Biotech Final - Lecture 4

Question 1

What is the significance (i.e. what applications) of recombinant protein production?

Answer 1

1) Required for in-vitro function studies
2) Required for determining 3D structure of protein
3) Determine functional architechture
4) Provide functional clues
5) Determine molecular details of catalysis

Question 2

What are the basic (very basic) steps of affinity chromatography?

Answer 2

1) Incubate crude sample with affinity chromatography resin
2) Wash away non-specific molecules
3) Elute using competitive ligand

Question 3

What was the lab objective? (Lab 4)

Answer 3

1) Isolate His-PTEN using nickel affinity chromatography

2) Analyze efficiency of chromatography using SDS-PAGE analysis

Question 4

The principle of affinity chromatography is based on what?

Answer 4

On the exploitation of the specific, reversible binding between the protein of interest and a specific molecule immobilized on an inert support.

Question 5

Affinity chromatography enables the purification of a biomolecule on the basis of its?

Answer 5

Biological function or individual chemical structure.

Question 6

Affinity chromatography is efficient because?

Answer 6

1) The technique is highly selective, allowing for high resolution
2) Allows for purification based on biological characteristics
3) Can be completed in a single step
4) Can be used to separate active biomolecules from denatured or functionally different forms
5) Can isolate substances present at low concentrations in large volumes of crude sample with lots of contaminants

Question 7

What are the different interactions between ligand and target molecule that are used in affinity chromatography?

Answer 7

1) Electrostatic
2) Hydrophobic
3) VdW
4) Hydrogen bonding

Question 8

To elute the target molecule, one must reverse the interaction. How can this be done?

Answer 8

1) Specifically
i) Competitive ligand
2) Non specificallly
i) Changing pH
ii) Changing the ionic strength (salt)
iii) Changing the polarity

Question 9

What are the critical features of a pertinent ligand for affinity chromatography?

Answer 9

1) Biospecific ligand that can be covalently attached to a chromatography matrix
2) The coupled ligand must be specific to the target molecule
3) After washing, the binding between ligand and target molecule must be reversible

Question 10

Different ligands used in affinity chromatography include?

Answer 10

1) Enzymes
2) Antibodies
3) Lectin
4) Antibodies
5) Nucleic acids
6) Metal ions
7) Glutathione

Question 11

Metal ions target which type of molecules (or tag)?

Answer 11

poly-His fusion proteins

Question 12

What is a affinity chromatography matrix?

Answer 12

The matrix is an inert support to which a ligand can be directly or indirectly coupled.

Question 13

What are the properties required for an efficient and effective chromatography matrix?

Answer 13

1) Extremely low non-specific adsorption
2) Stability under a range of conditions
3) Good flow properties for rapid separation
4) An open pore structure (for larger biomolecules)

Question 14

Hydroxyl groups on _______ residues are easily derivatized for ________ attachment of a ligand. It is an ideal platform for development of affinity media.

Answer 14

1) Sugar

2) Covalent

Question 15

What is adsorption?

Answer 15

The adhesion of molecules to a solid surface.

Question 16

What are spacer arms?

Answer 16

A spacer arm is interposed between the matrix and the ligand to facilitate effective binding.

Question 17

Spacer arms must be designed with what in mind?

Answer 17

1) Maximize binding

2) Avoid non-specific binding effects

Question 18

Why are spacer arms used?

Answer 18

The binding site of a target protein is often located deep within the molecule and an affinity medium prepared by coupling small ligands directly to the matrix may exhibit low binding capacity due to steric effects.

Question 19

What are the four typical elution methods?

Answer 19

All weaken the interaction between ligand and target molecule.

1) Change buffer composition
2) Change pH
3) Competition for binding with target
4) Competitive ligand binding (most common)

Question 20

In the affinity purification lab, what tag was fused to the protein, and where?

Answer 20

His6 tag fused to C-terminus of PTEN

Question 21

What is chelation?

Answer 21

The formation of presence of bonds between two or more separate binding sites within the same ligand and a single central atom.

Question 22

What amino acids form complexes with the chelated metals around neutral pH?

Answer 22

Histidine and cysteine.

Question 23

What makes the polyHis tag so great?

Answer 23

At neutral pH, the tag is unchanged and relatively small in size, thus it doesn’t affect secretion, compartmentalization, or folding of the fusion protein within the cell.

Question 24

How is an IMAC matrix made?

Answer 24

The matrix is made by coupling a metal chelate forming ligand to agarose. Before use, the matrix is loaded with a solution of divalent metal ions.

Question 25

Divalent metal ions include?

Answer 25

Nickel, Zinc, Copper, Calcium, Cobalt, Iron

Question 26

In this lab, what chromatography type did we use and what elution buffer was used?

Answer 26

We used nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography matrix for the isolation of His-PTEN and elution was performed using imidazole buffer.