduce non-specific binding to your affinity resin
  • Higher salt and detergent generally decreases non-specific binding BUT can also negatively affect binding of target protein
46
Q

What might cause low protein concentration?

A
  • Poor protein expression in bacteria – optimise growth/IPTG
  • Inefficient lysis – try other methods/combinations
  • Inefficient purification – reduce detergent/salt
  • Inefficient elution – optimise
  • Protein is insoluble – optimise expression conditions/use mammalian host
  • Protein degradation - proteins are prone to degradation throughout the process
  • Take samples for protein analysis at various stages
47
Q

Minimising Proteolysis/Protein Degradation

A
  • Major cause; protease enzymes released during cell lysis
  • Low temperature – keep reagents on ice, work in cold room
  • Work quickly
  • Include protease inhibitors
  • Include chelating agents (e.g. EDTA) – bind metal ions that are needed for protease activity
  • Include buffers to avoid acidification
  • Can monitor protein degradation with SDS-PAGE
48
Q

Quantitative Analysis of Protein Purification

A
  • No purification method is 100% effective
  • Important to monitor the efficiency of purification steps
  • Ultimate aim is high yield and high purity
  • Total protein (mg)
  • Total enzyme activity = total number of Units (U)
49
Q

Equation for Specific enzyme activity

A

Specific enzyme activity (U/mg) = enzyme activity (U) / total protein (mg)

50
Q

Equation for yield

A
  • yield (%) = (enzyme activity after purification step / enzyme activity in original sample) x 100
51
Q

Equation for Enrichment (or purification) factor

A

Enrichment (or purification) factor = specific activity after purification step / specific activity in original sample

52
Q

What does SDS-PAGE stand for?

A
  • Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
53
Q

Principle of SDS-PAGE

A
  • Separates proteins based on their size
  • Need to unfold proteins (denature)
  • Use sodium dodecyl sulphate (-’ve charge) and 2-mercaptoethanol or DTT (reduces disulphide bonds) and heat to 95C 5min
54
Q

How does SDS-PAGE work?

A
  • Protein samples are loaded into wells of gel, electric current is applied
  • -’vely charged proteins migrate towards positive electrode, mesh impedes proteins as they migrate
  • Proteins move through mesh of polyacrylamide depending upon size
  • After electrophoresis, proteins can be visualised using Coomassie Blue
  • Large proteins remain near top, smaller proteins migrate to bottom
  • Need to include molecular weight marker on gel
  • Can be used during protein purification to check purity of sample
55
Q

Using Western blotting (immunoblotting) following SDS-PAGE

A
  • Western blotting uses a specific antibody to detect a protein of interest after SDS-PAGE and transfer of proteins to a membrane
  • Primary antibody binds target protein
  • Secondary antibody with tag for detection (e.g. fluorophore) then binds to primary antibody – band is visualised
56
Q

Factors that affect protein migration

A
  • Proteins generally migrate based on mass
  • However, can migrate at a different size
  • Large post translational modification (e.g.ubiquitylation and glycosylation) cause proteins to migrate at higher mass
  • Small PTMs (e.g. phosphorylation) generally don’t affect protein migration
  • If not fully denatured, they might migrate as complexes (e.g. dimer)
  • Inefficient reduction of disulphide bonds
  • High content of basic amino acids can affect migration
57
Q

Principle of Bicinchoninic acid (BCA) assay

A
  • in alkaline solution, proteins reduce Cu2+ to Cu1+
  • Cu1+ complexes with BCA, forms BCA-Cu1+ complex (purple)
  • absorbance measured at 562nm
  • darker purple = more protein

AP0609 - Advanced Analytical Techniques (10 decks)

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