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)
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?
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
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)
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
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)
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
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
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)
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)
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)
12
Q
Ways to purify protein
A
- differential solubility
- ion exchange chromatography
- affinity chromatography
- size exclusion chromatography
- hydrophobic interaction chromatography
- isoelectric focusing
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
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-
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
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)
17
Q
Three common methods of salt removal/buffer exchange
A
- dialysis
- gel filtration chromatography
- diafilration
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
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)
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
21
Q
What is meant by the isoelectric point (pI)?
A
- the pH at which a protein has no net charge
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
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
Affinity chromatography
- 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
What are epitope tags and affinity tags used for?
- epitope tags used for protein detection
- afffinity tags used for purification
27
Principle of Gel Filtration Chromatography (GFC)
- 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
How does GFC work?
- 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
What are elution volumes?
- 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
How would you look for protein of interest after GFC?
- take a sample from each fraction and perform a Western blot (immunoblotting)
31
Column calibration
- 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
Protein complexes
- 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
Key factors affecting separation in GFC
- 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
Gel filtration resins
- 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
Ion Exchange Chromatography (IEC) - Protein Charge
- 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
What is overall/net protein charge determined by and how can it be changed?
- determined by the proportion of acidic and basic amino acids
- changed by increasing or decreasing pH
- this can be exploited during protein purification
37
IEC - Isoelectric Point
- 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
Cation exchange resin
- 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
Anion exchange resin
- 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
How does Ion Exchange Chromatography work?
- 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
Hydrophobic Interaction Chromatography (HIC)
- 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
HIC – Other Factors that Impact Elution
- 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
Isoelectric Focusing (IEF)
- 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
2-Dimenisonal Electrophoresis
- 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
Checking Expression and Purity of your Protein
- 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
What might cause low protein concentration?
- 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
Minimising Proteolysis/Protein Degradation
- 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
Quantitative Analysis of Protein Purification
- 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
Equation for Specific enzyme activity
Specific enzyme activity (U/mg) = enzyme activity (U) / total protein (mg)
50
Equation for yield
- yield (%) = (enzyme activity after purification step / enzyme activity in original sample) x 100
51
Equation for Enrichment (or purification) factor
Enrichment (or purification) factor = specific activity after purification step / specific activity in original sample
52
What does SDS-PAGE stand for?
- Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
53
Principle of SDS-PAGE
- 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
How does SDS-PAGE work?
- 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
Using Western blotting (immunoblotting) following SDS-PAGE
- 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
Factors that affect protein migration
- 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
Principle of Bicinchoninic acid (BCA) assay
- 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
