Lecture 12 - Protein Purification Flashcards
How do you overexpress a protein?
Amplify gene through PCR, then place gene inside plasmid containing a gene to destroy antibiotics. Then inject plasmid into cells (recombinant expression system) on plates containing antibiotics. Only cells with the plasmid inside will grow and express protein.
How does centrifugation purify proteins?
Differential centrifugation, as you increase speed, lighter and lighter elements will precipitate and sediment out. So slowly increase speed, and assay for protein at each step to remove the heavier elements to separate components.
List purification techniques
filtration
column chromatography
dialysis
salting out (precipitation
How does salting out work?
Because of differing solubilities, slowly add more and more salt to mixture, and remove precipitated unwanted proteins by centrifugation. After wanted protein precipitates, can centrifuge, separate, then resuspend in solution in physiological buffer.
How does dialysis work
Place bag with solution inside physiological solution, bag has holes of a certain size. Small proteins, salts, and other impurities pass through holes into solution to reach equilibrium. Change dialysis buffer to re-dilute unwanted small molecules
What are the 2 parts of column chromatography? And what are 3 methods
Stationary phase - gel matrix (porous solid) with specific chemical properties or pore size
Mobile phase - buffered solution with crude protein
1) Size exclusion
2) Ion exchange
3) Affinity
What is size exclusion chromatography
Use porous beads, small proteins enter beads and have speed retarded, large proteins don’t enter and elute first
What is ion-exchange chromatography?
use charge to separate proteins. Cation exchange= positively charged protein binding to negatively charged beads, Anion exchange = negatively charged protein binding to positively charged beads. If you can calculate isoelectric point, can manipulate the pH so that specific charge is acquired
Proteins bind to beads, can be eluted through increasing salt concentration.
What is affinity chromatography
Attach specific functional group that protein binds to to the beads, desired protein then binds to that group. If not sure, can fuse protein to a specific protein that has a known affinity for a functional group.
Proteins eluted through adding competitor, or mobile funcitonal groups
Can cleave expression tags or fusion proteins with proteases
What does UV absorbance tell us
total concentration of protein, absorbance based on concentrations of tryptophan and tyrosine
How does SDS-PAGE work?
Used purely as a identification technique, not a purification technique
SDS mixed in and heated with protein, unfolds protein and coats it in a negative charge, also add reducing agent to remove disulfide bonds. Place denatured samples in polyacrylamide gel (has cross-linked polymers - molecular sieve), apply charge, sample moves towards positive terminal (anode) with smallest samples moving fastest.
Run with standard proteins with known mass in order to interpolate mass, stain with dye to visualize protein movement
What is 2-D gel electrophoresis
2 steps - separation by piezoelectric point (isoelectric focusing) and then by mass
1) place in tube with pH gradient, low pH at top, high pH at bottom, then denature and reduce proteins (don’t apply uniform charge). Apply voltage and allow to separate by charge
2) run through gel electrophoresis to separate by size after soaking in SDS
Only proteins with identical piezoelectric point and mass will be unresolved (rare)
