A procedure used to purify your desired protein from E. coli is presented below

Question

A procedure used to purify your desired protein from E. coli is presented below a. Calculate the specific activity for the final product b. Calculate the percent yield of the final product based on the initial enzyme quantity c. Calculate the fold increase in purification for each step. d. Assume that the protein is pure after gel filtration chromatography. What percentage of the initial extract contained your protein? The Coomassie Blue-stained gel showing your purification is shown below: Cell pellet; 2 - Cell extract; 3 - Ammonium Sulfate; 4 - Heat denaturation; 5 - DEAE FT; 6 - DEAE Elution; 7 - CM-cellulose FT; 8 - CM-cellulose Elution; 9 - Bio-Gel A Elution; 10 - Marker e. According to the gel, which step was the most efficient? f. What about based on the purification factor? If there is a discrepancy between (e) and (f), what can explain that discrepancy?

Explanation / Answer

a)

The specific activity of an enzyme is the activity of an enzyme per milligram of total protein (expressed in mol min1mg1)

specific activity of final product 1120/35= 32 ug-min-1mg-1

b) % yield =1120/2700*100 =41.48%

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