Part:BBa_K5398610

pET-PC-SUMO-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.

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.coli BL21(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 ℃ 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.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.

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.coli BL21 (DE3) with pET-PC-SUMO-TyrVs.

Lanes 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.

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 h, 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 min 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.

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.

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.

Mathematical modeling analysis

Tyrosinase exhibits dual catalytic properties, capable of catalyzing the conversion of tyrosine to L-DOPA and L-DOPA to dopaquinone. We analyzed through mathematical modeling to determine how to maximize the oxidation of tyrosine to L-DOPA. We selected multiple sets of parameters for fitting, and the goodness of fit R² was 0.9962, indicating a good fitting effect. We incorporated appropriate fitting parameters into the established model and determined that the optimal reaction time is approximately 130 sec, at which point the production of L-DOPA reaches its peak. To demonstrate that the reaction can proceed stably under conventional conditions, we introduced perturbations in each reaction channel.Under different disturbance conditions, the trend of dopamine quantity changes is similar, and the yield fluctuation is small, our reaction system has strong environmental adaptability and stability.

Fig. 8 | The activity assay results of tyrosinase TyrVs

a.Data fitting results. b.Changes in the concentrations of various substances in the reaction system. c.Changes in the concentration of substances in the system after adding disturbances.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1418
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 174
  Illegal BamHI site found at 708
  
• 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.