Difference between revisions of "Part:BBa K5398610"
Jinmengliu (Talk | contribs) |
Jinmengliu (Talk | contribs) |
||
Line 23: | Line 23: | ||
<div class="module"> | <div class="module"> | ||
<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"> | ||
− | <p><b>Fig. 1 Synthesis scheme of L-DOPA and further oxidized product L-dopachrome.</b></p> | + | <p><b>Fig. 1 | Synthesis scheme of L-DOPA and further oxidized product L-dopachrome.</b></p> |
</div> | </div> | ||
</body> | </body> | ||
Line 30: | Line 30: | ||
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=== | ||
− | ==== | + | ====Plasmid Construction==== |
− | We considered cloning TyrVs into the pET-SUMO vector to | + | 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). |
− | <html lang=" | + | <html lang="zh"> |
<head> | <head> | ||
<meta charset="UTF-8"> | <meta charset="UTF-8"> | ||
<meta name="viewport" content="width=device-width, initial-scale=1.0"> | <meta name="viewport" content="width=device-width, initial-scale=1.0"> | ||
− | |||
<style> | <style> | ||
− | . | + | .module { |
− | + | border: 1px solid #ccc; /* 边框 */ | |
− | border: 1px solid # | + | padding: 20px; /* 内边距 */ |
− | padding: | + | 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 */ | ||
} | } | ||
− | . | + | </style> |
− | width: | + | </head> |
+ | <body> | ||
+ | <div class="module"> | ||
+ | <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/sumo-tyrvs.webp" width="400" height="auto" alt="Protein purification"> | ||
+ | <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> | ||
+ | </div> | ||
+ | </body> | ||
+ | </html> | ||
+ | ====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. | ||
+ | <html lang="zh"> | ||
+ | <head> | ||
+ | <meta charset="UTF-8"> | ||
+ | <meta name="viewport" content="width=device-width, initial-scale=1.0"> | ||
+ | <style> | ||
+ | .module { | ||
+ | border: 1px solid #ccc; /* 边框 */ | ||
+ | padding: 20px; /* 内边距 */ | ||
+ | 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 */ | ||
} | } | ||
− | + | </style> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</head> | </head> | ||
<body> | <body> | ||
− | <div class=" | + | <div class="module"> |
− | <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs- | + | <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-pre-expression.webp" width="400" height="auto" alt="Protein purification"> |
− | <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> | ||
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> | ||
− | + | ====Western blotting==== | |
− | <html lang=" | + | Western bolotting revealed that after induction with IPTG, TyrVs was primarily expressed in its soluble form. |
+ | <html lang="zh"> | ||
<head> | <head> | ||
<meta charset="UTF-8"> | <meta charset="UTF-8"> | ||
<meta name="viewport" content="width=device-width, initial-scale=1.0"> | <meta name="viewport" content="width=device-width, initial-scale=1.0"> | ||
− | |||
<style> | <style> | ||
− | . | + | .module { |
− | + | border: 1px solid #ccc; /* 边框 */ | |
− | border: 1px solid # | + | padding: 20px; /* 内边距 */ |
− | padding: | + | 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 */ | ||
} | } | ||
− | + | </style> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</head> | </head> | ||
<body> | <body> | ||
− | <div class=" | + | <div class="module"> |
− | <img src="https://static.igem.wiki/teams/5398/tyrvs/ | + | <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-western.webp" width="300" height="auto" alt="Protein purification"> |
− | <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 | + | <p> Lane 1-3:whole-cell lysate,pellet and supernatant from induced cells with 0.5 mM IPTG respectively.</p> |
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> | ||
− | + | 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. | |
− | <html lang=" | + | <html lang="zh"> |
<head> | <head> | ||
<meta charset="UTF-8"> | <meta charset="UTF-8"> | ||
<meta name="viewport" content="width=device-width, initial-scale=1.0"> | <meta name="viewport" content="width=device-width, initial-scale=1.0"> | ||
− | |||
<style> | <style> | ||
− | . | + | .module { |
− | + | border: 1px solid #ccc; /* 边框 */ | |
− | border: 1px solid # | + | padding: 20px; /* 内边距 */ |
− | padding: | + | 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 */ | ||
} | } | ||
− | + | </style> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</head> | </head> | ||
<body> | <body> | ||
− | <div class=" | + | <div class="module"> |
− | <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-mizuo.webp"> | + | <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/mizuo-tyrvs.webp" width="400" height="auto" alt="Protein purification"> |
− | <p | + | <p><b>Fig. 5 | 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> | |
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> | ||
====Enzyme activity test==== | ====Enzyme activity test==== | ||
− | We dialyzed the extracted SUMO-TyrVs for 24 hours | + | 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. |
− | <html lang=" | + | <html lang="zh"> |
<head> | <head> | ||
<meta charset="UTF-8"> | <meta charset="UTF-8"> | ||
<meta name="viewport" content="width=device-width, initial-scale=1.0"> | <meta name="viewport" content="width=device-width, initial-scale=1.0"> | ||
− | |||
<style> | <style> | ||
− | . | + | .module { |
− | + | border: 1px solid #ccc; /* 边框 */ | |
− | border: 1px solid # | + | padding: 20px; /* 内边距 */ |
− | padding: | + | 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 */ | ||
} | } | ||
− | . | + | </style> |
− | width: | + | </head> |
+ | <body> | ||
+ | <div class="module"> | ||
+ | <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-96.webp" width="400" height="auto" alt="Protein purification"> | ||
+ | <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> | ||
+ | </body> | ||
+ | </html> | ||
+ | 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. | ||
+ | <html lang="zh"> | ||
+ | <head> | ||
+ | <meta charset="UTF-8"> | ||
+ | <meta name="viewport" content="width=device-width, initial-scale=1.0"> | ||
+ | <style> | ||
+ | .module { | ||
+ | border: 1px solid #ccc; /* 边框 */ | ||
+ | padding: 20px; /* 内边距 */ | ||
+ | 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 */ | ||
} | } | ||
− | + | </style> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</head> | </head> | ||
<body> | <body> | ||
− | <div class=" | + | <div class="module"> |
− | <img src="https://static.igem.wiki/teams/5398/tyrvs/ | + | <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/new-abcd.webp" width="400" height="auto" alt="Protein purification"> |
− | <p | + | <p><b>Fig. 7 | The activity assay results of tyrosinase TyrVs</b></p> |
− | + | <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> | |
</div> | </div> | ||
</body> | </body> | ||
+ | </html> | ||
<br><br><br><br> | <br><br><br><br> | ||
Line 157: | Line 192: | ||
− | + | === 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
Contents
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>
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).
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.
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.
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.
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.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 309
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]