Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH- RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH- RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions (6 mM MnCl(2) in 20 mM Tris-HCl buffer, pH 7.5). The binding capacity of immobilized AEAd with M-MLVH- RT was determined to be equal to approximately 1 mg enzyme/g moist weight gel. Adsorption studies with immobilized AEAd and soluble M-MLVH- RT demonstrated that the formation of the respective complex was perturbed by ATP. Quality control tests of the purified M-MLVH- RT essentially showed a single band (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and absence of nucleic acids and contaminating nuclease activities.
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PMID:Purification of M-MLVH- RT on a 9-aminoethyladenine-(1,6-diamine-hexane)-triazine selected from a combinatorial library of dNTP-mimetic ligands. 2082 67

The solvent resistance capacity of Pseudomonas putida S12 was applied by using the organism as a host for biocatalysis and through cloning and expressing solvent resistant pump genes into Escherichia coli. P. putida S12 expressing toluene ortho mononooxygenase (TOM-Green) was used for 1-naphthol production in a water-organic solvent biphasic system. Application of P. putida S12 improved 1-naphthol production per gram cell dry weight by approximately 42% compared to E. coli. Moreover, P. putida S12 enabled the use of a less expensive solvent, decanol, for 1-naphthol production. The solvent resistant pump (srpABC) genes of P. putida S12 were cloned into a solvent sensitive E. coli strain to transfer solvent tolerance. Recombinant strains bearing srpABC genes in either a low-copy number or a high-copy number plasmid grew in the presence of saturated concentration of toluene. Both of the recombinant strains were more tolerant to 1% v/v of toxic solvents, decanol and hexane, reaching similar cell density as the no-solvent control. Reverse-transcriptase analysis revealed that the srpABC genes were transcribed in engineered strains. The results demonstrate successful transfer of the proton-dependent solvent resistance mechanism and suggest that the engineered strain could serve as more robust biocatalysts in media with organic solvents.
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PMID:Solvent resistance pumps of Pseudomonas putida S12: Applications in 1-naphthol production and biocatalyst engineering. 2614 10