Lecture 5

Question 1

The biologics market?

Answer 1

Typical (traditional) drugs are small molecules, such as aspirin or
ibuprofen. Biologic drugs are produced from biological resources and are typically proteins, such as antibodies or insulin (like IgG or insulin).

• These proteins need to be produced and purified before they can be sold or purchased ($418 billion in 2022)

Question 2

Salting in/out of proteins?

Answer 2

For a lot of proteins, there is an optimal salt concentration. For the vast majority of globular proteins, you can get a salt concentration in solution thats a bit low and proteins tend to be soluble. Then, there’s a solute concentration range in which proteins have their maximum solubility (over an interval of concentration). When this interval is exceeded, the protein can become insoluble and precipitate out.

Question 3

Salting in/out definition?

Answer 3

In general, the solubility
of proteins can be
affected by the
concentration of
dissolved salts, with an
increase, followed by a
decrease with increasing
salt concentration.

Question 4

Ammonium sulfate precipitation?

Answer 4

Ammonium sulfate, (NH4)2SO4, interacts strongly with water, leaving less water to surround proteins. The addition of this salt therefore leads to protein precipitation. Different proteins precipitate at different concentrations of this salt, so they can be enriched or purified using specific concentrations of (NH4)2SO4. By titrating, you can often get your protein of interest fairly pure!

Question 5

General chromatography setup?

Answer 5

There is a column in which liquids can go in the top and through the column either by gravity or pressure induced by a pump, and then drip out of the bottom of the column. Inside the column is a material in which proteins will behave differently from one another: this is the basis for the separation.

Question 6

What is the material in chromatography?

Answer 6

Material is normally called resin or beads

resin - thick, slurry
beads - small beads at microscopic level

Question 7

What does chromatography include?

Answer 7

A mixture of molecules, a material in which proteins behave differently (beads/resin), buffer applied to column, and finally separated proteins at the end.

Question 8

What does chromatography do?

Answer 8

Chromatography separates molecules and it is a commonly used protein purification method. It involves a stationary phase (usually beads in a column) and a mobile phase (buffer that runs through the column).

Question 9

Size exclusion chromatography?

Answer 9

Size exclusion (gel filtration) chromatography separates proteins based on their size.

Stationary phase: porous beads with channels in them

• Small molecules are able to go into the channels, thus taking them more time to get out of the column.
• Large molecules do not fit into the channels, leaving them to easily come out of the column.

Question 10

What does size exclusion chromatography allow us to do?

Answer 10

We can estimate the molecular weight of a protein based on its retention volume.

Question 11

Ion exchange chromatography?

Answer 11

Separates proteins by charge. Useful if molecules are the same or similar size. If, however, they have different charges, you use anion exchange beads (positively charged) to attach/bind to negatively charged proteins or cation exchange beads (negatively charged) to attach to positively charged proteins.

• The beads are named for what they interact with - not what they are!

Question 12

Anion exchange bead?

Answer 12

Negatively charged proteins (anions) can bind to these positively charged beads.

Question 13

Cation exchange bead?

Answer 13

Positively charged proteins (cations) can bind to these negatively charged beads.

Question 14

How is the other protein eluted in Ion exchange chromatography?

Answer 14

You can elute the protein by adding a salt like NaCl or changing the pH. Ion exchange chromatography separates proteins based on their charge.

Question 15

Affinity Chromatography?

Answer 15

Affinity chromatography separates proteins based on their ligands/what they bind to.

Ex. Antibody binds to a particulaer antigen, if you immobilize that antigen on a bead, then the antibody will go and bind to to that antigen on the bead, thereby purifying the antibody.

NOTE: whatever is on the bead has to be particular to the protein you are trying to obtain.

Question 16

Affinity chromatography steps?

Answer 16

1. A mixture of proteins in added to column
2. Wash away any excess proteins that don’t bind
3. Ligand solution is added to compete (elute) proteins from column.

Question 17

Affinity bead example?

Answer 17

Mannose Sugar:
- Manose binding proteins will bind to these mannose beads. Any excess proteins will wash through.
- Excess mannose can be added to compete with the bound protein which is eluted.

Question 18

Affinity Tags?

Answer 18

Ligand binding proteins that, when they fold up, are able to recognize a ligand. If those are produced in a recombinant protein, then that protein can be bound to affinity beads with that ligand on it.

When people are expressing proteins, such as mammalian proteins and bacteria, they want to purify them very easily, they can express the protein as a longer peptide chain that encodes affinity tags.

Question 19

Affinity tag definition?

Answer 19

When recombinant proteins are designed, a sequence is often added to one end of the protein that binds to a ligand that is available on beads.
These sequences are often called affinity tags.

The affinity tag will allow the recombinant protein to be purified easily on beads with the ligand to which the tag binds.

an affinity tag can also be added to a recombinant protein for expression.

Question 20

6x histidine?

Answer 20

A common affinity tag, 6x histidine binds to beads with a coordinated nickel.

• Imidazole is a small molecule
  that corresponds to the R group
  of His. So, it can displace the
  6xHis tagged protein from the
  beads allowing the protein to be
  eluted from the column.
• Stationary phase has immobilized nickel

Question 21

Electrophoresis?

Answer 21

Electrophoresis is the separation of charged molecules in an electric field
Most common method is SDS polyacrylamide gel electrophoresis (SDS-PAGE).

Question 22

Step 1 of electrophoresis?

Answer 22

Heat the protein in sample buffer.
Sample buffer contains the detergent SDS and a reducing agent (e.g. β-
mercaptoethanol).

Sodium dodecyl sulfate (SDS) is a negatively charged ionic detergent. It has three things here:
1. SDS, along with heat, contributes
to unfolding the protein.
2. SDS keeps the protein unfolded by binding to it
3. SDS gives the protein a uniform
negative charge.

Question 23

Sample buffer: the reducing agent?

Answer 23

Some cysteines that are oxidized to cystines (which have disulfide bond), causing loops in proteins. These need to be broken!

• Cystines can be reduced to cysteines by using the reducing agent Beta-mercaptoethanol (B-m).

Question 24

Step 2 of Electrophoresis?

Answer 24

Separate the protein out by size using a gel and an electric
field. Since the proteins are denatured (linear) and negatively
charged, they will migrate according to their length (size). Bigger ones will be at the top of the plate (harder to move) and smaller at the bottom (easier to move). Move from cathode to anode (+ to -)

Question 25

Result of electrophoresis?

Answer 25

Electrophoresis efficiently characterizes the size and purity of protein samples.

Question 26

Gel filtration chromatography estimates the molecular weight of a protein to be 100 kDa. However, when your co-worker analyzes the protein by reducing SDS-PAGE it is observed to only have a size of 25 kDa. Which statement below explains this result? A. Your co-worker is incompetent B. The protein has degraded prior to gel filtration chromatography C. The protein exists in solution as a homotetramer D. The protein aggregated during SDS-PAGE analysis E. All of the above

Answer 26

C! This protein exists in solution as a homotetramer. The protein is in its native state (folded) in gel filtration chromatography. The protein is denatured (unfolded and therefore dissociated if it had quaternary structure) in SDS-PAGE. It is a protein with quaternary structure that forms a homotetramer. The native tetramer would be 100 kDa, whereas the subunit sizes would be 25 kDa.

Question 27

X-ray Crystallography?

Answer 27

The 3D arrangement of atoms in a protein can be deduced by measuring the diffraction of X-rays in a protein crystal. ~214,000 protein structures are now available in the Protein Data Bank (PDB), ~90% from X-ray crystallography

Question 28

Cryo-electron microscopy?

Answer 28

Cryo-EM uses electrons to determine high-resolution protein structures Excellent for large proteins and complex structures such as the nuclear pore and no crystal is needed! - Resolution differences between 2013 and current is amazing - so much more detailed.

Question 29

Nuclear magnetic resonance (NMR)?

Answer 29

Nuclear magnetic resonance (NMR) spectroscopy uses superconducting magnets to measure the magnetic environment of nuclei. - This can be used to determine protein structure and motions (dynamics) - Assign NMR resonances

Question 30

Alzheimer's Disease?

Answer 30

The 3D structure of Aβ(1–42) fibrils relevant to Alzheimer's disease. - Current drug design targets the surface of the amyloid fibrils