Practicals 1 and 2

Question 1

Where is beta-galactosidase from?

Answer 1

Escherichia coli.

Question 2

Why was beta-galactosidase chosen for these practicals?

Answer 2

It’s biochemical properties can be studied with relative ease.

It is used ubiquitously (everywhere) in molecular biology to aid in screening for cloned products.

Question 3

What is the function of beta-galactosidase?

Answer 3

Recognises galactose in di-, oligo-, and poly- saccharides and catalyses the hydrolysis of the glycosidic bond to form beta-galactose.

Many beta-galactosidases act on lactose (these enzymes are called lactases).

Question 4

What is lactose?

Answer 4

A disaccharide of galactose and glucose where galactose is bound by a beta-glycosidic bond to the hydroxyl group on carbon 4 of glucose.

Bond is cleaved by beta-galactosidase.

Question 5

Which three biochemical tools are used in these experiments?

Answer 5

N-terminal poly-histidine tag.

Fluorescent protein tag.

Chromogenic substrate.

Question 6

What is the purpose of the N-terminal poly-histidine tag?

Answer 6

Affinity tag allows easy purification of beta galactosidase from a mixture of other proteins.
Poly-histidine tag (or hexa histidine-tag/6xHis-tag/trademark name = His-tag) is an amino acid motif in proteins that consists of 6 His residues.

The term “affinity” in “affinity tag” refers to the specific and strong binding interaction that the tag has with a particular molecule, often immobilized on a resin or column.

An amino acid motif is a specific sequence or pattern of amino acids within a protein that has a distinct structural or functional role.

Question 7

How is the His-tag usually added to the protein?

Answer 7

By cloning the functional gene downstream of the His tagged in a suitable expression vector.

Question 8

Which vector have we used?

Answer 8

Vector produced by Qiagen called pQE80 which attaches a His-tag to the N-terminus of the expressed protein (mCherry-beta-galactosidase).

Question 9

What is mCherry-beta-galactosidase?

Answer 9

A fusion protein of a pink fluorescent protein (mCherry) to beta-galactosidase.

Question 10

What is the purpose of mCherry?

Answer 10

To make the protein easy to see so that it is easier to follow what is happening during purification.

Question 11

When mCherry-beta-galactosidase is expressed in E.coli, how is this protein separated from all the other proteins?

Answer 11

The histidine residues of the His-tag have a high affinity for metals (particularly nickel) so will bind strongly to the nickel column (forming co-ordinate bonds). It is the imidazole group of histidine that binds to the column.

The pure protein can then be eluted using a chemical called imidazole that competes with histidine for the nickel and displaces the bound protein.

Eluted refers to the process of extracting one substance from a mixture, typically by washing it out with a solvent

Question 12

What are chromogenic substrates?

Answer 12

When acted upon by a specific enzyme, they undergo a chemical change that produces a visible colour.

Question 13

Why have we used chromogenic substrates here?

Answer 13

To allow for the direct detection of the purified active protein and enable us to undertake kinetic and inhibition studies.

Usually the reaction of lactose to glucose and galactose is difficult to detect so a modified substrate is often used.

Question 14

Which chromogenic substrates are used in these practicals?

Answer 14

X-gal and para-nitrophenol-galactose (pNPG).

Question 15

Both are artificial substrates in which the chromophore is linked to beta-galactosidase.
What happens once they are added to beta-galactosidase?

Answer 15

The chromophore is cleaved off and results in a coloured product.

Question 16

With X-gal, what does the spontaneous dimerization and oxidation of the product result in?

Answer 16

Formation of 5,5’-dibromo-4,4’-dichloro-indigo which is blue in colour.

Can be seen by eye.

Question 17

The chromogenic substance pNPG uses a different chromophore called paranitrophenol which is linked to beta-galactosidase by a heteroglycosidic bond.

When this substrate is added to beta-galactosidase what happens? What about at an alkaline pH?

Answer 17

p-nitrophenol is formed.
At an alkaline pH (pKa ~ 9) produces a phenolate anion with an intense yellow colour.

Question 18

How can the intense yellow colour be measured?

Answer 18

Easily in a spectrophotometer.

Done in session 3 and 4 to measure enzyme kinetics.

Question 19

Why is the E.coli cell lysate kept on ice?

Answer 19

Using ice ensures that the lysate remains as close to its natural state as possible. Inhibits bacterial growth, prevents loss of sensitive biomolecules that may be unstable at higher temperatures, prevents protein degradation.

Question 20

Why should you carefully pour 4mL of mixed protein solution into the column?

Answer 20

To avoid disrupting the top of the fragile packed column too much.

Question 21

Why mustn’t you let the column run dry?

Answer 21

A nickel column should not be allowed to run dry when eluting protein because it can disrupt the structure and binding properties of the resin.

Resin can crack/form air bubbles disrupting uniform flow.
Poor recovery of target protein as nickel ions can redistribute meaning protein binding is weakened.
Abrupt drying may expose bound proteins to air, which can cause some proteins to denature.

Question 22

Why is the flow through yellow and the inside of the column pink?

Answer 22

The fraction in the flacon tube doesn’t have any visible levels of beta-galactosidase because most beta-galactosidase is in the column.

Question 23

Why is the flow through passed through the column a second time?

Answer 23

To ensure maximum binding of the target protein to the resin.
In the first pass, not all of the target protein molecules may have had enough time or opportunity to interact with the binding sites on the resin.

Question 24

Why is the column washed by several buffers when eluting proteins?

Answer 24

During protein elution, washing the column with several different buffers is crucial for removing non-specifically bound proteins and impurities, while selectively eluting the target protein with high purity.

Buffer A = wash through unbound substances.
Buffer B = imidazole to knock off proteins weakly bound to column. Fainter yellow fraction because less protein than previous fraction.
Buffer C = higher concentration to compete with His-tag and knock beta-galactosidase off column into collection tube (elute His-tagged proteins).

Question 25

Why is glycerol added to beta-galactosidase before it is frozen?

Answer 25

To ensure the purified protein retains activity during subsequent freeze-thaw cycles.

Ice crystals could cause protein denaturation by disrupting hydrogen bonds.
Glycerol reduces the chances of aggregation by stabilizing the enzyme in solution.

Question 26

How can protein fractions be analysed?

Answer 26

Using X-gal on microtitre plate and observing intensity of colour change after 2 minutes.

Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Run samples alongside molecular weight markers.
Sample falls into upright gel as it is denser than buffer.
Beta-galactosidase = 150kDa

kilodalton
1 Da is roughly equivalent to the mass of a single hydrogen atom or 1/12 the mass of a carbon-12 atom (approximately
1.66 ×10^-24 grams).

Question 27

When buffer C is added, why is a smaller volume added than buffer B?

Answer 27

Eluting with a smaller volume means the solution of protein (beta-galactosidase) will be more concentrated.

Question 28

Why is it important for us to know the final protein concentration?

Answer 28

In order to calculate the enzyme specific activity in practical 3.

Question 29

What technique did we use to determine the final protein concentration?

Answer 29

Bradford assay - unknown samples are compared to a calibration/standard curve of absorbance against concentration.

Standard curve made up of known concentrations of BSA (Bovine Serum Albium) protein. Serial dilutions.

Question 30

What is Bradford reagent?

Answer 30

Changes colour from brown to blue in proportion to the concentration of protein.

Question 31

How is the colour change detected?

Answer 31

By a spectrophotometer/plate reader.

Question 32

Give the equation for working out volume of BSA for serial dilutions.

Answer 32

C1V1 = C2V2

Question 33

Why does contaminating E.coli proteins bind weakly to the column?

Answer 33

They happen to have a run of a few consecutive histidine residues.

Question 34

What causes Bradford reagent to change colour when proteins are added?

Answer 34

Proteins preferentially bind and stabilise blue (cationic) form of dye.

Question 35

Why is it important the concentration of samples falls within the range of the standard curve?

Answer 35

• Extrapolation outside range is risky as
  trend may not continue.
• Bradford reagent saturates at high protein
  concentrations.