Protein Purification Flashcards
what are the different sources of proteins?
Recombinant Protein Produced in Bacteria
Recombinant Protein Produced in Other Organisms
Endogenous protein from tissue
how are recombinant proteins produced?
gene of interest is cloned into an expression plasmid via a vector
- plasmid is transferred into host cells
- high levels of proteins can be produced in host cells
- protein can be purified for functional studies
what is used as an inducer for T7 promoter?
IPTG
what can be added to a plasmid that can make purification easier?
a tag - for example a His tag
bacterial expression in BL21:
there is a his-tag in this expression vector
what are the advantages of protein expression in E.coli?
- fast growth rate - can generate lots of protein-expressing bacteria very quickly
- can transform bacteria with plasmid DNA rapidly
- relatively cheap
what are the disadvantages of protein expression in E.coli?
- proteins may not fold correctly
- high concentration of protein can be insoluble - inclusion bodies
- lack some post-translational modifications - phosphorylation
what is the general protocol for bacterial protein expression? 6 steps
- transform BL21 E.coli with expression plasmid
- pick a colony to grow from - one that has been selected for using the antibiotic resistance gene
- then start expression with the IPTG
- centrifugation to pellet cells
- lysis of bacterial cells
- protein purification
what is the first step in protein purification?
- lyse bacterial cells without degrading or denaturing your protein of interest
- then centrifuge the cells - the supernatant contains soluble cellular material, including proteins
how is protein degradation prevented?
freeze thawing - prevents the proteases
what chemical is used to lyse the cell?
triton X - non-ionic detergent
what other technique can be used to break cells?
Sonication - this is when the cells are broken using high frequency sound waves
what is the second step in protein purification?
properties of the protein are exploited so that it can be purified
give examples of what properties can be exploited?
- size
- charge
- affinity tag - his-tag?
- hydrophobicity
what needs to be taken into consideration when thinking about a purification technique?
- the recovery of the protein
- purity of the protein
- functionality
- cost of method
how can proteins be tracked throughout the purification process?
- western blotting with immunoblotting
- can also use an assay to measure biological activity - this is used for enzymes in particular
SDS-PAGE and Western blotting separates proteins based on what?
their size
SDS stands for: sodium dodecyl sulphate polyacrylamide gel electrophoresis
need to denature the proteins prior to method - what is used to do this?
SDS
what is the first step?
protein samples are loaded into wells of gel and electric current is applied
- -vely charged proteins migrate to the positive electrode
Proteins move through the mesh of? What is it dependent on?
polyacrylamide - the rate of this depends on size - larger proteins nearer the top and smaller proteins at the bottom
what can be added to SDS-PAGE so that the proteins can be visualised?
coomassie blue
what is needed so that you can determine the molecular weight of the proteins?
reference proteins are used - they have a known molecular mass so unknown masses can be compared against them
what is used in immunoblotting?
- antibodies specific for the target protein
- secondary antibody with a tag for detection is then bound to the primary antibody - tag is commonly fluorescence
- bands are then visualised
what factors can affect protein migration?
Large Post Translational modifications
Proteins are not fully denatures, they migrate as complexes
Inefficient reduction of disulphide bonds
High content of basic amino acids can affect migration
large post translational modifications - cause proteins to…
migrate at higher mass
- small PTMs - generally don’t affect protein migration
if proteins are not fully denatured, they migrate as complexes, what will this mean?
this will mean they will be of a higher molecular weight
give examples of different purification techniques:
Differential solubility
Affinity Chromatography
Size exclusion chromatography
Ion exchange chromatography
Hydrophobic interaction chromatography
Isoelectric focusing
How is differential solubility usually used as?
often used as an initial purification step
What we use differential solubility for?
high salt concentration, polyethylene glycol (PEG), heat denaturation and altering pH
precipitated protein can be…
re-dissolved and subject to additional purification
polar water molecules interact with what?
hydrophilic regions of protein, increasing protein solubility
what will affect protein solubility?
anything that affects the proteins charge or protein-water interactions
what are the different types of differential solubility?
Ammonium sulphate precipitation
Salt Removal and Buffer Exchange
pH and protein solubility
Heat denaturation
Describe ammonium sulphate precipitation
- can be used as an initial purification step
- proteins fold such that charged amino acids (acidic/basic/polar) are on the surface of the protein (hydrophilic)
- the addition of high salt concentration leads to…the displacement of the water molecules and precipitation of the protein
- what’s this called?this is called “salting out”
- what happens during this?water bonds with salt ions instead of the proteins
- the proteins bind to each other and precipitate
- different proteins have different solubilities in aqueous solution
why is ammonium sulphate used?
- highly water-soluble
- relatively cheap
- available at high purity
- no permanent denaturation of proteins - the enzymes remain active
what are some considerations when using ammonium sulphate?
- salt may need to be removed prior to next purification step
- ion exchange chromatography
- salt may not need to be removed for other purification steps though
- gel filtration chromatography
- hydrophobic interaction chromatography
salt removal and buffer exchange:
precipitated protein can be re-dissolved in buffer but may need to remove residual salt
what are the three common methods for salt removal and buffer exchange
Dialysis
Gel filtration
Diafiltration
How does dialysis work?
- sample is placed in a bag with semi-permeable membrane
- choose permeability based on target protein
- pores are too small for passage of the protein but big enough for the passage of salt ions
- several changes of buffer eventually remove the salt from your sample
How does gel filtration work?
Separates sample components based on size
- resin has pore/holes that some components can enter
- load dissolved protein onto column - flush sample through with buffer
- small salt ions enter the pores of the resin, whilst larger proteins pass straight through
- effectively the opposite of gel filtration
How does diafiltration work?
- 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
proteins have an overall charge, dictated by the presence of amino acid side chains that can gain or lose H+ - this is dictated by…
the pH of the solution
charged amino acids are hydrophilic - they form what?
form hydrogen bonds with water to increase their solubility
what is the point at when there is no net charge called?
- this is the isoelectric point (pI)
at this point there is the least solubility of the protein due to the lack of interaction with water molecules - causing it to precipitate
what does heating cause?
normally causes denaturing of the proteins
what does the unfolding of proteins do?
exposes the normally hidden hydrophobic areas that tend to bind to each other
What does unfolding protein cause?
precipitation of the proteins
some proteins don’t unfold after heating - what is this called?
thermostable
if purifying a thermostable protein, the heat can be used to…
remove the other proteins
Heat denaturation:
this can be used to precipitate the target protein or the contaminating proteins
composed of an affinity molecule bound to a solid support of…
sepharose beads
when mixed with cell extract what happens to target proteins?
target protein should bind to affinity resin
beads can be centrifuged and washed to…
remove unbound extract components
Affinity chromatography
affinity matrix specifically recognises protein of interest - this could be a specific tag (His tag)
purified target protein can then be eluted from beads
Affinity chromatography
affinity matrix specifically recognises protein of interest - this could be a specific tag (His tag)
purified target protein can then be eluted from beads
Principle behind larger scale purifications:
- affinity resin can be packed into a column
- add cell extract, then several wash steps and then elute target protein
- wash steps with increasing NaCl concentrations
His-tag purification:
bacterial expression vector for the production of a His-tagged protein
what do His tags bind strongly to?
- Nickel (Ni2+)
- this can be immobilised on a solid support
what is His-tag eluted with after bound?
500mM imidazole
this competes for binding to the nickel)
other protein Tags:
biotin can also be used as a tag - binds strongly to streptavidin
some tags can be removed after purification
what are epitope tags used for?
they are used for protein detection
what are affinity tags used for?
they are used for the purification of proteins
size exclusion chromatography also known as
gel filtration chromatography
separates proteins based on size -
the smaller proteins remain nearer the top (they get stuck) and the larger proteins nearer the bottom (pass through a lot faster)
the column in gel filtration chromatography is packed with…
porous resin
can the sample be loaded with high salt buffer in gel filtration chromatography?
yes - the protein can be added straight after protein precipitation
how is the protein of interest located in gel filtration chromatography?
- if the protein has a known activity, a functional assay can be performed
- western blotting can also be performed of individual fractions
column parameters and equations (don’t need to known in a lot of detail):
What is used to calibrate a column?
a range of protein standards are used to calibrate a column
protein elution monitored by…
UV absorbance and elution volume matched to mass
elution volume rules
larger proteins elute first at the lower elution volumes - so the elution volume can correspond to a particular mass for that column with the use of a standard
what can cause problems during column calibration?
- the formation of large protein complexes with themselves or other proteins
- complexes generally intact when proteins in native state
- they can form dimers/trimers which affect the observed masses for that protein during the chromatography
what can be used to see what proteins are bound to each other?
- mass spec
- this can be significant in seeing the interactions between various proteins in a mix
column calibration example:
key factors that can affect separation in GFC?
- size/mass of proteinthe molecular radius, which is generally proportional to mass
- shape of the proteinglobular or fibrous
- length of the column usedthe longer the column the better the separation between each of the different proteins would be
- the amount of protein that is added to the columntoo much protein can cause broad elution peaks
- the resin material - this is what determines the pore size
- the resins are designed to have pores that allow separation of proteins within a particular mass range
- the mass range of a sample can be selected by using particular resins
ion exchange chromatography separates proteins based on what?Where does this come from?
based on their charge which comes from the ionisation of the amino acid side chains - there is the loss or gain of H+
what is the pKa value?
this is the value at which 50% ionisation occurs
how many amino acids have the ability to be ionised?
7
what is the isoelectric point (pI)?
this is the pH at which the protein has no net charge
what happens if the pH is below the isoelectric point?
- decreasing the pH increases the H+ ions
- leads to protonation of protein
- gives it a positive charge
what happens if the pH is above the isoelectric point?
- increasing the pH increases the amount of OH-
- causes the deprotonation of protein
- gives a negative charge to the protein
overview of isoelectric point?
what does a positively charged protein bind to? give 2 examples?
- cation
- CM-cellulose
- S-sepharose
what does a negatively charged protein bind to? give 2 examples?
- anion
- DEAE-sepharose
- Q-sepharose
how is the bound protein eluted?
- bound proteins are released with buffer containing increasing salt concentration
- salt ions compete for ionic interactions and displace proteins from resin
hydrophobic interaction chromatography (HIC) requires interaction between what
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 and hinders…
hydrophobic interactions
in HIC the sample is prepared and loaded into a column with high salt buffer - what does this do?
this displaces water and exposes hydrophobic patches, which the resin binds to
can HIC be carried out after ammonium sulphate?
yes
what is the relationship between salt concentration and protein binding to resin? what salt gradient is used?
- it is inversely proportional - the more salt that is added the less protein bound to the resin
- a decreasing salt gradient is used because of this
what factors can impact elution?
- the choice of salt in the buffer
- the inclusion of non-ionic detergents - this can reduce hydrophobic interactions
- temperature can also have an effect
- the pH of the solution can also affect solubility - proteins are least soluble/hydrophobic at their isoelectric point
Overview of HIC
isoelectric focusing:
at a proteins isoelectric point they will have no charge, the isoelectric point can be determined by a pH gradient and an electric field
this is a phenomenon called?
isoelectric focusing
what can affect the process of isoelectric focusing?
if the protein is phosphorylated then there will be the addition of a negative charge to the protein - this can affect the migration
what are the proteins separated by in 2-dimensional electrophoresis?
first separated by charge along a pH gradient - IEF separated based on pI
they are then placed on a gel strip at the top of a regular SDS-PAGE gel and electrophoresis is performed separated based on size
what can also be done after 2D electrophoresis?
-
western blotting with immunoblotting
- this allows the protein of interest to be located
- the pI and molecular weight of the protein can then be determined
checking expression and purity of your protein:
protein assay, what are the two common methods to measure protein concentration?
Bradford assay
Bicinchoninic acid assay (BCA)
bradford assay
- this uses the dye coomassie blue
- the colour of the reaction can then be measured
- the colour produced is proportional to the amount of protein in the sample
bicinchoninic acid assay (BCA) principle
- there is the addition of
copper - in alkaline solution the copper…
gets reduced by hydroxyl group (OH-) on the protein - this reduces the copper
a standard curve can then be used to quantify the exact amount of protein that is in each of the solutions
the reduced copper then complexes with BCA - this turns the solution what colour?
purple
the concentration of the protein in the sample will then be proportional to what?
the absorbance of the solution - the darker purple the solution the more protein in the solution
what is the major cause of protein degradation
protease enzymes released during cell lysis
chelating agents
include buffers to avoid acidification - this will further degrade the proteins
chelating agents - what do these do?
bind metal ions that are needed for protease activity
protease enzymes released during cell lysis - how is this reduced?
- low temperatures - reagents on ice
- work quickly
- protease inhibitors
can monitor protein degradation with what?
SDS-PAGE
quantitative analysis of protein purification?
- no purification is 100% effective
- important to monitor the efficiency of purification steps
- ultimate aim is high yield and purity
Enrichment factor: specific activity of the pooled fraction/specific activity of the starting material
% yield: enzyme activity after purification step/enzyme activity in original sample
How might you purify an enzyme from human tissue - coat resin with?
Enzyme inhibitor since enzyme will bind inhibitor and remain inactive
why can’t enzyme substrate be used?
enzyme would more likely react with substrate and binding would then be lost
How might you purify a specific transcription factor from human tissue?
If TF binds a specific promoter DNA sequence, then you could try immobilising this DNA sequence on beads - TF should bind
You cloned a gene of interest with a C terminal His-tag and expressed the protein in bacteria. How would you purify the protein?
Nickel-coated beads
Which of the following proteins is the smallest and why?
NASP in the middle because in GFC, smaller proteins elute later than bigger proteins. Big proteins pass straight through the column as they can’t enter the pores in the GFC resin
