Difference between revisions of "Part:BBa K5398610"
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         <img src="https://static.igem.wiki/teams/5398/mfp6-picture/.webp" width="400" height="auto" alt="Protein purification">
 
         <img src="https://static.igem.wiki/teams/5398/mfp6-picture/.webp" width="400" height="auto" alt="Protein purification">
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         <p><b>Fig. 1 Synthesis scheme of L-DOPA and further oxidized product L-dopachrome.</b></p>
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         <p><b>Fig. 1 | Synthesis scheme of L-DOPA and further oxidized product L-dopachrome.</b></p>
 
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In our project, TyrVs can catalyze the tyrosine residues in the TRn4-mfp5 protein, converting them into L-DOPA, thereby enhancing its adhesive properties. L-DOPA exhibits excellent adhesion, particularly in moist environments. This transformation process is similar to the mechanism used by marine organisms like mussels, which enhance their adhesion through L-DOPA.
 
In our project, TyrVs can catalyze the tyrosine residues in the TRn4-mfp5 protein, converting them into L-DOPA, thereby enhancing its adhesive properties. L-DOPA exhibits excellent adhesion, particularly in moist environments. This transformation process is similar to the mechanism used by marine organisms like mussels, which enhance their adhesion through L-DOPA.
 
===Characterization===
 
===Characterization===
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====Protein expression====
+
====Plasmid Construction====
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We considered cloning TyrVs into the pET-SUMO vector to potentially increase its expression levels. So we constructed the pET-SUMO-TyrVs vector, after culturing at 16°C for 20 hours, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis.
+
We considered cloning TyrVs into the pET-PC-SUMO vector to explore the potential for enhancing its expression level. We constructed the pET-PC-SUMO-TyrVs vector and transformed it into <i>E. coli</i>BL21(DE3).
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         <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/sumo-tyrvs.webp" width="400" height="auto" alt="Protein purification">
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        <p><b>Fig. 2 | Plasmid pET-PC-SUMO-TyrVs construction results.</b></p>
 +
<p>a.Expression plasmids of TyrVs. b.PCR results of pET-PC-SUMO-TyrVs. Line 1: Marker. Lines 2-3:Vector;Lines 4-5:Gene.</p>
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====Protein expression====
 +
a single colony
 +
from a freshly streaked plate of the cells was cultured in 5 mL of LB
 +
medium with 25 μg/mL Ampicillin at 37℃ overnight. The secondary
 +
cultures were prepared with 1% inoculum in 50 mL of LB medium with 25 μg/mL Ampicillin. Cultures were
 +
then incubated at 37℃ and 200 rpm until the optical density at 600 nm
 +
(OD<sub>600</sub>) reached 0.6–0.8. 1 mM IPTG was added to induce production of recombinant proteins and
 +
cultures were further cultivated at 16℃ and 200 rpm for 20 h. The cells
 +
were collected by centrifugation at 6000 ×g at 4℃ for 20 min.The recombinant cells were harvested by centrifugation and re-suspension in lysis buffer(10 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, pH 8.0)and lysed on ice by sonication.Sonicated samples were centrifuged at 12,000 ×g at 4 ◦C for 20 min to obtain insoluble and soluble fractions.After protein extraction, different proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue.
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            margin: 20px auto; /* 外边距，自动居中 */
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            text-align: center; /* 内容居中 */
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         <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-pre-expression.webp" width="400" height="auto" alt="Protein purification">
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         <p class="caption"><b>Fig. 1 Plasmid profile of pET-PC-SUMO</b><br></p>
+
         <p><b>Fig. 3 | Expression of recombinant TyrVs in <i>E. coli</i>BL21 (DE3) with pET-PC-SUMO-TyrVs.</b></p>
 +
<p>Lane 1: Marker. lanes 2 to 4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively;lanes 5 to 7: whole-cell lysate, supernatant and pellet from induced cells respectively.</p>
 
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We constructed the pET-SUMO-TyrVs vector, after culturing at 16°C for 20 hours, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis. The SUMO-TyrVs (52.2 kDa) was primarily present in the supernatant, indicating that it was expressed in a soluble form.
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====Western blotting====
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Western bolotting revealed that after induction with IPTG, TyrVs was primarily expressed in its soluble form.
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         <img src="https://static.igem.wiki/teams/5398/tyrvs/pre-expression.webp">
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         <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-western.webp" width="300" height="auto" alt="Protein purification">
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         <p class="caption"><b>Fig. 2 Protein pre-expression of SUMO-TyrVs(52.2 kDa).</b><br></p>
+
         <p><b>Fig. 4 | Western blotting analysis recombinant TyrVs in <i>E. coli</i>BL21 (DE3) with pET-PC-SUMO-TyrVs.</b></p>
−
Lane 7: TyrVs-Whole Cell Lysate(+IPTG). Lane 8: TyrVs-Supernatant(+IPTG). Lane 9: TyrVs-Pellet(+IPTG). Lane 10: TyrVs-Whole Cell Lysate(CK). Lane 11: TyrVs-Supernatant(CK). Lane 12: TyrVs-Pellet(CK).  
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<p> Lane 1-3:whole-cell lysate,pellet and supernatant from induced cells with 0.5 mM IPTG respectively.</p>
 
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Subsequently, 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.
+
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.
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            width: 800px; /* 模块宽度 */
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         <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-mizuo.webp">
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         <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/mizuo-tyrvs.webp" width="400" height="auto" alt="Protein purification">
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         <p class="caption"><b>Fig. 3 Protein expression of SUMO-TyrVs(52.2 kDa).</b></p>
+
         <p><b>Fig. 5 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.</b></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.
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<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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====Enzyme activity test====
 
====Enzyme activity test====
−
We dialyzed the extracted SUMO-TyrVs for 24 hours and then diluted it 10,000 times for the enzyme activity assay. Given that tyrosinase exhibits dual catalytic properties, capable of catalyzing the conversion of tyrosine to L-DOPA and L-DOPA to dopaquinone, we aimed to develop a model to determine how to maximize the oxidation of tyrosine to L-DOPA. Therefore, 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°C 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.
+
We dialyzed the extracted SUMO-TyrVs for 24 hours, followed by diluting it 10,000-fold for enzymatic activity assays. In a 96 Well Cell Culture Plates, we prepared different concentrations of tyrosine and L-DOPA solution, added the diluted SUMO-TyrVs, and measured the change in OD475 over the first 5 minutes using a microplate reader.  
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            margin: 20px auto; /* 外边距，自动居中 */
−
             text-align: center;  
+
            width: 800px; /* 模块宽度 */
 +
             text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 
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 +
        <p><b>Fig. 6 | The 96 Well Cell Culture Plates of tyrosinase TyrVs.</b></p>
 +
<p>a.The experiment of enzymatic reaction from tyrosine to dopaquinone. b.The experiment of enzymatic reaction from L-DOPA to dopaquinone. </p>
 +
    </div>
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</body>
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The data were processed to generate a Michaelis-Menten curve and a Lineweaver-Burk plot. The experiment of enzymatic reaction from tyrosine to dopaquinone was conducted at 37°C 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<sup>-1</sup>·s<sup>-1</sup>, 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<sup>-1</sup>·s<sup>-1</sup>, respectively.
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 +
            margin: 20px auto; /* 外边距，自动居中 */
 +
             width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 
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         <p class="caption"><b>Fig. 4 Tyrosinase TyrVs kinetic parameters</b></p>
+
         <p><b>Fig. 7 | The activity assay results of tyrosinase TyrVs</b></p>
−
      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.
+
<p>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. </p>
 
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==== Reference ====
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=== Reference ===
  
 
<br>#TAN D, ZHAO J P, RAN G Q, et al. Highly efficient biocatalytic synthesis of L-DOPA using in situ immobilized <em>Verrucomicrobium spinosum</em> tyrosinase on polyhydroxyalkanoate nano-granules [J]. <em>Appl. Microbiol. Biotechnol.</em>, 2019, 103(14): 5663-78.
 
<br>#TAN D, ZHAO J P, RAN G Q, et al. Highly efficient biocatalytic synthesis of L-DOPA using in situ immobilized <em>Verrucomicrobium spinosum</em> tyrosinase on polyhydroxyalkanoate nano-granules [J]. <em>Appl. Microbiol. Biotechnol.</em>, 2019, 103(14): 5663-78.

Revision as of 01:40, 29 September 2024

A tyrosinase enzyme TyrVs

Introduction

Tyrosinase is a copper-containing oxidoreductase that possesses two catalytic activities, and is involved in the first few steps of melanin synthesis from l-tyrosine. As shown in Fig. 1, tyrosinase catalyzes the ortho-hydroxylation of l-tyrosine to l-DOPA via its monophenolase (MP) activity, and consecutively oxidizes l-DOPA to l-dopaquinone via the diphenolase (DP) activity, thereby consuming oxygen. l-dopaquinone is not stable and will be further non-enzymatically oxidized to l-dopachrome (a red-colored product) in the presence of O2.TyrVs refers to a tyrosinase enzyme derived from Verrucomicrobium spinosum, which plays a critical role in the hydroxylation of tyrosine residues into L-Dopa. This enzyme has shown efficient activity, particularly in the context of biological adhesion, as demonstrated in studies co-expressing mussel foot protein 3 with TyrVs. </p>

Protein purification

Fig. 1 | Synthesis scheme of L-DOPA and further oxidized product L-dopachrome.

Usage and Biology

In our project, TyrVs can catalyze the tyrosine residues in the TRn4-mfp5 protein, converting them into L-DOPA, thereby enhancing its adhesive properties. L-DOPA exhibits excellent adhesion, particularly in moist environments. This transformation process is similar to the mechanism used by marine organisms like mussels, which enhance their adhesion through L-DOPA.

Characterization

Plasmid Construction

We considered cloning TyrVs into the pET-PC-SUMO vector to explore the potential for enhancing its expression level. We constructed the pET-PC-SUMO-TyrVs vector and transformed it into E. coliBL21(DE3).

Protein purification

Fig. 2 | Plasmid pET-PC-SUMO-TyrVs construction results.

a.Expression plasmids of TyrVs. b.PCR results of pET-PC-SUMO-TyrVs. Line 1: Marker. Lines 2-3:Vector;Lines 4-5:Gene.

Protein expression

a single colony from a freshly streaked plate of the cells was cultured in 5 mL of LB medium with 25 μg/mL Ampicillin at 37℃ overnight. The secondary cultures were prepared with 1% inoculum in 50 mL of LB medium with 25 μg/mL Ampicillin. Cultures were then incubated at 37℃ and 200 rpm until the optical density at 600 nm (OD600) reached 0.6–0.8. 1 mM IPTG was added to induce production of recombinant proteins and cultures were further cultivated at 16℃ and 200 rpm for 20 h. The cells were collected by centrifugation at 6000 ×g at 4℃ for 20 min.The recombinant cells were harvested by centrifugation and re-suspension in lysis buffer(10 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, pH 8.0)and lysed on ice by sonication.Sonicated samples were centrifuged at 12,000 ×g at 4 ◦C for 20 min to obtain insoluble and soluble fractions.After protein extraction, different proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue.

Protein purification

Fig. 3 | Expression of recombinant TyrVs in E. coliBL21 (DE3) with pET-PC-SUMO-TyrVs.

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

Western blotting

Western bolotting revealed that after induction with IPTG, TyrVs was primarily expressed in its soluble form.

Protein purification

Fig. 4 | Western blotting analysis recombinant TyrVs in E. coliBL21 (DE3) with pET-PC-SUMO-TyrVs.

Lane 1-3:whole-cell lysate,pellet and supernatant from induced cells with 0.5 mM IPTG 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. 5 | 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.

Enzyme activity test

We dialyzed the extracted SUMO-TyrVs for 24 hours, followed by diluting it 10,000-fold for enzymatic activity assays. In a 96 Well Cell Culture Plates, we prepared different concentrations of tyrosine and L-DOPA solution, added the diluted SUMO-TyrVs, and measured the change in OD475 over the first 5 minutes using a microplate reader.

Protein purification

Fig. 6 | The 96 Well Cell Culture Plates of tyrosinase TyrVs.

a.The experiment of enzymatic reaction from tyrosine to dopaquinone. b.The experiment of enzymatic reaction from L-DOPA to dopaquinone.

The data were processed to generate a Michaelis-Menten curve and a Lineweaver-Burk plot. The experiment of enzymatic reaction from tyrosine to dopaquinone was conducted at 37°C 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-1·s-1, 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-1·s-1, respectively.
Protein purification

Fig. 7 | 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.








Reference


#TAN D, ZHAO J P, RAN G Q, et al. Highly efficient biocatalytic synthesis of L-DOPA using in situ immobilized Verrucomicrobium spinosum tyrosinase on polyhydroxyalkanoate nano-granules [J]. Appl. Microbiol. Biotechnol., 2019, 103(14): 5663-78.
#YAO L, WANG X, XUE R, et al. Comparative analysis of mussel foot protein 3B co-expressed with tyrosinases provides a potential adhesive biomaterial [J]. Int. J. Biol. Macromol., 2022, 195: 229-36.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 309
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]