Özet:
The thermostable TaqI restriction endonuclease from Thermus aquaticus was cloned into Escherichia coli for overproduction and single step affinity chromatography purification. The gene was cloned into pMAL-c2 vector, resulting in the cytoplasmic expression of a MBP-TaqI restriction endonuclease fusion protein. In the first part of this study, TaqI restriction endonuclease gene was amplified using Taq DNA polymerase and cloned into E. coli. Although the expected size fusion protein was produced, no endonuclease activity was obtained. Growth condition manipulations and expression in different strains did not result in the production of an active enzyme. Subsequent sequencing of the gene indicated a mutation at position 410 that might abolish activity. In the second part, cloning was repeated after amplification of the gene with Pfu DNA polymerase which has proofreading activity. A colony coding for a protein with the expected size and activity was identified. Upon optimization of induction time, higher tiled was obtained when the culture was induced at an absorbance of 0.9. Expression in three different strains of E. coli, indicated total recovery was higher in TB1 cells at 300,000 Units/liter of culture volume as compared to 100,000 Units/liter in XL1-Blue cells and 150,000 Units/liter in ER2508 cells.
