Lecture 7 - Cellular and molecular techniques Flashcards
(24 cards)
What are the three steps in function investigation using mutation?
- Design primers to introduce a mutation
- Confirm mutation using sequence
- Express a protein to check function
What can we do with DNA vs RNA?
DNA can be used to transfect cells and RNA can be used to inject oocytes
What can we do with the purified protein produced from cells (2)?
Crystallography studies
Liposome reconstitution
Why use E.Coli cells for protein expression vs human mammalian cells?
The higher the complexity of the systems, the longer it takes to double/reproduce the cell
How to arabinose and IPTG induce over-expressioin
They bind to the inhibitor of the gene to allow the polymerase to bind to the DNA to translate it
How are his-tags used to purify proteins?
- Cells are crushed, concentrated, spun and run through a column
- Proteins with his-tag will attach to the column
- Column is washed with buffer
- Protein is eluted using a molecule with a higher affinity to NTA/nickel (imidazole)
What are the four characteristics of a protein for it to crystallise?
Pure/homogenous
Stable
Soluble
Voluminous
How is the his-tag removed
Thrombin digest
What are the two crystallography studies that can be done with purified protein?
Binding assay
Isothermal titration calorimetry
What does isothermal titration calorimetry measure?
Thermodynamic parameters in a solution between a ligand and a protein
What type of assay is used to examine the binding of a ligand or substrate?
Fluorescence based assay, intrinsic F or introduced F
What can be measured in a liposome reconstitution assay with GLTph?
The uptake of glutamate and the movement of sodium which is required for the uptake.
What is protein crystallography?
The visualisation of protein structures at atomic state resolution
What does protein crystallography allow us to do? (2)
Understand conformational changes in proteins
Develop drugs to specifically bind to proteins
What EMR does protein crystallography use?
X-rays
Why do we require crystals for amplification of signal in x-ray crystallography?
Crystals have many unit cells and there is more scattering
How does a synchrotron produce EMR
By accelerating electrons almost to the speed of light
Are hydrophobic proteins membrane bound or soluble generally?
Membrane bound
Why are membrane proteins difficult to perform x-ray crystallography for?
They are difficult to crystallise because of the difficulty of removing them from the membrane
What is required to solubilise the membrane proteins?
Detergent
What is the human glutamate transporter
An excitatory amino acid transporter
What information do we have on the human glutamate transporter (4)?
DNA sequence
Amino acid-sequence
Hydropathy plot
Biochemical techniques - 2D topology
What is used to determine function and structure of EAATs instead?
GltPh
What information have we gained from crystallography of the homologue?
Transmembrane domains/topology
Residues implicated in substrate and ion-binding (in C-terminus)
