Protein Purification Flashcards
Describe the flow of genetic information to primary sequence
The genetic information is stored in DNA, then transcribed into mRNA, And finally translated into protein primary sequence.
What does the primary sequence of a protein determine
The primary sequence of a protein determines its ordered or disordered native 3-dimensional structure, hence its function
What are recombinant proteins
By manipulation of genetic codes, we can produce whatever amino acid sequence. We can use lab-cultured cells / organisms to produce large quantities of recombinant proteins
Applications of recombinant proteins (3)
- Truncated proteins (part of a large protein)
- Fusion protein (two or more proteins linked in one peptide chain)
- Tagged protein (protein fused with a short peptide for identification or purification)
What are the three key macromolecules for protein biosynthesis
1) mRNA
2) Ribosome complete with auxiliary factors such as the elongation factor and GTP as the energy source
3) aminoacyl-tRNA
how are peptide bonds formed and broken down
formed by condensation
Broken down by hydrolysis
What are the sources of proteins
Natural sources- tissues, seeds, eggs, cultured cells, microorganisms, media etc. Only naturally abundant proteins are available this way
Recombinant proteins- produced in cultured genetically modified organisms. Wild-type, mutant any engineered proteins can all be made available in this way, and in large amount
How are proteins extracted? How do you choose the extraction method
Need to break up the cell. There are many ways:
Osmotic shock
french press
sonification
Digestions
homogenization
The best way is the most convenient for your purpose
What are the practical considerations during extraction
Condition of the solution must be mild to keep the protein of interest in its native state:
pH
Temperature
Reducing agent
EDTA
Ionic strength
proteolysis
Glycerol or sucrose
Describe the soluble and insoluble part of extract after centrifugation
Soluble part is the supernatant/ crude extract.
Insoluble part is the pellet, which is cell debris, higher density, forms a pellet in the bottom
How can you measure the protein concentration in the supernatant/ crude extract
Bradford or Biuret method to monitor the efficiency of cell lysis
What are the effects of salt on protein solubility
In the optimal amount of salt, solubility is maximized
What is salting out and salting in
Salting in: solubility is very low in the absence of salt. Solubility increases when a small amount is present
Salting out: solubility decreases with excess concentration of salt or other solute
how can fractionation be achieved by salting out
Two proteins may salt out at different concentrations of salt. As one protein become insoluble, you can then centrifuge out the precipitate from the soluble portion.
What does amino acid composition determine
Determines charges on proteins
what is the isoelectric point
pH at which the protein has a net neutral charge
What do salts do to protein aggregation
Salts keep proteins separated from each other
When is solubility lowest
solubility is lowest around pi
Describe protein interactions at the following pH and pI
pH>pI
pH=pI
pH<pI
Proteins attract through hydrophobic interactions when pH=pI
Proteins repel at other
Describe the stationary and mobile phase of chromatography
Stationary: beads contained in the column
Mobile: aqueous solution supplied from the inlet, flows through the beads and filters the outlets
Describe a typical chromatography system
A typical chromatography
system also has a pump, an in-line UV absorption detector, as well as a computer for controlling the system and for displaying the chromatogram
Describe the UV absorption of proteins
UV absorption at 280 nm
Mainly by aromatic residues
Real-time monitoring
Describe Biuret & Bradford assays
time consuming
Fractions collected first, then assayed
Describe activity assays
Can tells if it is the target protein
What is Coomassie Blue G-250
Dyes free proteins red-brown
dyes protein-bound blue
Absorbance at 595 nm is measured for quantification of protein concentration (Bradford)
What is a chromatogram
What is an SDS-PAGE
Polyacrylamide gel electrophoresis
Smaller proteins move faster
Stain with Coomassie blue
Sodium dodecyl sulfate (SDS) denatures proteins by alkyl chain binding to hydrophobic core
Sulphate groups cover protein with negative charges
Large proteins are impeded more by the pores in gel matrix
T/F it is rare that proteins other than the enzyme bind the substrate with similar affinity
True
Describe affinity chromatography
Protein mixture is added to a column containing a polymer-bound ligand specific for the protein of interest
Unwanted proteins are washed through the column
Protein of interest is eluted by ligand solution
High affinity of the Ni-NTA resins for 6xHistidyl-tagged proteins or peptides is due to what two things
High affinity of the Ni-NTA resins for 6xHistidyl-tagged proteins or peptides is due to:
1) Ni ions are anchored to agarose beads
2) two empty valence of Ni
What is histagged protein
Have a stretch of 6 or more consecutive histidine residues that interact tightly with Ni ion that is bound to a NTA bead
Naturally occurring protein does not have this feature and does not bind Ni ion tightly
What is the specificity of the Ni-affinity chromatography reaction? What purity does it create?
Extremely high, allows protein purification to a purity of 95% in a single step
4 steps of Ni2+-affinity chromatography
- Prepare and equilibrate the column with a binding/ running buffer at pH~7 and 500mM NaCl
- Load crude sample. the protein with engineered polyhistidyl tag will be bound to the beads. Other proteins will not be bound.
- Wash away non-bound sample components with the running buffer (wash with same solution)
- Elute his-tagged protein with an elution buffer with 300 mM imidazole and 500 mM NaCl. Collect the effluent. (elute with different solution)
Why do we use imidazole to liberate the bound protein
Imidazole ring is part of the histidine structure and a perfect competitor for binding the metal ion and liberating the bound protein
How are TAG-proteins eluted
Either by a compeititve ligand or by cleavage
Why is it called cation exchange chromatography
.
5 steps of anion exchange chromatography
Common steps in affinity and ion-exchange chromatography
- Prepare the column and equilibrate with a binding buffer
- Load sample in the binding buffer
- Wash the column with the binding buffer
- Elute the bound protein with the elution buffer or a series of elution buffer with a concentration gradient
Describe gel filtration chromatography (aka size exclusion, molecular sieving)
Larger molecules travels less into the pores and therefore elutes earlier
There are porous beads in the column, and small proteins enter and are slowed down
Gel filtration procedure
- Prepare a column by filling with beads and flooding with a running buffer
- Load the protein mixture on the top
- Keep supplying the running buffer on the top
BIGGER ELUTES FASTER
What is a gel filtration chromatogram
Why do larger molecules migrat faster in gel filtration
The beads for gel filtration are made of porous
material
Detour effect:
- Huge molecules travel through gaps between effects
- Medium molecule detour into large enough cavities
- Tiny molecules detour into all cavities
What type of column will result in better separation for GF
Bigger the column will result in better separation. The wider the molecules can spread
Why can we only estimate relative molecular weights from chromatography
shape affects the pore-entering possibility of a molecule, elongated molecules are less likely to enter pores
If proteins have a known round shape, then MW = RMW
What the partition coefficient (Kav)? When do bigger molecules, smaller molecules, and intermediate molecules elute?
Independent of column size as long as we use the same type of beads
(Ve- Vo)/(Vt-Vo)
MW range for G-200, G-100, G-25
– G-200 good for 5,000-600,000 Daltons
– G-100 good for 4,000-150,000 Daltons
– G-25 good for 1,000-5,000 Daltons
How to choose running buffer
Retains solubility of the sample
Has 100 mM or more salt to suppress charge-charge interaction between proteins
Column has to be equilibrated by the running buffer by at least Vt
Which chromatography should be used first
most specific (powerful) chromatography should be applied first
1st: Affinity
2nd: Ion Exchange
Last: Gel filtration
How can we concentrate protein solutions
Protein solution can be concentrated by ultra-filtration. A filter with a cutoff size (5000 Dalton for example) is used. The protein solution is passed through this very slow filter in a nitrogen pressure cell or a centrifugation tube
Describe the storage of proteins
Protein solution can be stored at -80C for an extended period of time. At first, 10% glycerol is usually added to the protein solution as the cryoprotectant. Then the sample is transferred into many small sealed tubes, and the tubes are dropped into liquid nitrogen for rapid cooling. Finally, the cooled sample tubes are stored in a -80C freezer.
There are other ways of storing proteins. The above-mentioned one is the most popular.
