Protein purification

Question 1

Why purify

Answer 1

Solve function or structure, medical or industrial importance

Question 2

hABCA1

Answer 2

HDL synthesis, purify via his tag, find sites of pathogenic mutations

Question 3

Attaining protein

Answer 3

recombinant plasmid or a native protein

Question 4

purification of a native protein

Answer 4

cell source, disruption, debris removal, initial + high resolution purification

Question 5

Things to consider

Answer 5

how much, how pure, cost/time, loss of activity tolerated.

stability, temp, pH, ionic strength, protease sensitivity, ligand binding

Question 6

Prokaryotic cells

Answer 6

bacterial plasmid, insert eukaryotic DNA, transform cell

Question 7

plasmid

Answer 7

Circular DNA molecule of bacterial species.

Amplify DNA and add fragment so sticks in plasmid via DNA ligase

Question 8

vector

Answer 8

promotor, induction, selectable marker, copy number, fusion protein, tag removal, post translational modification, where in cell

Question 9

pET expression system

Answer 9

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

Question 10

Cell lysis

Answer 10

osmotic disruption, chopping, grinding, high pressure, lysozyme cell destruction

Question 11

Clarification

Answer 11

centrifuge -> remove insoluble by aggregating membranes, nucleolus, proteins

Question 12

Salting in

Answer 12

add a little salt -> repulsive electrostatic charge between proteins = more soluble
sodium chloride

Question 13

Salting out

Answer 13

strip water off/out of proteins, hydrophobic particles interact -> precipitation (less soluble). Most efficient at pI of target
ammonium sulfate

Question 14

types of liquid chromatography

Answer 14

ion exchange, size exclusion, affinity, hydrophobic interaction

Question 15

liquid chromatography components

Answer 15

pump, two buffers, mixer, column, detector, recorder, fractionator

Question 16

Affinity chromatography

Answer 16

derivatised bead (attach different things Ni2+)

Question 17

washing

Answer 17

wash off unbound particles

Question 18

eluting

Answer 18

selective bound molecules are recovered

Question 19

ion exchange chromatography

Answer 19

below pI = +ve = cation exchanger
above pI = -ve = anion exchanger
bound (least charged) proteins disbond first
low to high salt

Question 20

size exclusion chromatography

Answer 20

small molecules enter aqueous space within beads = takes longer to elute (can use to estimate size)

Question 21

hydrophobic interaction chromatography

Answer 21

absorption of proteins to hydrophobic columns.

high to low salt

Question 22

Monitoring purification

Answer 22

total protein conc of each fraction-A280, volume/conc of target protein- (where, how pure, how much)

Question 23

What is measured during purification

Answer 23

1 activity, 2 protein, 3 specific activity (1/2),4 recovery (1final/1initial), 5 enrichment (3final/3initial)

Question 24

gels

Answer 24

SDS -> monomers, polyacrylamide=effects migration rate.

Question 25

Western blotting

Answer 25

add tag antibodies will bind to (primary or secondary) then wash

Question 26

mass spec

Answer 26

ionise -> analyser -> detector

can use tandem mass spec = separate specific fragment

Question 27

endopeptidases

Answer 27

smaller peptides, cleave by specific proteins (can use mass spec to find full aa sequenc3)

Question 28

chemical cleavage

Answer 28

cyanogen bromide - alter methionine residues

Question 29

problems with expression

Answer 29

no protein, insoluble, degradation

Question 30

how to avoid aggregation

Answer 30

codon optimisation (match host cell tRNA)
insoluble inclusion body
alter expression conditions (buffer, temperature, reducing agent)

Question 31

Expression systems

Answer 31

Yeast - secrete proteins, PTMs, slow
Insect cells - closer to mammalian, different PTMs
Human cells - expensive, lower yield, properly processed