Protein Purification Flashcards
There are many reasons to purify protein. What are the 3 reasons discussed in class?
Structure determination
Affinity assays
Functional characterization
What natural sources are rich in proteins? What happens if they are not?
Sperm whale muscles
Pancreas
Bacteria
If protein is not abundant, large amounts of starting material is required
How can proteins be overexpressed?
Via genetically modified systems i.e. E coli, baculovirus, etc
Describe the process of protein expression in E Coli
- Make plasmid containing the GOI
- Transform Plasmid in E. Coli
- Grow cells in shaker
- Adjust temperature and add inducer for expression of protein
- Harvest cells
What are 4 properties of E. Coli plasmids?
DNA pol Origin of Replication
Antibiotic resistance (for selection)
Regulated transcription promotor, usually controlled by an inducible operator like the lac operon
A multiple cloning site
Which E coli strains are engineered for protein expression? Why are they engineered?
BL21
JM109
C41
They lack proteases that would normally degrade overexpressed protein, and may contain DE3 gene encoding a special polymerase
How are plasmids incorporated into E Coli?
Heat shock
Electroporation
What are the 3 pros and 2 cons of Protein expression via E coli?
Pros:
Easy and fast
Cheap
Can use minimal medium (for incorporation of NMR isotopes)
Cons:
Heterologous expression (lack mammalian chaperones etc)
Fast translation rate may lead to aggregates “inclusion bodies”
What is Pichia Pastoris?
A yeast strain that can use methanol as a sole carbon source
How can P. pastoris be converted to secretion mode?
By attaching the alpha mating factor in medium
When would you use P. pastoris over eg E Coli?
For secreted glycosylated protein expression
How can you use S. frugiperda to express protein?
Isolate clonal cells Sf9 or Sf21 from moths, which are susceptible to infection by baculoviruses (large lytic viruses)
Large “bacmids” need to be generated by homologous recombination with smaller E. Coli plasmids
Co-transfected in insect cells with plasmids containing viral genes
(expensive and lengthy)
What mammalian cells do you use to express protein?
Human embryonic kidney cells (HEK293)
Or Chinese Hamster Ovary cells
When would you use Mammalian cells to express protein?
Useful for protein overexpression in tissue culture or suspension culture
Idela environment for mammalian protein expression (contains all necessary factors for proper folding)
How do you get transfected cells with plasmids to express transiently?
A strong promoter such as Cytomegalovirus
What is an affinity tag and how could it be useful?
A region in the gene coding for a protein domain that can easily be filtered out during a purification assay (eg His6, GST, biotin)
Why wouldn’t you put an affinity tag at the start or end of a gene?
Could affect function of the protein
If you’re adding an affinity tag, what else should you add to make sure the protein behaves properly?
Protease site, to remove the affinity tag after purification (Rhinovirus 3C, TEV, Thrombin)
What is codon optimization and when is it important?
Different codons code for the same amino acid, but some codons are favored in certain organisms.
In the case of heterologous transfections, you may need to optimize your gene to reflect the relevant concentration of tRNA
How are cells collected after protein expression is completed?
Low-speed centrifugation
What are the main components of resuspension medium and what is their function?
Buffer (maintain pH) - HEPES or Tris
Salts to maintain solubility (salting in effect)
Detergents (to extract membrane-bound proteins) - CHAPS triton or deoxycholate
Urea to solubilize protein aggregates
Protease inhibitors, EDTA
What are 5 ways to lyse cells?
Sonication (good for bacteria and mammalian cells)
French cell press (bacteria and yeast)
Freezing-grinding (yeast)
Lysozyme treatment (gram-negative bacteria)
Dounce Homogenizer (mammalian cells)
How is debris removed from cells?
High speed centrifugation
What are the two components to Chromatography, and the key principle that makes it useful?
Mobile phase (buffer) in continuous flow Stationary phase (resin) with desired chemical properties
The different interactions between the proteins and the solid phase allows for the separation (elution) in the mobile phase
