Expression vectors pt 1
What is one example of an expression vector?
pETSUMO, pETm60, pQLinkH, p11 (other answers acceptable)
What is one benefit of using solubility tags?
solubility, yield, help folding
What is the point of dialyzing your purified sample?
To remove contaminants
Give one example of an epitope tag
FLAG, HA, Myc, V5, etc (other answers are acceptable)
Name one other protein purification strategy apart from affinity purification
Size exclusion, ion exchange, heparin, salt precipitation, etc (other answers acceptable)
What are solubility tags?
Very soluble protein normally expressed in system
What are the two modes of action of antibiotic selection markers?
Bactericidal (kill), Bacteriostatic (slow growth/prevent cell division)
Name one method to figure out which fraction your protein eluted in.
SDS-PAGE
(Bradford and other reasonable answers also accepted)
Name the type of interaction between GST and glutathione
Hydrogen bond
What are two ways you can elute an ion exchange column?
Change salt, change pH
Name one reason why you would use yeast cells to express your protein over HEK/CHO cells?
faster growth, still get post translational mods (not as many), large yield, cheaper
Why would you choose to use the T7lac promoter over the lac promoter alone?
Better yield, not as leaky
Name one method you could use to further clean up your dialyzed protein sample (after affinity purification).
Second pass through affinity column, SEC (other reasonable answers also accepted)
Name the type of interaction between histidine and metal ions
Coordination
Why are size exclusion columns larger than other columns?
Better separation
Name 3 examples of promoters used in bacteria
lac, trp, Ptac, T7, T3, T5, pL, T7lac
Why don’t we use DH5alpha cells to express protein?
DH5alpha cells still have proteases, its mutations are specifically to remove endonucleases and recombinase
Name two ways to lyse cells
Sonication, lysozyme, french press, beads (other answers also acceptable)
Name two metal ions that a His tag binds specifically to
Ni, Co, or Cu
What are two benefits to affinity purification over other strategies?
Higher purity/yield, tag can improve protein stability and solubility, tag can allow for convenient detection (other reasonable answers are also acceptable)
Name an antibiotic, its class, and how it acts
Kan - aminoglycoside - bactericidal
Strep - aminoglycoside - bactericidal
Amp - b-lactam - bacteriacidal
Erythromycin - macrolide - bacteriostatic
Can you name the example of a negative selection marker I gave?
Thymidine kinase and ganciclovir
What are two reasons for why you would induce protein purification at a lower temperature?
Slow the growth rate of cells
Reduce production of endogenous proteins
Improve yield of recombinant protein
(other reasonable answers also accepted)
Draw the structure of histidine
Correct structure of histidine
What are two downsides of affinity purification compared to other strategies?
Expensive, tag may need to be removed, tag may alter native state of protein (other reasonable answers are also acceptable)