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)
Why are biuret & lowry no longer used to measure protein concentration?
How does the Bradford Assay measure protein concentration? What are its advantages? Disadvantages?
How does the Warburg-Christian method measure protein conc? What are the adv & disadv?
Biuret = low sensitivity, Lowry = time consuming
Coomassie blue binds to aromatic residues & arginine & undergo absorbance shift from 465nm brown to 595nm blue. Highly sensitive & rapid. Interferences with strong basic buffers.
Measure the absorption of 280nm light with Tyr & Trp residues & calculate with Beer-Lambert. Adv = moderate sensitivity & rapid. Dis = interference with purines, pyrimidines & NAs
How can you measure the protein concentration with ELISA?
How does SDS page exclude proteins based on their mass?
How can you produce a graph?
Add primary and secondary antibodies (with substrate enzyme) and the intensity of the colour corresponds to concentration. Directly measure colour intensity (OD) against amount of protein to get calibration curve
Add SDS molecules to the unfolded peptide chains (gives them a strong negative charge) and thiols (to remove disulfide to denature) so in gel electrophoresis the speed of their movement only depends on their mass.
Produce a graph of mass against distance = calibration curve
What are the 2 methods of analysing proteins?
How can they be used to determine their mass?
How do these mass specs occur?
What is great about this method?
Electrospray ionisation mass spec and matrix-assisted layer desorption/ionisation mass spec
Find mass from m/z ratio
Generate high voltage electric field & sample evaporates into ions & enter mass spec for m/z measurement
High precision (3dp) and high accuracy
Enzyme assays are used to see if the protein’s function is an enzyme. What are the 3 key components of an assay?
What are the different types of assays?
What are the units of enzyme activity? What is the international unit?
Define activity & specific activity
Reaction mixture, incubation & termination
Continuous (disappearance of substrate/appearance of product directly measured)
Discontinuous (product separated from substrate)
Coupled (indirect measurement- substrate & product not measurable so link/couple another enzyme into the assay to produce a measurable product)
Micromoles/min
Amount enzyme catalyses 1micromole of substrate per min under defined conditions of temp & pH
Activity = total units of enzyme in solution
Specific activity = number enzyme units per mg of total protein
What are the differences between homologous and heterologous protein production?
Where can affinity tags genes be introduced on recombinant proteins genes?
Ni-NTA agarose tags bind to what residues on proteins?
How can purification be simplified?
What else can tags be used for?
Homologous = gene sequence to be inserted recombinantly is from the same species Heterologous = expression of recombinant proteins in cells that don't occur naturally
At N or C terminus or both
Histidines
Using tags in an affinity step & then cleave the protein-tag complex at the end
Detection
What are the differences between homologous and heterologous protein production?
Where can affinity tags genes be introduced on recombinant proteins genes?
Ni-NTA agarose tags bind to what residues on proteins?
How can purification be simplified?
What else can tags be used for?
Homologous = gene sequence to be inserted recombinantly is from the same species Heterologous = expression of recombinant proteins in cells that don't occur naturally
At N or C terminus or both
Histidines
Using tags in an affinity step & then cleave the protein-tag complex at the end
Detection
What are some advantages & disadvantages of heterologous protein production in E.coli?
What happens to misfolded proteins in E.coli?
How can you facilitate soluble heterologous proteins in bacteria?
How can you take misfolded aggregates into soluble proteins with correct folding?
+ = well characterised, suitable fermentation technology, can generate high yield, economical
- = protein folding issues, no post transl modifications, mass>80kDa difficult, adverse public perceptions
Leads to folding intermediates that are inefficiently processed by chaperones & aggregate together = inclusion body formation
Slower protein production, lower temperature
Co-production of molecular chaperones & folding modulators
Fusion partner proteins- tags increase protein solubility & stability
Prepare inclusion bodies & dissolve them, then refold with rapid dilution or by urea
How could you overcome problems of source availability?
Source safety?
Modifications?
What are the + and - for recombinant insulin?
Chymosin
Biological cleaning agents
High level of expression with recombinant technology- high specific activity in cell extract & fewer steps for purity
Recombinant technology procedures have strict regulations
Adding tags to target protein enhances stability & solubility & can lead to improve quality as well as for purification
+ cures, increased purity, improves shelf life, more rapidly absorbed
- injected, side effects
+ cheaper, GMO free, vegetarian
- lack of side reactions & affects flavour
+ large yield, non-intimate use, eco friendly
- low value, sensitivity issues
