Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization.
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Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization.
DNA polymerases are present in all organisms and enzymes important that synthesize DNA molecules. They are used in various fields of science, especially as an important component for the in vitro synthesis of DNA, known as PCR. modern diagnostics, molecular biology and genetic engineering of DNA polymerase needs which demonstrate improved performance.
This study aims to get NeqSSB-TaqS new DNA polymerase fusion of domain Stoffel Taq DNA and single-stranded DNA binding protein from Nanoarchaeum equitans as to significantly improve the properties of the DNA polymerase. Stoffel Taq DNA coding sequence specific DNA polymerase and DNA-binding proteins Nanoarchaeum equitans (NeqSSB-like protein) fused. A new recombinant genes obtained were cloned into pET-30 Ek / LIC vector and introduced into E. coli for expression. Purified recombinant enzymes and enzymatic properties including the activity of DNA polymerase, PCR amplification level, thermostability, processivity and resistance to inhibitors, were tested.
The results of the target protein around 18 mg / l after 24 hours of IPTG induction. The specific activity of the polymerase is 2200 U / mg. Recombinant NeqSSB-TaqS exhibited a higher level extensions (1000 bp template 20 s), processivity (19 nt), thermostability (half-life 35 minutes at 95 ° C) and a higher tolerance for PCR inhibitors (0.3 to 1.25 % of whole blood, 0.84 to 13.5 ug heparin lactoferrin and 4.7 to 150 ng) of Taq DNA polymerase Stoffel.
In addition, our research shows that NeqSSB-TaqS DNA polymerase has a high degree of flexibility in relation to the Mg 2+ ions (from 1 to 5 mM) and KCl or (NH4) 2SO4 salt (more than 60 mM and 40 mM, respectively ). NeqSSB-TaqS using DNA polymerase Taq DNA polymerase can not be a better choice in many applications of PCR.
Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization.
Data from self-made Taq DNA polymerase prepared for screening purposes.
DNA analysis is the main procedure in genetic engineering. This time the analysis is often done by PCR with Taq DNA polymerase. Although the price of the last enzyme is quite low, the demand for analysis of various results in a lot of spending money that is not affordable for many laboratories. Meanwhile, many screening duties do not require highly purified enzyme.
Taking into account the unique nature of the enzyme that allows for little simplifies the production without the use of expensive techniques such as column chromatography or the length and / or dialysis. The following data routine use of Taq DNA polymerase is prepared in accordance with the protocol developed in our laboratory are presented.
protocols only take a few hours to realize and does not need to be a qualified technician or costly equipment. But give an enzyme preparation suitable for most screening purposes. Taq DNA polymerase isolated stock can be stored as ammonium sulfate suspension in the refrigerator for a long time, not less than 6 months. Working enzyme solution prepared from stock suspension on demand, no more than once a month and can be stored well in the refrigerator.
Port plant homolog of various animal genes involved in the metabolism of phosphorus, telomere biology and other cellular processes. Compared to experiment with many other multicellular organisms, research in the model plant Arabidopsis thaliana take advantage of a short generation time and increasing warehouse of genetic tools and GMOs, including a large collection of T-DNA knockout and line activation. Available to the public availability of thousands of transgenic Arabidopsis lines provide a unique opportunity to address a number of important biological questions.
