ABSTRACT
Reteplase is a segment of tissue plasminogen activator used for the removal of thrombi in blood vessels. In the present study the cloned reteplase gene was used for its expression in competent E. coli. The recombinant plasmid, pET15b/reteplase (rpET-BL21), was transformed into competent E. coli strain BL21 (DE3) cells. Overnight culture of the transformed bacteria was induced by the addition of isopropylthio-ß-Dgalactoside (IPTG) to the final concentrations of 0.25, 0.5, 1 and 1.5 mM. Also, the effects of different temperatures(25, 30, 37 and 39°C), shaking speeds (100, 170 and 190 rpm), and various glucose concentrations (0.25, 0.5, 0.75 and 1 mM) on the expression of reteplase were examined. Samples were analyzed by SDS-PAGE. Maximum amount of protein production was obtained by the addition of 1 mM IPTG at 37°C, 100 rpm of shaking speed in the absence of glucose.
ABSTRACT
Taq DNA polymerase is widely used in laboratories and for this reason many investigators have focused their attention on understanding the role of various regions and amino acids in this enzyme. O-helix is a part of taq polymerase suggested to play an important role in the enzyme fidelity. The influence of Asn666 in this helix on the enzyme function has never been investigated, and therefore by using nested PCR, a portion of taq DNA polymerase gene containing Asn666Glu mutation was amplified. This DNA was digested with Eco RI restriction enzyme to confirm the presence of Asn666Glu mutation. After digesting this product and the wild type taq-pET-15b plasmid with NheI and BamHI restriction enzymes, they were ligated and used for the transformation of E. coli DH5α competent cells. The obtained colonies were screened for the presence of the mutated taq polymerase gene using EcoRI, NdeI and BamHI restriction enzymes. In conclusion, with the use of the obtained recombinant plasmid it is possible to study the role of this amino acid on taq DNA polymerase function.
