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Vlatakis G. Skarpelis G. Stratidaki I. Bouriotis V. Clonis Y. D. 《Applied biochemistry and biotechnology》1987,15(3):201-212
The resolution of restriction endonucleases from the same microorganism is conventionally achieved by lengthy fractionation
protocols. We now report effective single-step procedures that exploit dye-ligand chromatography for the resolution and purification
of restriction enzymes. After suitable initial screening, we demonstrated that resolution of two restriction activites can
be achieved in one chromatographic step, and further purification can subsequently be effected using selected dye-adsorbents.
Accordingly, we resolved in one step, Hpa I from Hpa II, Hind II from Hind III, and Sac I from Sac II. Furthermore, a three-step
Chromatographic procedure has been developed to purify EcoRV suitable for commercial exploitation, as judged by the “overdigestion”
and “cut-ligate-recut” quality control tests. 相似文献
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Partitioning of restriction endonucleases between two liquid aqueous phases can be strongly influenced by group-specific ligands included in the two-phase system. Three restriction endonucleases, namely EcoR I, EcoR V and BamH I, were partitioned within an aqueous dextran-polyethylene glycol (PEG) system. The enzymes could be extracted into the upper PEG phase by using either triazine dyes or herring DNA as affinity ligands. The influence of the endogenous bacterial nucleic acids, concentration of polymerbound dye and concentration of sodium chloride on the system were examined. A partial purification of EcoR I (up to 52-fold) and EcoR V (up to 37-fold) was achieved using a combination of affinity partitioning and ion-exchange chromatography, providing an extremely fast and economical method for the isolation of restriction endonucleases free from contaminating nuclease activities. 相似文献
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The use of sequence-specific DNA affinity adsorbents for the isolation of restriction endonucleases EcoRI and SphI to near homogeneity has been reported. However, the high cost of these adsorbents is a limiting factor for their wider application. This paper reports the application of sequence-specific DNA affinity ligands containing recognition sequences for 34 restriction endonucleasesas group-specific ligands in the isolation of restriction endonucleases. Crude samples of six restriction endonucleases, namely BshFI, BamHI, SmaI, SacII, PvuII and SalI, were shown to bind to these adsorbents and could be eluted at different KCl concentrations. High purification factors and recoveries were obtained. Restriction endonuclease BshFI, an isoschizomer of HaeIII, from the microorganism Bacillus sphaericus was purified to near homogeneity employing a two-step procedure which involves DNA-cellulose chromatography and oligonucleotide- ligand affinity chromatography. The enzyme exists as a monomer with an apparent relative molecular mass of 34 000 as determined by both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion chromatography. 相似文献
4.
Liang ZX Tsigos I Bouriotis V Klinman JP 《Journal of the American Chemical Society》2004,126(31):9500-9501
The hydride transfer catalyzed by thermophilic alcohol dehydrogenase (htADH) exhibits sharply different kinetic and activation parameters from that catalyzed by the more flexible psychrophilic alcohol dehydrogenase (psADH). In addition, the hydride transfer in htADH is affected by mutating two distal residues that are suggested to be responsible for the decreased local protein flexibility in htADH. These observations provide support for the view that protein dynamics is tightly coupled to the hydrogen-transfer processes in these enzymes. 相似文献
5.
Kouyianou K. Mitsikas D. A. Kotsifaki D. Providaki M. Bouriotis V. Kokkinidis M. 《Crystallography Reports》2022,67(7):1218-1223
Crystallography Reports - DNA methyltransferase M.BseCI from Geobacillus stearothermophilus (EC 2.1.1.72), an enzyme with 579 residues, methylates the N6 atom of the 3'-adenine in the sequence... 相似文献
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