Difference between revisions of "Part:BBa K5398600:Experience"
 
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         <p><b>Fig. 2 | Expression of recombinant TyrVs in <i>E. coli</i>BL21(DE3) with pET-PC-SUMO-TyrVs.</b></p>
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         <p><b>Fig. 2 | Expression of recombinant TyrVs in <i>E. coli</i> BL21(DE3) with pET-PC-SUMO-TyrVs.</b></p>
 
<p>Lane 1: Marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells respectively.</p>
 
<p>Lane 1: Marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells respectively.</p>
 
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         <p><b>Fig. 3 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.</b></p>
 
         <p><b>Fig. 3 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.</b></p>
 
<p>Lane 1: Marker; Lane 2: Lysis Buffer; Lane 3: Supernatant; Lane 4: 20 mM Imidazole; Lane 5: 50 mM Imidazole; Lane 6: 150 mM Imidazole. </p>
 
<p>Lane 1: Marker; Lane 2: Lysis Buffer; Lane 3: Supernatant; Lane 4: 20 mM Imidazole; Lane 5: 50 mM Imidazole; Lane 6: 150 mM Imidazole. </p>
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         <p><b>Fig. 4 | The activity assay results of tyrosinase TyrVs</b></p>
 
         <p><b>Fig. 4 | The activity assay results of tyrosinase TyrVs</b></p>
 
<p><b>a-b.</b> Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from tyrosine to dopaquinone experiments. <b>c-d.</b> Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-DOPA to dopaquinone experiments. </p>
 
<p><b>a-b.</b> Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from tyrosine to dopaquinone experiments. <b>c-d.</b> Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-DOPA to dopaquinone experiments. </p>

Latest revision as of 11:49, 1 October 2024

Applications of BBa_K5398610

We use TyrVs to oxidize tyrosine residues in TRn4-mfp5(BBa_K5398020) to L-DOPA,thereby endowing it with adhesion ability.

Characterization

To validate the functionality of the tyrosinase TyrVs, we designed bacteria expressing TyrVs.We constructed the pET-SUMO-TyrVs vector, after culturing at 16°C for 20 h, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis.

Protein purification

Fig. 2 | Expression of recombinant TyrVs in E. coli BL21(DE3) with pET-PC-SUMO-TyrVs.

Lane 1: Marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells respectively.

We purified SUMO-TyrVs using a HiTrap Ni-NTA column. The purified protein was verified by SDS-PAGE and was found to be present in the 50 mM imidazole elution fraction.
Protein purification

Fig. 3 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.

Lane 1: Marker; Lane 2: Lysis Buffer; Lane 3: Supernatant; Lane 4: 20 mM Imidazole; Lane 5: 50 mM Imidazole; Lane 6: 150 mM Imidazole.

We conducted tests on the reactions from tyrosine to dopaquinone and from L-DOPA to dopaquinone. The experiment of enzymatic reaction from tyrosine to dopaquinone was conducted at 37℃ with an enzyme concentration of 0.1 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 456.8 μmol/L and 0.31 μmol/L·s, respectively. The experiment of enzymatic reaction from L-DOPA to dopaquinone was conducted at 37°C with an enzyme concentration of 0.2 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 8787 μmol/L and 0.86 μmol/L·s, respectively.
Protein purification

Fig. 4 | The activity assay results of tyrosinase TyrVs

a-b. Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from tyrosine to dopaquinone experiments. c-d. Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-DOPA to dopaquinone experiments.