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Search results for: Recombinant MMLV RT Proteins

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#28400277   2017/04/08 To Up

High sensitive one-step RT-PCR using MMLV reverse transcriptase, DNA polymerase with reverse transcriptase activity, and DNA/RNA helicase.


Hiroyuki Okano, Misato Baba, Tomomi Yamasaki, Ryota Hidese, Shinsuke Fujiwara, Itaru Yanagihara, Takeshi Ujiiye, Tsukasa Hayashi, Kenji Kojima, Teisuke Takita, Kiyoshi Yasukawa

2890 related Products with: High sensitive one-step RT-PCR using MMLV reverse transcriptase, DNA polymerase with reverse transcriptase activity, and DNA/RNA helicase.

25 reactions1000 units200 units100 µg200 units4 x 10,000 Units 10,000 Units 1000 units25 reactions96 tests

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#27871370   2016/10/11 To Up

Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase.

Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4pol from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.
Hiroyuki Okano, Yuta Katano, Misato Baba, Ayako Fujiwara, Ryota Hidese, Shinsuke Fujiwara, Itaru Yanagihara, Tsukasa Hayashi, Kenji Kojima, Teisuke Takita, Kiyoshi Yasukawa

1048 related Products with: Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase.

5 X 1000 U10 nmol1000 units50 mg200ul1ml5 X 1000 U10 nmol1000 units50 assays100 μg

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#25959458   2015/05/07 To Up

Preparation and characterization of the RNase H domain of Moloney murine leukemia virus reverse transcriptase.

Moloney murine leukemia virus reverse transcriptase (MMLV RT) contains fingers, palm, thumb, and connection subdomains as well as an RNase H domain. The DNA polymerase active site resides in the palm subdomain, and the RNase H active site is located in the RNase H domain. The RNase H domain contains a positively charged α-helix called the C helix (H(594)GEIYRRR(601)), that is thought to be involved in substrate recognition. In this study, we expressed three versions of the RNase H domain in Escherichia coli, the wild-type domain (WT) (residues Ile498-Leu671) and two variants that lack the regions containing the C helix (Ile593-Leu603 and Gly595-Thr605, which we called ΔC1 and ΔC2, respectively) with a strep-tag at the N-terminus and a deca-histidine tag at the C-terminus. These peptides were purified from the cells by anion-exchange, Ni(2+) affinity, and Strep-Tactin affinity column chromatography, and then the tags were removed by proteolysis. In an RNase H assay using a 25-bp RNA-DNA heteroduplex, WT, ΔC1, and ΔC2 produced RNA fragments ranging from 7 to 16 nucleotides (nt) whereas the full-length MMLV RT (Thr24-Leu671) produced 14-20-nt RNA fragments, suggesting that elimination of the fingers, palm, thumb, and connection subdomains affects the binding of the RNase H domain to the RNA-DNA heteroduplex. The activity levels of WT, ΔC1, and ΔC2 were estimated to be 1%, 0.01%, and 0.01% of full-length MMLV RT activity, indicating that the C helix is important, but not critical, for the activity of the isolated RNase H domain.
Kosaku Nishimura, Kanta Yokokawa, Tetsuro Hisayoshi, Kosuke Fukatsu, Ikumi Kuze, Atsushi Konishi, Bunzo Mikami, Kenji Kojima, Kiyoshi Yasukawa

1924 related Products with: Preparation and characterization of the RNase H domain of Moloney murine leukemia virus reverse transcriptase.

5 250 ug250 ug250 ug1 mg250 ug25 100μg100ug Lyophilized100ug0.1 mg100 ug

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#25450388   2014/10/17 To Up

Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation.

We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by which those mutations increase the thermal stability of the RT. The three single-mutants (E286R, E302K, and L435R), an RNase H-deficient MMLV RT (carrying the RNase H-inactivating mutation D524A), a quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) and the wild-type enzyme (WT) were produced in Escherichia coli. All RTs exhibited similar dissociation constants (Kd) for heteropolymeric DNA/DNA (2.9-6.5 nM) and RNA/DNA complexes (1.2-2.9 nM). Unlike the WT, mutant enzymes (E286R, E302K, L435R, D524A, and MM4) were devoid of RNase H activity, and were not able to degrade RNA in RNA/DNA complexes. These results suggest that the mutations, E286R, E302K, and L435R increase the thermostability of MMLV RT not by increasing its affinity for T/P but by abolishing its RNase H activity.
Atsushi Konishi, Tetsuro Hisayoshi, Kanta Yokokawa, Verónica Barrioluengo, Luis Menéndez-Arias, Kiyoshi Yasukawa

2278 related Products with: Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation.

5 mg100 mg250 ug 25 G1 g25 mg1 mg10 mg1 mg500 mg10 g

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#23149716   2012/11/13 To Up

Enzymatic characterization of human immunodeficiency virus type 1 reverse transcriptase for use in cDNA synthesis.

The aim of this study is to explore the advantages of using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in cDNA synthesis. Recombinant HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT were produced in an Escherichia coli expression system. In the incorporation of dTTP into poly(rA)-p(dT)(15) (T/P), the K (m) values for dTTP of HIV-1 M RT and HIV-1 O RT were 8 and 12 % of that of Moloney murine leukemia virus (MMLV) RT, respectively, and the K (m) values for T/P were 25 and 23 % of that of MMLV RT, respectively. Compared with MMLV RT, HIV-1 M RT and HIV-1 O RT were less susceptible to formamide, which is frequently used for cDNA synthesis with a G + C-rich RNA to improve specificity. The high substrate affinity and low susceptibility to formamide of HIV-1 RT might be advantageous for its use in cDNA synthesis.
Atsushi Konishi, Mayu Shinomura, Kiyoshi Yasukawa

1855 related Products with: Enzymatic characterization of human immunodeficiency virus type 1 reverse transcriptase for use in cDNA synthesis.

100ug10ìg100ug1mg96 wells (1 kit) 100ul50 96T 100ul10 10ug100μg

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#23026053   2012/09/28 To Up

Kinetic analysis of reverse transcriptase activity of bacterial family A DNA polymerases.

Some bacterial thermostable, wild-type or genetically engineered family A DNA polymerases have reverse transcriptase activity. However, difference in reverse transcriptase activities of family A DNA polymerases and retroviral reverse transcriptases (RTs) is unclear. In this study, comparative kinetic analysis was performed for the reverse transcriptase activities of the wild-type enzyme of family A DNA polymerase (M1pol(WT)) from Thermus thermophilus M1 and the variant enzyme of family A DNA polymerase (K4pol(L329A)), in which the mutation of Leu329→Ala is undertaken, from Thermotoga petrophila K4. In the incorporation of dTTP into poly(rA)-p(dT)(45), the reaction rates of K4pol(L329A) and M1pol(WT) exhibited a saturated profile of the Michaelis-Menten kinetics for dTTP concentrations but a substrate inhibition profile for poly(rA)-p(dT)(45) concentrations. In contrast, the reaction rates of Moloney murine leukemia virus (MMLV) RT exhibited saturated profiles for both dTTP and poly(rA)-p(dT)(45) concentrations. This suggests that high concentrations of DNA-primed RNA template decrease the efficiency of cDNA synthesis with bacterial family A DNA polymerases.
Kiyoshi Yasukawa, Atsushi Konishi, Mayu Shinomura, Eriko Nagaoka, Shinsuke Fujiwara

2059 related Products with: Kinetic analysis of reverse transcriptase activity of bacterial family A DNA polymerases.

100ug100 µg100ug100ug100ug1 module100 assays1 module1 kit

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#19330487   2009/03/29 To Up

A novel and simple method for high-level production of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) in Escherichia coli.

A novel protocol for producing recombinant Moloney murine leukemia virus (MMLV-RT) in Escherichia coli is reported. The optimized coding sequence for mature MMLV-RT was cloned into pET28a and over-expressed as an N-terminal His6-tagged fusion protein. An enterokinase (EK) recognition site was introduced between the His6-tag and MMLV-RT to release tag-free enzyme. Optimal expression of soluble His6-MMLV-RT was achieved by chaperone co-expression and lower temperature fermentation. The His6-tagged enzyme was first purified by Ni(2+) affinity chromatography. The bound enzyme was then eluted by EK digestion and the eluate was purified on an anion-exchange Q column to remove DNA and EK. Twenty-one milligram MMLV-RT was obtained from 1 l of bacterial culture.
Yu Chen, Weiguo Xu, Qiming Sun

2311 related Products with: A novel and simple method for high-level production of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) in Escherichia coli.

400Tests100 200 1 mL100 500 250 ug100 1 mL

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#18724716   // To Up

[Prokaryotic expression and purification of moloney murine leukemia virus reverse transcriptase and verification of the activity].

To produce the reverse transcriptase of moloney murine leukemia virus (MMLV-RT) through gene recombination, MMLV-rt gene was amplified by polymerase chain reaction (PCR) with specifically designed primers bearing restriction enzyme sites. Five mutation sites increasing the solution of the target protein were introduced through Site-directed mutation. After verification by sequencing, the gene was cloned into the expression vector pET15b to construct the recombinant plasmid pET15b-MMLV-rt. Purified MMLV-RT was obtained by affinity chromatography (Ni3+-NTA beads). Molecular weight and purity of MMLV-RT were analyzed with SDS-PAGE. Enzyme activity was characterized with RT-PCR. We successfully constructed the recombinant plasmid pET15b-MMLV-rt and obtained the MMLV-RT fusion protein with 6His on the N-terminus. Recombinant protein was purified through Ni3+-NTA beads based affinity chromatography, the purity of which was 96%. The Activity of the enzyme was high. MMLV-RT of 96% purity was obtained with the prokaryotic expression technique, which serves as the basis for mass production of this enzyme.
Xiansong Wang, Xuemei Ma, Yi Sun

1866 related Products with: [Prokaryotic expression and purification of moloney murine leukemia virus reverse transcriptase and verification of the activity].

100ug2.5 mg1 mg25 1,000 tests25 reactions1 mL50 ug 25 mg100ul1000 tests

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#18443380   // To Up

Molecular assessment of the potential combination therapy of cytokines with biphalin and AZT for Friend leukemia virus infection in vitro.

Biphalin, a dimeric enkephalin analog, is under investigation as a potential, long-lasting medication of pain associated with chronic diseases, like cancer or AIDS. The role of cytokines, and splenocytes in anti-Friend leukemia virus (FLV) activity of biphalin, a synthetic opioid, and AZT was investigated in vitro. Mouse splenocytes inhibited FLV replication in Mus dunni (Dunni) cells when they were added to the cell culture. This inhibitory effect of splenocytes also was evident when cells were combined with biphalin and AZT as measured using a focus-forming assay. Under cell-free conditions, recombinant interferon gamma (IFNgamma), interleukin 2 (IL-2) and IL-4 directly inhibited the FLV reverse transcriptase (RT) activity by 27% to 36%. IFNgamma at 0.005 pg to 500 ng inhibited FLVRT activity by 61% to 80%. Acombination of 250 ng IFNgamma and 50 mug biphalin resulted in a 94% reduction of FLVRT activity, as compared with 61% inhibition by IFNgamma alone. The combination of AZT and IFNgamma, IL-2 or IL-4 also induced a stronger suppression of FLV RT activity than either cytokine or AZT used alone. In addition, cloned RT from Moloney murine leukemia virus (MMLV) was directly sensitive to inhibition by biphalin. Thus, the anti-FLV effects of splenocytes in combination with biphalin and AZT in cell culture are likely mediated to a large degree by the direct effect of cytokines. This antiviral activity of splenocytes or cytokines combined with chemotherapy, biphalin, and/or AZT, could be used as a complementary therapy to current approaches for retroviral infection and benefit acquired immunodeficiency syndrome (AIDS) patients. In conclusion, biphalin applied primarily as a new medicine for chronic pain treatment in AIDS patients may play a significant beneficial role as a component of antiviral HIV multidrug therapies.
Jie-Liu Tang, Andrzej W Lipkowski, Steven Specter

1204 related Products with: Molecular assessment of the potential combination therapy of cytokines with biphalin and AZT for Friend leukemia virus infection in vitro.

10 0.25 mg1 mg1 mL1001 mL 5 G5 200 1 mL

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