Open AccessDevelopment of a recombinant Taq DNA polymerase enzyme expressed using a synthetic gene and its comparison with a commercial enzyme
Author(s) -
Yuliana,
Uus Saepuloh,
Suryani Suryani
Publication year - 2021
Publication title -
asia-pacific journal of molecular biology and biotechnology/asia pacific journal of molecular biology and biotechnology
Language(s) - English
Resource type - Journals
eISSN - 2521-9839
pISSN - 0128-7451
DOI - 10.35118/apjmbb.2021.029.2.05
Subject(s) - thermus aquaticus , taq polymerase , hot start pcr , microbiology and biotechnology , recombinant dna , dna polymerase , biology , amplicon , polymerase , polymerase chain reaction , gene , polymerase chain reaction optimization , biochemistry , nested polymerase chain reaction
Taq DNA polymerase is a thermostable enzyme widely used for DNA amplification in the PCR technique. It was initially characterized and isolated from thermophilic bacteria, Thermus aquaticus. It was difficult to developed in this enzyme using a native host system. Therefore, the development of the recombinant Taq DNA polymerase expressed using a synthetic gene is important to improve production efficiency. In this study, we developed the in house Taq DNA polymerase recombinant based on a codon-optimized using E. coli expression system. We cloned 2685 bp of the Taq DNA polymerase gene in the pET151/D-TOPO vector. The gene was synthesized and the expression was analyzed with SDS-PAGE technique which indicated with a 100.9 kDa specific target protein. The concentration and activity of this purified enzyme were 5.17 mg/mL and 4.647 U/µL, respectively. The application of this enzyme to the PCR technique showed that this enzyme could amplify the target genes from 200 bp to 3500 bp amplicons with a minimum DNA concentration template 10 ng/µL. This assumes that the in house recombinant Taq DNA polymerase based on synthetic genes is successfully expressed, purified, and was functional and comparable to the commercial Taq polymerase.