Flashcards in Biotech Final - Lecture 4 Deck (26)
What is the significance (i.e. what applications) of recombinant protein production?
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
What are the basic (very basic) steps of affinity chromatography?
1) Incubate crude sample with affinity chromatography resin
2) Wash away non-specific molecules
3) Elute using competitive ligand
What was the lab objective? (Lab 4)
1) Isolate His-PTEN using nickel affinity chromatography
2) Analyze efficiency of chromatography using SDS-PAGE analysis
The principle of affinity chromatography is based on what?
On the exploitation of the specific, reversible binding between the protein of interest and a specific molecule immobilized on an inert support.
Affinity chromatography enables the purification of a biomolecule on the basis of its?
Biological function or individual chemical structure.
Affinity chromatography is efficient because?
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
What are the different interactions between ligand and target molecule that are used in affinity chromatography?
4) Hydrogen bonding
To elute the target molecule, one must reverse the interaction. How can this be done?
i) Competitive ligand
2) Non specificallly
i) Changing pH
ii) Changing the ionic strength (salt)
iii) Changing the polarity
What are the critical features of a pertinent ligand for affinity chromatography?
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
Different ligands used in affinity chromatography include?
5) Nucleic acids
6) Metal ions
Metal ions target which type of molecules (or tag)?
poly-His fusion proteins
What is a affinity chromatography matrix?
The matrix is an inert support to which a ligand can be directly or indirectly coupled.
What are the properties required for an efficient and effective chromatography matrix?
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)
Hydroxyl groups on _______ residues are easily derivatized for ________ attachment of a ligand. It is an ideal platform for development of affinity media.
What is adsorption?
The adhesion of molecules to a solid surface.
What are spacer arms?
A spacer arm is interposed between the matrix and the ligand to facilitate effective binding.
Spacer arms must be designed with what in mind?
1) Maximize binding
2) Avoid non-specific binding effects
Why are spacer arms used?
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.
What are the four typical elution methods?
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)
In the affinity purification lab, what tag was fused to the protein, and where?
His6 tag fused to C-terminus of PTEN
What is chelation?
The formation of presence of bonds between two or more separate binding sites within the same ligand and a single central atom.
What amino acids form complexes with the chelated metals around neutral pH?
Histidine and cysteine.
What makes the polyHis tag so great?
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.
How is an IMAC matrix made?
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.
Divalent metal ions include?
Nickel, Zinc, Copper, Calcium, Cobalt, Iron