Protein purification Flashcards
Why purify
Solve function or structure, medical or industrial importance
hABCA1
HDL synthesis, purify via his tag, find sites of pathogenic mutations
Attaining protein
recombinant plasmid or a native protein
purification of a native protein
cell source, disruption, debris removal, initial + high resolution purification
Things to consider
how much, how pure, cost/time, loss of activity tolerated.
stability, temp, pH, ionic strength, protease sensitivity, ligand binding
Prokaryotic cells
bacterial plasmid, insert eukaryotic DNA, transform cell
plasmid
Circular DNA molecule of bacterial species.
Amplify DNA and add fragment so sticks in plasmid via DNA ligase
vector
promotor, induction, selectable marker, copy number, fusion protein, tag removal, post translational modification, where in cell
pET expression system
uses T7 transcription signal from host cells T7 RNA polymerase -> lots of proteins synthesised.
IPTG induction in expression cell - binds to inactivate lac repressor -> activation of expression
Cell lysis
osmotic disruption, chopping, grinding, high pressure, lysozyme cell destruction
Clarification
centrifuge -> remove insoluble by aggregating membranes, nucleolus, proteins
Salting in
add a little salt -> repulsive electrostatic charge between proteins = more soluble
sodium chloride
Salting out
strip water off/out of proteins, hydrophobic particles interact -> precipitation (less soluble). Most efficient at pI of target
ammonium sulfate
types of liquid chromatography
ion exchange, size exclusion, affinity, hydrophobic interaction
liquid chromatography components
pump, two buffers, mixer, column, detector, recorder, fractionator
Affinity chromatography
derivatised bead (attach different things Ni2+)
washing
wash off unbound particles
eluting
selective bound molecules are recovered
ion exchange chromatography
below pI = +ve = cation exchanger
above pI = -ve = anion exchanger
bound (least charged) proteins disbond first
low to high salt
size exclusion chromatography
small molecules enter aqueous space within beads = takes longer to elute (can use to estimate size)
hydrophobic interaction chromatography
absorption of proteins to hydrophobic columns.
high to low salt
Monitoring purification
total protein conc of each fraction-A280, volume/conc of target protein- (where, how pure, how much)
What is measured during purification
1 activity, 2 protein, 3 specific activity (1/2),4 recovery (1final/1initial), 5 enrichment (3final/3initial)
gels
SDS -> monomers, polyacrylamide=effects migration rate.
Western blotting
add tag antibodies will bind to (primary or secondary) then wash
mass spec
ionise -> analyser -> detector
can use tandem mass spec = separate specific fragment
endopeptidases
smaller peptides, cleave by specific proteins (can use mass spec to find full aa sequenc3)
chemical cleavage
cyanogen bromide - alter methionine residues
problems with expression
no protein, insoluble, degradation
how to avoid aggregation
codon optimisation (match host cell tRNA)
insoluble inclusion body
alter expression conditions (buffer, temperature, reducing agent)
Expression systems
Yeast - secrete proteins, PTMs, slow
Insect cells - closer to mammalian, different PTMs
Human cells - expensive, lower yield, properly processed
