protein purification

Question 1

proteins differ in 5 ways list them

Answer 1

SSHIC- Size, stability, charge, hydrophobicity [number + location of hydrophobic A.A.) Iso-electric point

Question 2

what is the isoelectric point of a molecule

Answer 2

pH - where the protein gas no net charge [not + or -)

Question 3

why must protein purification be done

Answer 3

to determine the protein structure and mechanism of action

Question 4

proteins can be separated based on x and y

Answer 4

physical and chemical properties

Question 5

what enzyme converts Arachidonic acid—> prostaglandin [what is the function of the prostaglandin]

Answer 5

cyclooxygenase 1/2 - inflammation [can cause major pain]

Question 6

what type of chromatography is used in protein purification

Answer 6

the liquid mobile phase, solid stationary phase

Question 7

column chromatography is x method

Answer 7

separation

Question 8

what are the 2 phases in column chromatography and describe their locations

Answer 8

stationary phase -packed into 1 column- usually solid
mobile -gas or liquid flows over the stationary phase
- mobile phase carries material need to be separated over stationary phase

Question 9

separation takes place based on ?

Answer 9

the materials interaction between the stationary phase relative to interaction with mobile phase

Question 10

chromatography columns can be made from x or y

Answer 10

glass or plastic

Question 11

the stationary phases initially appears what colour?, and needs to be ? it generally has 2 parts

Answer 11

• appears white
  -hydrated by mixing with a mobile phase or water for more than 2 hours
  -inert and reactive groups

Question 12

sources of proteins
proteins can be x, y or z

Answer 12

cells [mammals or bacteria] and blood, saliva and tears
native [purified from their natural source], recombinant and over-expressed

Question 13

all protein extraction methods use x and y

Answer 13

buffers and stabilising conditions i.e. low temp

Question 14

column chromatography can separate proteins based on which 4 methods

Answer 14

ion exchange, gel filtration, affinity and hydrophobic interaction

Question 15

what is affinity

Answer 15

ability of proteins / molecules to recognise specific chemical shapes and structures

Question 16

a small molecule or a x is y on a solid stationary phase- what relevance does this have to the protein separation

Answer 16

ligand affinity - immobilized
when the mobile phase which contains the protein passes through the stationary phase the protein interacts with the ligand and binding occurs- proteins with different ligand binding sites and sizes will bind @ different parts of the column - this can be elution by changing buffer concentration to increase competitor for ligand in the buffer or by changing the pH

Question 17

what is a ligand

Answer 17

The molecule that binds reversibly to a specific
molecule/group of molecules that you want to purify

Question 18

what are 3 characteristics when selecting a ligand

Answer 18

must selectively and reversibly binds and have chemically modifiable groups - so can bind -> column without destroying it’s binding activity

Question 19

give e.g. of protein ligand for
- Enzyme
-Antibody
-Lectin
-Hormone/Vitamin
-Glutathione
-Poly His fusion proteins

Answer 19

co-factor
antigen
polysaccharide
receptor
Glutathione S transferase
Metal ions

Question 20

matrix

Answer 20

place for ligand attachment - it should be chemically and physically inert

Question 21

spacer arm

Answer 21

used to improve binding between ligand and target molecule as it overcomes any effects of hinderances

Question 22

Ligand attachment matrix

Answer 22

covalent attachment of a ligand -> a suitable pre-activated matrix to create affinity medium

Question 23

pre-activated matrices

Answer 23

matrices which have been chemically modified -> facilitate binding of specific types of ligand

Question 24

list the 4 elution options

Answer 24

Change of buffer pH
– Alters the ionisation of charged groups on the ligand or bound protein
– changes the binding affinity
– pH changes must be within the range of stability of the matrix, ligand
and target protein
* Ionic strength elution
– Again, changes the ionisation of charged groups
* Competitive elution
– Use of a selective eluent, increasing concentration of a competitor
molecule, that competes for binding to the protein or to the ligand
* Use of chaotropic agent
– Used when the binding between target protein and ligand is very tight
and all other elution methods fail
– Chaotropic agent e.g. Urea or guanidine hydrochloride can be used to
unfold the protein, thereby causing dissociation from the ligand
– Should be avoided if possible as may cause irreversible denaturation
of the protein

Question 25

define a chaotropic agent- give e.g. of one

Answer 25

agent that disrupts the folding of proteins and denatures the protein
ammonium sulfate- its high salt concentration prevents proteins from forming H bonds

Question 26

What does IMAC stand for here the metals are immobilized on which phase- describe the valence of these metals
proteins bind to metals via?

Answer 26

immobilized metal affinity chromatography- immobilized on solid stationary phase i.e copper, Zn, Ni- all have divalent variance
histadine, tryptophan and cysteine A.A.

Question 27

how does elution of metals occur in IMAC X3

Answer 27

change ph of buffer
increase imidazole/another competitor buffer
addition of EDTA buffer- strips column of metal ions

Question 28

recombinant proteins can be genetically engineered to ? [His-tag]
what is His-tag used for

Answer 28

add 6 extra histidine to N or C terminus of a protein
used to purify protein via binding to NI-NTA or IDA-NTA

Question 29

what does imidazole do- it is a part of? - it is used in what type of elution

Answer 29

binds to Ni or other divalent cations through nitrogen’s
- part of histidine
- used in competitive elution of His-tagged [nickel affinity chromatography ]proteins from metal ions

Question 30

recombinant GFP purification from E.coli uses what type of purification chrom

Answer 30

recombinant His-tag affinity chromatography

Question 31

GFP purification stands for? - comes from? what was it used for

Answer 31

Green Fluorescent protein [comes from jelly fish] - used to add His-tag

Question 32

protein G and Protein A are bacterial proteins from where? -> they have high affinity for?

Answer 32

bacterial proteins
G- from Streptococci
A- from Staphylococcus aureus
for Fc region of polyclonal and monoclonal antibodies

Question 33

proteins A and G can be coupled to ? serves as… and this can be used for?

Answer 33

cross-linked agarose - and will serve as ligands for antibody purification