Question

After activation of Lysozyme, each tube was added PMSF (serine protease inhibitor, active period ~45 min, prevent Taq degradation during purification)

Explain why heating the sample to 75 C for 30 min is an excellent first step in the purification of Taq DNA polymerase from E. coli

Answer 1

Taq DNA polymerase is a thermostable enzyme isolated from thermophile Thermus aquaticus. Its optimum temperature for polymerase activity is 75⁰C. It is thermostable and does not get degraded till 95⁰C. Hence, it is used for PCR, which employs a range of temperatures from 55-95⁰C.

Heating the E.coli lysate at 75⁰C will denature all proteins except, TAq polymerase. This step is an excellent method to be rid of all unwanted contaminating proteins except Taq polymerase. The sample can be heated at 75⁰C for 30 min, cooled on ice for 20 min and centrifuged to be rid of all contaminating E. coli denatured proteins. Thus, the Taq polymerase can be easily purified.