Purifying proteins & tagging Flashcards
Expression vectors contain an operon, what does this allow?
How is the desired DNA fragment regulated in an expression vector?
How would you produce an epitope tagged protein?
What can epitope tags be used for?
Allows insert DNA to be transcribed/translated
Sequences that regulate it/turn on and off
Insert DNA encoding peptide for epitope tag into gene for protein of interest- then introduce gene into cell
Immunolocalisation (antibodies bind to tag) and purification
What is GST?
What binds to GST?
How do you tag proteins with GST?
Glutathione-S-transferase:
Glutathione: has Cys-Gly dipeptide attached at its carboxyl carbon
- Combine gene for target protein & gene for GST to form gene for fusion protein. 2. Insert into cell (of which is translated)
- Prepare cell extract with fusion protein in mixture (lyse cells)
- Put mixture into column
- Separates as GST binds to glutathione on medium so other proteins flow out
- Add solution of free glutathione- so fusion protein eluted
Protein purification tags can be added with an enzyme from a particular ‘organism’, what is it?
How does it do this?
As well as a protein purification tag gene, what else could be inserted?
This also allows to insert mutations (known as site-directed mutagenesis)- how can this occur?
How can this be done with PCR?
DNA ligase from T4 bacteriophage
Blunt end ligation
Any DNA sequence including restriction enzyme sites
- Cut out/cleave sequence in recombinant vector with restriction endonuclease & re-ligate DNA back together (with sticky end ligation)
- Insert DNA fragment with mutation
- Denature & anneal oligonucleotide primers with the mutation on the plasmid
- DNA polymerase incorporates mutagenic primers in plasmid
- Non mutated DNA template is digested & newly synthesised strands are annealed
- End up with mutated plasmid with nicked strands- can transform to dsDNA in cells
How does Cas-9/CRISPR insert a recombinant fragment into DNA from the plasmid?
What 4 main things can CRISPR cause to DNA/genome?
How does it block gene expression?
How does it enhance gene expression?
- Insert plasmid into nucleus & sgRNA complex forms.
- In a double strand break- both strands DNA broken & recombinant fragment inserted between, so non-homologous ends join.
In single strand break (nick)- recombinant fragment inserted into 1 strand & then other strand repaired homology-directedly.
Repair, disruption, repression & activation
RNA polymerase is blocked by the tightly bound complex.
Activator binds to complex- allowing RNA polymerase to bind after the complex to have access to gene.
What are the 6 functions of proteins?
What do we need protein purification for?
What 2 frameworks are used in protein purification?
Which organisms produce a higher yield of proteins?
What are the main 4 steps of protein purification?
Structure, storage, transport, communication, enzymes, defence
Function & activity studies, structural studies, isolate pharmaceutically active proteins, industry, biotechnology
1- from natural sources (cells, organs etc)
2- recombinant technologies (expression vectors)
Bacteria most, then yeast, then animal cells
- Choose framework & purification source/host
- Extract, solubilise & choose solvents
- Protein separation procedures (chromatography)
- Analyse protein purity, properties & function
For a protein source/host/expression system, what do you need to consider?
E.coli, yeast, insect & mammalian cells are all expression systems, how do they compare?
E.g growth times, complexity & cost of medium, expression levels, post modifications & folding
Characteristics of protein, source availability, yield, safety consumer perception & regulatory constraints
E.coli = fastest growth, uncomplex & low cost growth medium, high expression level, no post translation modifications & might need refolding
Yeast = rapid, low complexity & cost medium, low-high expression levels, might need refolding, post trans modifications
Insect cells = slow, complex, high cost medium, low-high exp level, good folding & post trans modifications
Mammals = slow, complex, high cost medium, low exp level, proper folding, all post trans modifications including gamma-carboxylation
What are the key steps of making a cell extract?
1- culture cells
2- cell breakage
3- centrifugation
4- preservation
US - liquid shear
Agitation - liquid shear
Pressure - liquid shear french press
Grinding- solid shear- pestle & mortar
Pressure - solid shear- hughes press
Dessication - air, vacuum, freeze/solvent drying
physical - lysis- osmotic shop, freeze thaw
chemical - lysis -cationic & anionic detergents
enzymatic- lysis - lysozymes
After cell breakage the cell extract is centrifuged, what is centrifuged and at what speed?
What is the supernatant & pellet?
What happens if the protein of interest ended up in the pellet?
Which layer is taken on for differential sedimentation (i.e has higher chance of finding protein of interest?)
How does differential sedimentation work?
After centrifugation, how are membrane associated/integral proteins released & made soluble?
cytoplasm, 150,000g (low speed)
Supernatant = cytosol (enzymes & proteins) Pellet = particulate fraction (particles & organelles)
Perform further protein extraction & purify
Higher speed centrifuge SN1 to give 2nd pellet P2 (with medium size particles) and SN2. Ultracentrifugate SN2 to end up with small particles in P3.
Ultrasonication, detergent solubilisation, organic solvent extraction, digestion of phospholipid
How can you prevent denaturation of the proteins?
Prevent inactivation?
Proteolysis?
Why should a buffer not be used outside of its buffering capacity?
Buffer exchange & dialysis- how can ions be removed from proteins?
Which protease inhibitors can you used for cysteine-dependent proteases, metal activated proteases, & serine proteases?
Use neutral buffers, do initial steps on ice, avoid organic solvents & chaotropic agents
reducing agents (dont like oxidising) add chelators EDTA (dont like metal ions) add cofactors (stabilise)
Speed & low temp in initial stages, add cocktail of protease inhibitors
Most buffers drop dramatically 1pH either side of pKa
In a dialysis tube & depending on the size of the pores- ions exchange with buffer until equilibrium reached so that protein is present outside the tube in the buffer solution
Cysteine reagents, EDTA, Leupeptin & PMSF
What are the protein properties which are a basis for separation?
What variables affect properties/interactions of proteins?
How does salting out work in precipitating out the protein? What agent can be used?
Size, shape, net charge/charge distribution, hydrophobicity, solubility, ligand & specific epitope
Interactions, temperature, ionic strength, ion types, solvent polarity & pH
Proteins in water are surrounded by a hydration shell, so increasing salt concentration can strip away shell causing proteins to aggregate with hydrophobic interactions & precipitate. Ammonium sulphate
What property of proteins does size exclusion/gel filtration separate proteins?
What is the matrix?
How are the proteins separated in the column?
Size & shape
Cross-linked polymer with pores of selected size
Large proteins are too large to enter the pores so are eluted first.
Smaller proteins go through the pores & elute later, so goes from large to small
What does ion-exchange chromatography exploit in separating proteins?
What is the matrix?
If the matrix was anionic (-ve) how would the proteins be separated?
What is the affinity of the protein for the charged groups on the matrix affected by?
What is the charge if a protein if the pH is below its pI? How can it be eluted?
Net charge
Polymer with bound charged groups either anionic (cation exchangers) or cationic (anion exchangers)
Very positive proteins would be eluted later as would be stuck at top of column, and very negative would be eluted first.
pH and concentration of competing free salt ions (gradually increased to elute bound proteins)
Positive (so binds to negatively charged medium & can be eluted with positive counter ions)
How does hydrophobic interaction chromatography exploit protein characters?
What is the matrix?
What is the mobile phase?
Why is the binding done in high salt concentration?
How can you elute the proteins?
Hydrophobicity
Synthetic polymer with bound hydrophobic groups which is surrounded by water (hydrophobic effect)
Hydrophobic patch on proteins surrounded by water
As salt interacts with water, so less water for ‘shielding off,’ and hydrophobic groups binds with water shielding around but less water.
With a lower salt concentration
What does affinity chromatography exploit?
What is the stationary phase?
What happens to the proteins?
How do you elute the proteins?
Binding to specific ligands
Ligands
Some proteins bind specifically to the ligand in stationary phase, and those that don’t bind are washed through
Solution containing free ligand/or a competing molecule
What is the flowchart for an automated purification system?
What does a stationary phase typically consist of?
Buffer->Pump->Sample & gradient mixer->column->monitor (PC recorder)->fraction collector
Insert material (agarose, cellulose)
