Techniques for Crystallisation Flashcards
How can proteins be stabilised for crystallisation?
Using ligands, Fab fragments or detergent.
What are the advantages and disadvantages to using bacteria for recombinant expression?
Quick doubling time, but no DS bonds or PTMs, inclusion bodies can form easily from overexpression and toxicity induced death can occur.
What are the advantages of using mammalian cells for recombinant expression?
Can have PTMs, DS bonds and complexes.
What mammalian cells are often using for recombinant expression?
Chinese hamster ovary cells
Why is the PTM glycosylation often removed when added in eukaryotic expression systems?
Because it is highly flexible - this is not good for crystallisation of a protein.
What proteins are often expressed in baclovirus infected insect cells recombinantly?
Complex eukaryotic proteins e.g. Kinases and multi-protein complexes.
What is multi-BAC
Multiple reading frames are expressed all at once of a complex.
Name 4 common affinity tags.
His6, His8, GST, MBP
Why are GST and MBP sometimes favourable?
They are larger so assist in folding the protein correctly.
Why does a cleavage site near the affinity tag need to be encoded?
Because the tags often have variable charges (His) and this hinders crystallisation.
Which protease removes affinity tags?
TEV protease.
What is used to elute proteins with a His tag from a cell lysate?
Imidazole
What can be done to a protein to make it more crystallisable?
Bioinformatics can be used to identify disordered and stable regions.
Introduce mutations to improve thermal stability (without removing function).
What is the last step before crystallisation?
Size exclusion chromatography - ensures one species and oligomeric state.
What is SEC combined with to ensure correct oligomer of protein is eluted?
MALS - this measures the mass of a protein as its eluted.
Do you want most the interactions to be between the hydration layers or protein-protein interactions?
Hydration layer
When is a protein least soluble?
At its isoelectric point (PI) - because protein has no charge.
How can the PI of the protein be used in crystallography?
The solution can be concentrated to the PI of the protein to help precipitation.
What are 2 common precipitants?
Ammonium sulphate and polyethylene glycols (PEG).
What is the solubility phase correlation?
This relates the precipitant concentration (c) to the protein concentration (p).
What region do the precipitant and protien concentrations need to be for spontaneous homogeneous nucleation?
Metastable region of graph
If precipitant concentration is low, is the protein likely to precipitate?
No
If precipitant concentration is high, what happens?
Proteins will aggregate/small crystals form.
Describe the Batch crystallisation technique.
Sealed glass box is used - wait for water to evaporate and leave a crystal.
