Lecture 1 Flashcards
(45 cards)
Define protein purification.
Purification of a target protein from a complex mixture of proteins and other cell materials.
What happens in the Edman cycle?
The N-terminal of a protein reacts with PITC forming a Phenylthiocarbamoyl derivative. Then under acidic conditions, the molecule splits, forming an AZT-amino acid (Azidothymidine) and the original protein, which can then be reused in the next Edman degradation.
What can happen to an AZT-amino acid when separated via HPLC?
A PTH (phenylthiohydantoin)-amino acid will form as the terminal amino acid is cleaved.
What is HPLC?
High performance liquid chromatography.
What 3 things can we discover from the amino acid sequence of a purified protein?
- The homology of the protein.
- cDNA determination and genetic over-expression.
- prediction of secondary/tertiary structure from AA sequence.
What is homology?
A similarity often attributable to common origin (Merriam-Webster).
What can be used to discover the homology of an amino acid sequence?
BLAST (basic local alignment search tool).
2 reasons being able to determine cDNA important in protein purification?
It allows you to engineer bacteria that can over-express protein of interest.
Can harvest DNA from bacteria and use to Transfect other types of cell.
An example of transfection?
How is its product normally produced?
Transfection of amyloid precursor protein in human neuroblastoma cells.
β and γ secretase enzymes cut Amyloid Precursor Protein (APP), producing Amyloid Beta (Aβ).
Where are +ve residues typically likely to be found in a protein?
On the exterior.
Where are hydrophobic residues likely to be found in a protein?
On the interior.
What technique allows you to see the position of individual atomic nuclei within a molecule?
NMR (nuclear magnetic resonance).
What technique allows you to see an electron density map?
X-ray crystallography.
2 biomedical benefits to protein purification?
Inhibitors/modulators can be modelled by understanding 3D structure of active site.
Enables enzyme replacement therapy via intravenous introduction of purified recombinant protein.
An example of a molecule that could be inhibited to aid inhibition of Alzheimer’s?
BACE1 (Beta site Amyloid precursor protein Cleaving Enzyme 1).
What causes Gaucher’s disease?
Toxic accumulation of glucocerebrosides in lysosomes, caused by deficiency of glucocerebrosidase IV.
4 methods of purification?
Salt precipitation, size exclusion chromatography, protein assays, SDS-PAGE.
What must happen before protein purification?
Extraction and clarification of sample.
What is clarification?
ADD DEFn W/DICTIONARY.
What 4 things should be considered when deciding on type of material to extract protein from?
Species, (i.e. size, relative expression, possible cross reaction with antibody test?)
Abundance, (highest activity/g)
Stability, (speed of degradation?)
Secretion (can it be secreted via signal peptide - easier to purify).
What can be a potential problem when obtaining protein via bacterial secretion?
Some bacteria also secrete highly active proteases.
Outline of secretion pathway for a protein in cell?
Ribosomes on ER synthesise protein, packaged and labelled at Golgi, before being released at plasma membrane via exocytosis.
5 methods for homogenisation?
Blending, Agitation with abrasives, Ultrasonics, Liquid extrusion, Explosive decompression (nitrogen bomb).
When is blending used? With what apparatus?
Advantage and potential disadvantage and solution?
Used with animal tissues.
Waring blender (metal exterior to conduct heat) or rotor-stator.
Large scale. Generates heat, but can be pre-cooled and performed on ice.
