Lecture 4

Question 1

What is another name for the Ni-2+ chromatography technology that we’re using?

Answer 1

immobilized metal affinity chromatography

Question 2

How much time does it take to get from the G0 sample to the G3 sample?

Answer 2

3 hours

Question 3

How much of the G3 sample is used?

Answer 3

15 mL is harvested and centrifuged

Question 4

What are the four intellectual steps to optimize protein purification?

Answer 4

development of a suitable assay procedure; selection of the best source material; solubilization of the desired protein; development of a suitable series of fractionation steps

Question 5

What are the advantages of yeast/bacterial expression systems? (5)

Answer 5

inexpensive; minimal equipment requirements; large yields; control of expression; can be constitutive or induced

Question 6

What are the disadvantages of yeast/bacterial expression systems? (3)

Answer 6

post-translational modification; stability/solubility; small yields

Question 7

What can you use to decrease extract volume?

Answer 7

(NH4)2SO4, which also removes nucleic acids

Question 8

What do you do to remove small molecules and (NH4)SO4?

Answer 8

dialysis

Question 9

What are constitutive genes?

Answer 9

genes that are expressed continuously regardless of environment

Question 10

What method do we use to cause cell wall lysis?

Answer 10

Slow freeze/quick thaw method

Question 11

When we slowly freeze cells, what happens?

Answer 11

ice crystals form inside cells, some cell walls are punctured

Question 12

When we quickly thaw the cells, what happens?

Answer 12

cells with damaged cell walls burst open because of osmotic shock

Question 13

What does the addition of the lysozyme do? (2)

Answer 13

degrades neighboring cell walls; chain reaction of lysing begins

Question 14

Chromatography has what two phases?

Answer 14

mobile phase and stationary phase

Question 15

What are the three factors you need to consider during fractionation steps in protein purification?

Answer 15

purity; yield; activity

Question 16

Purity is expressed as a

Answer 16

percentage

Question 17

Yield is expressed as a

Answer 17

mass/amount

Question 18

During purification, what roughly happens to yield, purity, activity, and specific activity?

Answer 18

yield and activity decrease, purity and specific activity increases

Question 19

What are the five common types of chromatography?

Answer 19

gel filtration/size exclusion; ion-exchanger; affinity; hydrophobic interaction; partition

Question 20

In gel filtration chromatography, which molecules elute first?

Answer 20

the large ones

Question 21

Ion-exchange chromatography separates on the basis of

Answer 21

charge-charge interactions

Question 22

Partition chromatography separates on the basis of

Answer 22

polarity or water solubility

Question 23

Which atom in the histidine ring contributes to the Ni2+ linkage?

Answer 23

the N atom

Question 24

What do we use to elute out His6-tagged proteins off the column?

Answer 24

imidazole

Question 25

Which fraction contains the void volume?

Answer 25

W1

Question 26

What is the purpose of the wash step when developing the column?

Answer 26

This is to help remove more contaminates which are larger than rGFP.

Question 27

If we were purifying a histidine-tagged phosphatase, how would we monitor its presence during the purification procedure?

Answer 27

We would need to add PPP, then crude extract and sodium carbonate which would yield a pink color. This would take about 30 minutes.

Question 28

What are the five options for source material?

Answer 28

primary tissue; tissue culture (in situ); yeast expression system; bacterial expression system; in vitro expression system

Question 29

Which source materials are expensive?

Answer 29

primary tissue (tissue from a living organism, which can be hard to obtain); tissue culture (in situ - culturing tissue based on primary tissue)

Question 30

Which source materials require a cloned gene?

Answer 30

yeast, bacterial, and in vitro expression systems

Question 31

Cloned genes usually use what purification method?

Answer 31

tagged method

Question 32

What is the underlying principle behind any purification step?

Answer 32

exploiting the differences between your molecule and all other molecules

Question 33

Hydrophobic interaction chromatography separates on the basis of

Answer 33

differences in hydrophobic properties