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