Difference between revisions of "Part:BBa K5398600"

(→‎Characterization)
Line 22: Line 22:
 
<body>
 
<body>
 
     <div class="module">
 
     <div class="module">
−
         <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/fanyingtu.webp" width="800" height="auto" alt="Protein purification">
+
         <img src="https://static.igem.wiki/teams/5398/design/design-new/part-fig1.png" width="800" height="auto" alt="Protein purification">
 
         <p><b>Fig. 1 | Synthesis scheme of L-DOPA and further oxidized product dopachrome.</b></p>
 
         <p><b>Fig. 1 | Synthesis scheme of L-DOPA and further oxidized product dopachrome.</b></p>
 
     </div>
 
     </div>

Revision as of 17:18, 1 October 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 tyrosine. As shown in Fig. 1, tyrosinase catalyzes the ortho-hydroxylation of tyrosine to L-DOPA via its monophenolase (MP) activity, and consecutively oxidizes L-DOPA to dopaquinone via the diphenolase (DP) activity, thereby consuming oxygen. dopaquinone is not stable and will be further non-enzymatically oxidized to 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.

Protein purification

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

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.






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]

Reference


[1] 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.
[2] 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. Biomacromolecules, 2014, 15(9): 3278-3289.</p>