Protein - Analysis and Sequencing

Question 1

How are we able to distinguish/separate proteins?

Answer 1

Polypeptide diversity

There are an unlimited amount of theoretical possibilities of polypeptides - in reality they are limited in size and composition

Question 2

Why do we need to purify proteins?

Answer 2

The design of drugs that inhibit/alters the function of a protein,
The re-design of proteins with novel properties
Use of enzymes in the bio-pharmaceutical industry
Understanding of the causes of disease

Question 3

What are the two broad methods of purification?

Answer 3

Protein is common/abundant:
Use a convenient and well understood species e.g. rubisco

Protein is expressed at low levels and very specific:
Isolate from specific organism or express in each to handle species e.g. HIV proteins

Both: require breaking open the cell and fractionate by different methods

Question 4

What factors do we need to control to keep proteins stable?

Answer 4

pH - buffers prevent denaturation

Temperature - most denature at high temperatures
Protein purification tends to be at 0 degrees

Degradative enzymes (proteases/nucleases) - can be released when cells are disrupted to liberate the protein

Adsorption to surfaces - proteins can denature by contact on air-water

Question 5

Give an overview of the fractionation process of purification of proteins?

Answer 5

1. Break up the cell either mechanically or chemically (acquire the homogenate)
2. Remove most of the contaminating material by differential centrifugation (acquire the pellet and supernatant)
3. Isolate the protein by column chromatography - the unique combination of properties of the protein (e.g. mass, size, charge, shape) enable its separation
4. Check the purity by electrophoresis

Question 6

How can we track proteins through the process of purification?

Answer 6

Quantified by assays

Absorbance - some are coloured and others absorb UV-ligth (absorbance is proportional to amount of protein)

Visualisation - Using dye

Activity - identifying characteristic of an enzyme

Question 7

What are some of the most sensitive assay techniques?

Answer 7

Immunoassays that use antibodies in response to an antigen
RIA - Radioimmunoassay
ELISA - Enzyme-linked immunosorbent assay

Question 8

Describe ELIZA?

Answer 8

1. Immobilise the first antibody on a solid support
2. Incubate with a protein containing sample
3. Add a second antibody that is covalently linked to an assayable enzyme
4. Wash and assay enzyme activity

The amount of substrate converted to product indicates amount of protein present

Question 9

What are the physiochemical properties of proteins to distiguish during purification procedures?

Answer 9

Solubility - salting out
Ionic charge - ion exchange chromatography, electrophoresis & isoelectro focusing
Polarity - hydrophobic interaction chromatography
Size - gel filtration chromatography & SDS-PAGE
Binding specificity - Affinity chromatography

Question 10

Describe salting out?

Answer 10

As more salt is added, the solubility of the protein decreases = salting out
The salt allows the proteins to clump together acting as a barrier between different charged groups
Centrifuge to acquire the correct protein

Proteins precipitate at different concentrations of ammonium sulphate
Good first purification step

Question 11

What is chromatography?

Answer 11

There is a mobile and stationary phase in efforts to separate molecules
Solutes pass through the column, interacting with the stationary phase and are retarded to differing extents

Question 12

What does ion exchange chromatography do?

Answer 12

Property - Ionic charge

Charged molecules bind to oppositely charged groups chemically linked to a solid matrix (e.g. cellulose or agarose)
Amino acids have different charges due to their R groups and the pH they are in = different isoelectric points

Question 13

What are the two types of ion exchange chromatography?

Answer 13

Cationic exchange - a cation protein will bind to anionic groups in the cation exchange resin - CM: carboxymethyl

Anionic exchange - an anionic protein will bind to cationic groups in the anionic exchange resin - DEAE: diethylaminoethyl

Question 14

How can the charge of a protein be affected within ion exchange chromatography?

Answer 14

pH alters the the charge and the position of the pI in relation to pH changes the binding

Altering the pH at extreme ends could denature the protein, so changing salt concentration e.g. increasing NaCl - can displace proteins via competition through higher charge densities

Question 15

Describe hydrophobic interaction chromatography?

Answer 15

It purifies non-polar molecules
The matrix is substituted with octyl/phenyl groups
At high salt concentrations non-polar groups interact with the hydrophobic groups in the matrix

Question 16

What does gel filtration/size exclusion chromatography do?

Answer 16

Property - Size

Molecules are fed into a column of gel beads containing pores
Small molecules - diffuse through the pores (takes longer)
Larger molcules - go around the beads (come out first)

Question 17

How can we control gel filtration chromatography?

Answer 17

Different size beads and therefore pores are used to allow different sized proteins to be separated

There is a resolution range for the size of the pores to determine which proteins can be separated
Within this range, there is an inverse linear relationship between the volume at which a protein elutes and the log of the Mr

If we mimic the native conditions of a protein we can work out it’s native size or its quaternary structure

Question 18

What are the elutes and graphs in gel filtration chromatography?

Answer 18

The elute - as they come out if there is 3 for example: void - partial interaction - total interaction

On an elution graph:
Before the peak = high molecular mass contaminants
After the peak = low molecular mass contaminants

Question 19

What does affinity chromatography do?

Answer 19

Property - binding specificity

Adding a chemical tag/ligand to a protein e.g. histidine
The tag binds to a resin and we can elute the protein by altering the pH or ionic strength

Question 20

What is electrophoresis?

Answer 20

The migration of ions through an electric field
The electric field is passed through a gel - normally polyacrylamide
This forms a mesh and acts as a molecular sieve

Called PAGE - polyacrylamide gel electrophoresis

Question 21

What are the types of electrophoresis?

Answer 21

Native PAGE
SDS PAGE
Isoelectric focusing

Question 22

Describe Native PAGE?

Answer 22

Property - Size and Charge

Size - smaller proteins move faster than larger proteins
Charge - proteins with a larger charge move faster that proteins with smaller charge of the same sign

Negative to positive electrode, so pH is adjusted so proteins are negative (move down)

Question 23

How can different proteins be affected in native PAGE?

Answer 23

If a protein is quaternary it will remain in tact - not separate
Proteins of a similar size but different charges - move different distance

Question 24

Describe SDS PAGE?

Answer 24

Property - size (smaller = faster)

Sodium Dodecyl Sulphate (negatively charged detergent) binds 1 per 2 amino acids
It breaks the non-covalent structures = denaturation subunits/quaternary structure
The R groups become masked = equal charge across whole protein

Separation only depends upon the sieving effect of the acrylamide gel

Question 25

What else can be used in SDS PAGE?

Answer 25

Addition of reducing agent allows presence/absence of disulphide bonds to be identified (as many quaternary structures are stabilised by these)

Mercaptoethanol reduces disulphide bonds in proteins
e.g. antibody split into its heavy and light chains

Question 26

What can we deduce from SDS PAGE?

Answer 26

The relative mobilities of proteins vary approximately linearly with the log of their molecular mass
Low mass proteins elute first - the smaller the mass the greater the mobility

Single band by PAGE = pure protein

Question 27

What is isoelectric focusing?

Answer 27

Property - charge

Proteins are poured along a gel with a pH gradient built in and the proteins are place anywhere within
Each protein will gain / lose protons according to the pH of the liquid phase in what its in
They stop moving when they reach a pH in the gel when their charge becomes zero – at their iso-electric point (pI)

Proteins of a similar size with different pI values will move different distances

Question 28

As gel electrophoresis is slow what can be used instead?

Answer 28

Capillary Electrophoresis

Thin capillary tubes, rapidly dissipate heat and can use high electric fields - reducing separation times to a few minutes

Question 29

What is 2D gel electrophoresis?

Answer 29

Isoelectric focusing combined with SDS-PAGE

The sample is subjected to IEF in one direction and then subjected to SDS-PAGE in the perpendicular direction

Useful for proteomics

Question 30

How is electrophoresis visualised?

Answer 30

If radioactive - autoradiograph

If antibody present - immunoblotting or Western blotting

Question 31

What does ultracentrifugation do?

Answer 31

Separates macromolecules by mass

Svedbergs (S) - sedimentation velocity per unit of centrifugal force

Adding CsCl = forms a density gradient under high fravitational field