Rapid purification of high activity<i> Taq</i> DNA polymerase expressed in transformed<i> E. coli</i> cells.

Abstract

A simplified method is described here for the preparation of a thermostable Taq DNA polymerase enzyme from Escherichia coli (E. coli) strain DH5a carrying the pTTQ18 expression vector transformed with the Taq polymerase gene. Standard purifications were done with 1 litre batch cultures of E. coli cells and produced yields in excess of 100 000 units of Taq polymerase. Optimal induction times with isopropyl b-D-thiogalactopyranoside were determined at varying times of 11, 14 and 18 hours and found to be optimal at 11 hours. Induction times greater than 11 hours resulted in complete degradation of enzyme. The resulting enzyme preparation from 11 hour incubation of E. coli cells with IPTG gave a 94 kDa protein band on an 8% SDS-polyacrylamide gel consistent with the size of commercially available Taq polymerase.


Transactions of the Zimbabwe Scientific AssociationVolume 72 1998, pp. 23-26