OPOI is recombinantly expressed and purified, and the procedure was tracked usin

Question

OPOI is recombinantly expressed and purified, and the procedure was tracked using a Purification Table like we have learned about in class.

Ion-exchange

peak 2

Ion-exchange

peak 3

a) Fill in the “Yield” and “Purification Level” columns above

b) Whatvolumeofproteinmusthavebeenaddedtoion-exchangepeak2toarrive

at this specific activity?

Step Protein Concentration (mg/mL) Total Protein (mg) Total Activity (Units) Specific Activity (Units/mg) Yield (%) Purification level (fold) Lysate 2.5 200 0.08 0.002 Ion-exchange Peak 1 8.7 104 0.01 0.002

Ion-exchange

peak 2

5.3 64.1 5.5 2.1

Ion-exchange

peak 3

1.73 20.8 0.01 0.012

Explanation / Answer

a)
Lysate :
Yield % = 100 %
Purification fold = 1

Ion - Exchange Peak 1:
Yield % = (total activity at each step / total activity in the Lysate) * 100 =( 0.01 / 0.08 )*100 = 12.5%
Purification fold = specific activity at this step / specific activity at lysate = 0.002 / 0.002 = 1

Ion - Exchange Peak 2 :
Yield % = total activity after that step / total activity before that step = (0.01 / 0.01 ) * 100 = 100%
Purification fold = 0.012 / 0.002 = 6

Ion - Exchange Peak 3 :
Yield % = (0.01 / 0.08 )*100 = 12.5%
Purification fold = 0.012 / 0.002 = 6

b) specific activity = total activity / total protein
   2.1 = 5.5 / total protein
total protein = 5.5 / 2.1 = 2.61904 mg
2.61904 mg of protein must have been added to Ion exchange peak 2 to arrive at this specific activity.

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