DNA polymerases are found in a diverse range of organisms, prokaryotes, eukaryotes, viruses and bacteriophage. T7 DNA polymerase is a replicative enzyme from E. coli bacteriophage T7. It relies on the thioredoxin binding domain (TBD) of phage gene 5 protein (gp5) and E. coli thioredoxin (Trx) for processive replication of phage DNA. Although T7 DNA polymerase is processive, it is also thermolabile. In order to design a thermostable and processive DNA polymerase, the structural stabilities of the TBD and Trx were studied in respect to their binding affinity and affect on enzyme processivity. An artificial operon was designed for coexpression of subunits of T7 DNA polymerase. By means of a 9??His-tag at the amino terminus of gp5, T7 DNA polymerase complex was purified by one-step nickel-agarose chromatography, with subunits gp5 and Trx co-eluting in a one to one molar ratio. Purified T7 DNA polymerase was assayed for polymerase activity, processivity and residual activity and compared to the commercial T7 DNA polymerase. The two enzymes were not identical with commercial T7 DNA polymerase being less processive at 37??C. Mass spectrometry of the two enzymes identified a mutation of Phe102 to Ser in the Trx subunit (TrxS102) of commercial T7 DNA polymerase. The Ser102 mutation, was found near the carboxyl terminal helix of Trx. TrxS102 was less stable than wild type Trx. In the study of the TBD structural stability, a hybrid polymerase was constructed by inserting the TBD motif into the homologous position in the Stoffel fragment of Taq DNA polymerase. The hybrid enzyme was coexpressed with Trx from an artificial operon; however, the TBD inserted retained a mesophilic binding affinity to Trx. The chimeric polymerase required 100 molar excess of Trx for processive polymerase activity at 60??C. TBD structural deformation at elevated temperatures was hypothesized to be the cause of the change in the subunit stoichiometry. Mutagenesis of TBD would be required to increase its thermostability. An efficient, rapid high throughput mutagenesis method (SLIM) was invented and would be appropriate for further studies.
| Identifer | oai:union.ndltd.org:ADTP/282435 |
| Date | January 2005 |
| Creators | Chiu, Joyce, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Joyce Chiu, http://unsworks.unsw.edu.au/copyright |
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