Protein Purification Flashcards
(41 cards)
purity required for therapeutic use of proteins
99%+
purity required for most physico-chemical purposes
high purity 95-99%
purity requirement for antigens for antibody production, sequencing
moderate <95%
name the three stages of protein purification strategy
- Extract the proteins
- Capture - isolate and stabilise
- Purification + polishing
locations where the protein may be expressed
Extracellular expression: protein is secreted onto culture medium.
Intracellular expression: either in the cytoplasm (soluble or insoluble) or in the periplasmic space
how to extract proteins of intracellular expression
cytoplasm: cell lysis, remove debris to recover supernatant (soluble) / harvest inclusion bodies (insoluble)
periplasmic space: cell wall disruption. cell removal, recover sample
briefly describe key features of gram positive and gram negative cell walls
gram negative: like e.coli
Outer and inner wall.
thin peptidoglycan layer connected to the per plasmic side of outer membrane by lipoproteins
gram positive: thick peptidoglycan layer outside periplasmic space. inner membrane.
what breaks down peptidoglycan in cell walls?
lysozyme
main methods to open up E.coli (gram negative bacteria)
Chemical lysis: using lysis buffer. contains detergents and protease inhibitors
or enzymatic: eg. using lysozyme
mechanical disruption: high pressure, osmotic shock, freeze-thaw cycles etc
what is the purpose of adding protease inhibitors before cell disruption?
cell lysis can release various proteases that could degrade the protein of interest. adding inhibitors protect these proteins during extraction.
which expression systems may result in the production of inclusion bodies?
bacterial and yeast expression systems
how is the lysate (obtained after cell lysis) clarified?
usually be centrifugation
what is protein precipitation? when does it happen?
happens after cell lysis and centrifugation.
a method to concentrate proteins by making them insoluble so they fall out as solid precipitate.
“salting out” proteins by changing solubility (often using ammonium sulphate), as salt competes for water
reversible process
what is dyalysis for? explain
to remove small unwanted molecules (usually salt) from a protein solution
happens after the protein precipitation, protein is redissolved in clean buffer, placed in a dialysis membrane in solvent
by what method are proteins captured for step 2 of the three phase purification strategy?
column chromatography
outline how column chromatography works to separate proteins. (define the two phases involved)
separates proteins by physiochemical properties
liquid phase = mixture of dissolved proteins
stationary phase = porous solid matrix, sometimes called resins.
liquid phase passes through stationary phase, proteins interact in certain ways to be separated
briefly describe the the four main steps involved in column chromatography
Equilibrate - pass buffer through medium to match buffer conditions
load sample with target protein - pass it through
wash - removes other molecules
elute - to extract target protein from the stationary phase
what is the most common type of chromatography used for the purification of proteins, antibodies and other biomolecules?
affinity chromatography
explain affinity chromatography of tagged recombinant proteins.
protein expressed as a fusion with an “affinity tag”
when passed through column, ligands in the resin specifically bind the tag. contaminants washed away.
why is there a protease cleavage site next to the affinity tag (between the tag and the protein)?
so that the tag can be removed after purification
what is IMAC?
Immobilising Metal Affinity Chromatography
the use of IMAC usually requires the desired protein to have what feature?
a histidine tag (His-tag)
Explain how IMAC works. what is the protein eluted with?
column resin loaded with metal ions like Ni+ or Co+
histidine residues on the protein bind tightly
elution by imidazole (outcompetes histidine)
how are antibodies purified by affinity chromatography?
protein A/G affinity chromatography.
proteins A or G in the resin binds specifically to Fc regions of antibodies.
