Difference between revisions of "Part:BBa K3853014"
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<p style="text-align:center"><b>Fig. 2 SDS-PAGE of MnP(E74M).</b> <i>lane 2 for MnP(E74M)</i></p> | <p style="text-align:center"><b>Fig. 2 SDS-PAGE of MnP(E74M).</b> <i>lane 2 for MnP(E74M)</i></p> | ||
− | <P>For assaying the mRNA expression of MnP (E74M), qRT-PCRs were performed. We did data analysis using a variation of the Livak method. To determine the relative expression of MnP (E74M) vs. reference gene <i>ACT1</i>, total RNA was prepared from an equal volum of yeast solution. In Fig. , MnP (E74M) reached its peak to a fold difference of 0.11 after 2% methanol inducing for 36 h. The C<sub>T<sub> values for the MnP (E74M) and the reference gene *ACT1* were then used to calculate the fold difference with the following equation:</P> | + | <P>For assaying the mRNA expression of MnP (E74M), qRT-PCRs were performed. We did data analysis using a variation of the Livak method. To determine the relative expression of MnP (E74M) vs. reference gene <i>ACT1</i>, total RNA was prepared from an equal volum of yeast solution. In Fig. 3, MnP (E74M) reached its peak to a fold difference of 0.11 after 2% methanol inducing for 36 h. The C<sub>T<sub> values for the MnP (E74M) and the reference gene *ACT1* were then used to calculate the fold difference with the following equation:</P> |
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Revision as of 22:18, 21 October 2021
MnP (E74M)
Manganese peroxidase (MnP) is the key enzyme in our degrading system. In order to improve its catalyzing ability, we tried rational design. And according to the computational redesign results, 6 mutants were chosen and tested, including their relative enzyme activity and the effect of temperature/pH/organic solvents on them. MnP(E74M) is one of the most promising mutant of MnP. We use BBa_K3853057 to construct the expression system to express and purify the protein.
Biology
Manganese peroxidase (MnP), a glycosylated heme enzyme derived from the white-rot fungus Phanerochaete chrysosporium, can oxidize Mn2+ to Mn3+ under the action of H2O2. Mn3+ can be released outside the enzyme under the action of a chelate such as malonic acid and can oxidise a wide range of phenolic and non-phenolic compounds as a common substrate. The Mn3+-malonic acid chelate can be detected at 469 nm by oxidation of 2,6-dimethyloxyphenol (2,6-DMP), which is also the main enzyme activity detection method for MnP. MnP (E74M) is obtained by mutating the glutamate at position 74 of wild-type MnP (BBa_K3853000) to methionine.
Usage
We mutated the glutamate at position 74 of wild-type MnP to methionine through single-point mutation in order to improve the stability of wild-type MnP. We use BBa_K3853057 to construct the expression system to express and purify the protein.
Characterization
1. Identification
After receiving the synthetic plasmid, we electrotransformed it into Pichia pastoris, and selected monoclonal colonies for colony PCR to verify the successful transformation.
Fig. 1 Agarose gel electrophoresis of PCR products of monoclonal colonies of MnP (E74M). lane 2 for MnP(E74M)
2. Proof of the expression
After the expressed protein was re-dissolved by ammonium sulfate precipitation, it was verified by running gel, and the target protein band was observed by SDS-PAGE (Fig. 2).
Fig. 2 SDS-PAGE of MnP(E74M). lane 2 for MnP(E74M)
For assaying the mRNA expression of MnP (E74M), qRT-PCRs were performed. We did data analysis using a variation of the Livak method. To determine the relative expression of MnP (E74M) vs. reference gene ACT1, total RNA was prepared from an equal volum of yeast solution. In Fig. 3, MnP (E74M) reached its peak to a fold difference of 0.11 after 2% methanol inducing for 36 h. The CT values for the MnP (E74M) and the reference gene *ACT1* were then used to calculate the fold difference with the following equation:</P>
<p>[1] Wariishi, H., Valli, K. & Gold, M. H. Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators. The Journal of biological chemistry 267, 23688-23695 (1992).</p>
Sequence and Features
References