Protein purification and characterisation

Question 1

True or false: Key metabolic processes are common to many organisms

Answer 1

True. It is seen from the biochemistry (proteins)

Question 2

Proteins are how many percent of all cellular systems?

Answer 2

60-70%

Question 3

Proteins consist of linear polymers called…

Answer 3

polypeptides

Question 4

Proteins are … and never branched

Answer 4

Linear

Question 5

What are the bonds called that link amino acids together?

Answer 5

Peptide bonds

Question 6

What are the individual amino acids within a protein called?

Answer 6

Residues

Question 7

What is the smallest known protein of only nine residues called?

Answer 7

Oxytoxin

Question 8

The largest protein titin is over 25000 residues and is found in the…

Answer 8

muscles

Question 9

How many residues do proteins generally have?

Answer 9

100 to 1000

Question 10

In the absence of stabilising forces how many residues are needed to adopt stable 3D structure in water?

Answer 10

40

Question 11

Edman degradation is done by systematically removing the amino acids one at a time, why is it done?

Answer 11

To determine protein sequence

Question 12

What are the four levels of protein structure called?

Answer 12

Primary, secondary (alpha helix, beta sheets), tertiary (domains), quaternary (two or more forming complexes)

Question 13

Experiments have shown that the final 3D tertiary structure is determined by the

Answer 13

primary structure, the amino acid sequence

Question 14

The shape of the protein determines its…

Answer 14

function

Question 15

T/F: Proteins play a central role in all biological processes

Answer 15

T

Question 16

What is the name of the type of protein that catalyses

Answer 16

Enzyme

Question 17

What is the name/example of the type of protein that transports and stores

Answer 17

Haemoglobin, myoglobin

Question 18

What is the name of the type of protein that acts as mechaninal support

Answer 18

Collagen

Question 19

What is the name of the type of protein that acts as immune protection

Answer 19

Immunoglobin

Question 20

What is the name of the type of protein that transmits nerve pulses

Answer 20

Rhodopsin

Question 21

What is the name of the type of protein that controls growt and differentiation

Answer 21

Growth factor

Question 22

What is an example of a protein function?

Answer 22

Regulate and repair DNA

Question 23

Structure of a protein defines

Answer 23

interactions
site specificity
hydrodynamics
association

Question 24

A protein has non-covalent bonds too, what are these called?

Answer 24

Ionic bonds
Hydrogen bonds
Van der Waals forces

Question 25

T/F: A protein always folds to its most stable state which has the lowest possible energy

Answer 25

T

Question 26

What does the hydrophobic effect mean?

Answer 26

That the nonpolar molecules want to "stay inside" and be shielded from water interactions

Question 27

Protein folding is a --- process

Answer 27

spontaneous highly cooperative hard to predict lowest energy

Question 28

What do chaperones do?

Answer 28

Help to fold proteins

Question 29

Examples of post-translational modifications

Answer 29

Addition of: - phosphate groups - methyl group - hydroxyl groups - lipids - acetyl groups - ubiquitin Formation of disulfide bonds

Question 30

Proteins can aggregate. The environment has a strong effect. What are the factors that affect in the environment?

Answer 30

pH Temperature Ionic strength other molecules

Question 31

In what ways can we characterise a protein?

Answer 31

Using its binding activity 3D structure

Question 32

Why are proteins negatively charged at high and positively charged at low pH?

Answer 32

Because of their composition in charged amino acids, proteins will have an isoelectric point. At low pH the protein is protonated therefore positively charged. At high pH the protein is deprotonated therefore negatively charged.

Question 33

What is an isoelectric point?

Answer 33

The isoelectric point is the pH at which the protein net charge is 0

Question 34

How to find out the isoelectric point of a protein?

Answer 34

Experimentally: isoelectric focusing, potentiometric titration Theoretically: from sequence and the knowledge of the various amino acids pKa + the N and C terminal contributions. The theoretical pI may differ from the experimentally determined one, because folding (3D structure) is not taken into account. In the 3D structure, the accessibility of the different amino acids would depend on their location.

Question 35

You want to purify a protein from bovine muscle tissue. Unfortunately, you do not have any suitable protocol, but you know a lot about the protein: - The protein is found in the cytoplasm of muscle cells (cattle) - Molar mass: 145 kDa - Isoelectric point: 5 - Amino acid composition shows a relatively high content of glutamic acid and histidine Design a purification procedure that also utilizes the above tasks! (and the internet)

Answer 35

1) Here you need to disintegrate and homogenise using solubilizing agent (detergent, salt, etc.) and mechanical distruption of tissue. 2) Ion exchanger. Proteins can be purified with both cations and anions but one may be more efficient for your particular protein. If you know the isoelectric point (pI) of your protein, start with a cation exchanger for your capture step if pI>7, and with an anion exchanger if pI<7. 3) SEC for a polishing step An interesting alternative 1) similar to above 2) precipitate your protein at pH5. It could be that your protein and only a few have such a low pI. Selective precipitation at pH5 will results in a pellet enriched with your protein. 3) polishing step with SEC for example -

Question 36

Design a protein purification from the bacterium E. coli?

Answer 36

Here the lysis is much simpler, a lysis buffer and sonication or freeze-thawing cycles

Question 37

How do you check that your purification leads to an increasingly cleaner protein? After all, it would be terrible if the whole purification procedure led to you enriching the contaminants and throwing away your target protein. Therefore, it is important to check how things are going after each purification step. Suppose you are cleaning up an enzyme.

Answer 37

Here we want to understand the general guiding principles, in other words what do we expect. Each step should lead to: i. Fewer and fewer total protein content (you are removing other proteins but your enzyme) ii. Increasing specific activity (specific activity = Units/mg protein; 1 Unit = 1 micromole formed product per minute). Any steps could work. Note1: Activity is measured using a specific substrate Note 2: Purity could be estimated from an image analysis of some standard gel electrophoresis. For example, measuring the bands intensities and calculating a relative % to the total intensity from all the bands. ImageJ (an open source software) has very good routine for that.