Soft robots are known for their intelligence, flexibility, and adaptability, showing great potential in the field of ocean exploration. However, these robots are easily damaged in the complex and changeable marine environment. If not repaired in time, it can greatly decrease their lifespan and increase salvage and repair costs. However, current self-healing materials have shortcomings that limit their practical application, such as severe requirements for the healing process, long healing times (hours), and are not environmentally friendly. Considering the existing problems, our project tried to develop a new type of self-healing material for underwater soft robots.

Then, we noticed squid ring teeth (SRT) proteins which have high elastic modulus and toughness due to their special sequence. We optimized the tandem repeat polypeptides with n repetitions (TRn) derived from squid ring teeth proteins and make them self-assemble into supramolecular β-sheet-stabilized networks (Fig. 1), enhancing their hydrogen-bonded nanostructure and network morphology and giving their excellent healing properties (2–23 MPa strength after 1 s of healing). Such healing performance creates new opportunities for bioinspired materials design, especially in self-healing materials for soft robotics and personal protective equipment.

Fig. 1 | The sequence and structure of tandem repeat polypeptides with n repetitions (TRn) derived from squid ring teeth proteins.

In our project, we used the biosythetic protein TRn5 as a component of special materials to realize self-healing. We anticipated the engineered bacteria could successfully express TRn5 and make it into materials with self-healing ability.

First, we used AlphaFold3 to predict the structure of TRn5. Then, we constructed pET-29a(+)-TRn5 (Fig. 2a) and respectively amplified TRn5 and pET-29a(+) vector using PCR. The PCR produced the pET-29a(+)-TRn5 parts ready for In-fusion Cloning (Fig. 2b,c).

Fig. 2 | The plasmid map of pET-29a(+)-TRn5 and 1% agarose gel electrophoresis of the PCR amplified pET-29a(+)-TRn5 parts.

a. The plasmid map of pET-29a(+)-TRn5. b. 1% agarose gel electrophoresis of the PCR amplified TRn5 (K5398001) (527 bp). c. 1% agarose gel electrophoresis of the PCR amplified pET-29a(+) plasmid (5170 bp).

Next, we made an In-fusion Cloning of purified PCR amplified TRn5 and the pET-29a(+) vector parts and transferred the recombinant plasmid into E.coli DH5α. According to the results of colony PCR and Sanger sequencing, we concluded that the pET-29a(+)-TRn5 plasmid was constructed successfully (Fig. 3).

Fig. 3 | Verification of recombinant plamid pET-29a(+)-TRn5.

a. 1% agarose gel electrophoresis of colony PCR of using T7 and T7 ter primers. b. The result of sequencing the TRn5 of the recombinant plasmid.

We expressed the protein in E.coli BL21(DE3) using LB medium and incubated the cells at 23℃ for 16 h and 37℃ for 5 h respectively. Then, we denatured TRn5 with 8 M urea for 12 h and renatured it by dialysis.

We found the TRn5 predicted by AlphaFold had β-sheet composed of five β-strands and were connected by flexible chains as we expected (Fig. 4). This provided solid foundation for our next experiment.

Fig. 4 | TRn5 predicted by AlphaFold3.

Using SDS-PAGE to assess the expression of TRn5, we found that most TRn5 (17.58 kDa) existed in precipitate as stated in previous research and the TRn5 expression level at two temperatures had little difference (Fig. 5).

Fig. 5 | SDS-PAGE of expression products of TRn5.

Lane 1: marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from uninduced cells at 23℃, respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells at 23℃, respectively; Lanes 8-10: whole-cell lysate, supernatant and pellet from uninduced cells at 37℃, respectively; Lanes 11-13: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively.

As a result, when we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC), we found a great protein loss as shown in SDS-PAGE because the TRn5 expression level was too low to verify (Fig. 6).

Fig. 6 | SDS-PAGE of expression products of TRn5 purified by IMAC.

Lane 1: marker; Lanes 2-11: induced cell samples at 23℃; Lane 2: pellet; lane 3: sample washed with denaturing buffer with 8 M urea; Lane 4: sample after dialysis overnight; Lane 5: sample after being bound to Ni-NTA resin; Lane 6: sample eluted with 20 mM Tris-HCl; Lanes 7-11: samples eluted with 20, 50, 150, 300, 500 mM imidazoles.

Based on the results of production of TRn5, we learned that incubation at 37℃ for 5 h was an efficient method to express TRn5. At the same time, due to the failure of purification of TRn5 by IMAC, we concluded TRn5 was not suitable for this purification strategy. Therefore, after a comprehensive review of the existing literature, we found that the protonation of His residued in the sequence of TRn was important for solubility and the acidification of TRn resulted in conformational switch, causing the protein to self-assemble and facilitating self-healing ability. Therefore, we attampted to use a new protocol to purify TRn5.

Design and Build

We set 37℃ for 5 h as our incubation condition and implemented a novel protocol containing 5% acetic acid for the purification of TRn5. We then obtained protein samples of TRn5 by freezedrying 24 h, dissolved protein samples in 5% acetic acid again and dried them to get TRn5 protein films.

We separated TRn5 by SDS-PAGE, and distinct and clear bands of TRn5 (17.58 kDa) were observed (Fig. 7).

Fig. 7 | SDS-PAGE of expression products of TRn5 using a new protocol.

Lane 1: marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively; Lane 5: sample washed with 5% acetic acid.

We obtained protein samples of TRn5, whose final yield was about 150.4 mg/L bacterial culture (Fig. 8).

Fig. 8 | The protein sample freeze-dried by a lyophilizer.

To test the self-healing ability of TRn5 protein film, we did a punctured simulation. After putting the punctured film at room temperature for 12 h, we saw the hole defect healing (Fig. 9). So it was proved that the materials made of TRn5 have self-healing properties to some extent. However, the self-healing efficiency of TRn5 could not meet the requirements of damaged underwater soft robots, which required higher self-healing efficiency.

Fig. 9 | Self-healing of TRn5 protein film after puncture damage.

a. A hole defect was left by a needle through the film. b. Puncture damage was healed.

To pursue more excellent self-healing materials, we deeply studied literature on TRn and squid ring teeth protein. We found there exists a positive correlation between the number of repeat units and self-healing properties of squid-inspired proteins in an article, which means the more repeat units the proteins have, the better self-healing properties it will be. Therefore, we need to produce the squid-inspired biosynthetic proteins with various long tandem repeats (TRn). However, it would lead to excess burden of chassis and low yield finally. To solve these problems above, we learnt the td intron, also called RNA cyclase ribozyme, which can form a circular mRNA, allowing ribosomes to repeatedly translate the sequence derived from squid ring teeth protein and produce proteins with different repeat numbers. Thus, we adopted the cmRNA strategy to obtain materials with exceptional self-healing properties.

We designed a circular mRNA on which the ORF of TRn5 is between the 3' and 5' intron of td gene from T4 phage. The ribosome binding sequence (RBS) and start codon ATG were placed downstream of TRn5. Consequently, the regulatory sequences were located upstream of TRn5 only after circularization of the mRNA. To purify the mixed TRn protein which repeated for different times, a His tag was incorporated into the TRn5. If the mRNA is circularized, the ribosome could circle the cmRNA, producing proteins with different repeats (Fig. 10).

Fig. 10 | Design of a circular mRNA based on td flanking introns.

We synthesized plasmid pET-29a(+)-cmRNA(TRn5) and transferred it into E.coli BL21(DE3) to express and obtain TRn through the successful methods that TRn5 previously used before. At the same time, we built some mathematical and biological models to examine the stability of the proteins.

After the protein samples were subjected to SDS-PAGE (Fig. 11), we found that TRn sample was a mixture of proteins of various sizes that formed a ladder on the gel due to uncertainty of the round number that the ribosome traveled. According to the protein marker, we supposed that the size of the proteins ranged from about 8 to 96 kDa, indicating that the ribosome could travel along the cmRNA at most 6 rounds.

Fig. 11 | SDS-PAGE of cmRNA expressed at 37℃ and 16℃.

Lane 1: marker; Lanes 2-5: whole-cell lysate, supernatant, pellet and diluted pellet from induced cells at 37℃, respectively; Lane 6: marker; Lanes 7-9: whole-cell lysate, supernatant and pellet from induced cells at 16℃, respectively.

We did a punctured simulation and tested the self-healing ability of materials. We clearly saw the hole defect healing at a quick pace (Fig. 12). Compared with the self-healing effciency of TRn5 protein film (12 h of healing), this self-healing efficiency of TRn produced by RNA cyclase ribozyme mechanism is higher because nearly 90% healing were completed in 1 h. Meanwhile, it should be noticed that the hole defect of TRn protein film is bigger than that of TRn5 protein film.

Fig. 12 | Self-healing of TRn protein films after puncture damage.

Using mathematical modeling to simulate the individual hydrogen bond network (Fig. 13 and Fig. 14), we can see that as the number of repetitions increases, the point-line ratio which can reflect the stability of the network structure gradually decreases, indicating that the network gradually changes from loose to tight. In microscopic protein structures with self-healing functional materials, networks with dense lines can often recover faster and show stronger self-healing ability. Therefore, the more times circular mRNA is translated, the higher the biological activity and functional stability of the protein will be.

Fig. 13 | Intermolecular hydrogen bond networks in different proteins.

TRn with n=5, 10, 15, 20, 25 and 30 from top to bottom, left to right.

Fig. 14 | Results of intermolecular hydrogen bond network.

Subsequently, we simulated the overall hydrogen bond network (Fig. 15 and Fig. 16). we got consistent conclusions. The more times protein repeats, and the more stable the hydrogen bond network between protein molecules will be, which makes the interacting protein molecules more resistant to deformation when facing external perturbations, and have higher self-healing adaptability.

Fig. 15 | Simulation of hydrogen bond networks between TRn molecule.

TRn with n=5, 10, 15, 20, 25 and 30 from top to bottom, left to right.

Fig. 16 | Results of overall hydrogen bond network.

To verify the validity of our mathematical modeling above, molecular dynamics were simulated and the energy in the system was calculated (Fig. 17). We found that as TRn repetitions increased, the energy of the system became lower and lower, so the protein became more stable, which was consistent with the results we obtained through mathematical modeling, and to a certain extent, it showed the accuracy of the mathematical modeling results.

Fig. 17 | Unit energy of different TRn.

We succeeded in producing the tandem repeat polypeptides with n repetitions (TRn) derived from squid ring teeth proteins by RNA cyclase ribozyme mechanism. Simultaneously, we verified our materials with excellent self-healing ability from function tests and mathematical modeling. In brief, all of these findings indicate that the cmRNA is a good strategy to produce squid-inspired biosynthetic proteins with various long tandem repeats.

In the future, we will further research the production of the long tandem repeats by RNA cyclase ribozyme mechanism and improve the self-healing ability of our materials. We hope that our materials can finally realize the self-healing capability in 1 sec, which we saw in papers before.

If you want to know more about our work in wet lab experiments and modeling, you can click the links below.

Wet Lab Experiments https://2024.igem.wiki/nau-china/projectresult

Modeling https://2024.igem.wiki/nau-china/model

The adhesion of material with self-healing capabilities to the substrate material is a crucial part of our project. After extensive literature review, we discovered that the tandem repeat polypeptides with 4 repetitions(TRn4) can bind to the tandem repeat polypeptides with n repetitions(TRn) through hydrogen bonding derived from β-sheets. Additionally, the mussel foot protein type 5 (Mfp5), which is rich in tyrosine residues, can be oxidized to Levodopa(L-DOPA) by tyrosinase TyrVs and then adheres to the substrate material of the soft robot.

Based on the above, we designed a fusion protein TRn4-mfp5 for the adhesion of self-healing proteins to the substrate material of soft robots.

Fig. 18 | The adhesion principle of Mfp5.

We used the pET-21a(+) vector to express the protein in E. coli BL21(DE3) strains, with 0.5 M IPTG induction.

Fig. 19 | The plasmid map of pET-21a(+)-His-SUMO-TRn4-mfp5.

We planned to express the protein in E. coli BL21(DE3) using LB medium. We conducted incubation at 16°C for 20 h or at 37°C for 4 h.

Additionally, we used both LB and TB media for cultivation to identify optimal conditions for protein expression. This comparison was aimed at determining which medium would support higher yields and better solubility of the fusion protein.

We used SDS-PAGE to analyze the protein expression under the conditions of 16°C for 20 h or 37°C for 4 h (Fig. 20).

Fig. 20 | Comparison of fusion protein expression in different temperature.

Lane 1: Protein ladder; Lanes 2-7 (LB 37°C 4 h): Lane 2: Total liquid (IPTG); Lane 3: Supernatant (IPTG); Lane 4: Precipitate (IPTG); Lane 5: Total liquid; Lane 6: Supernatant; Lane 7: Precipitate; Lanes 8-13 (TB 16°C 20 h): Lane 8: Total liquid (IPTG); Lane 9: Supernatant (IPTG); Lane 10: Precipitate (IPTG); Lane 11: Total liquid; Lane 12: Supernatant; Lane 13: Precipitate; Lane 14: Protein ladder.

We found that the protein expressed better under the 16°C for 20 h condition, as indicated by the stronger bands in Fig. 20. This suggests that lower temperature incubation may enhance protein solubility and proper folding, resulting in improved yield.

This is the SDS-PAGE analysis after cultivation in LB and TB media. (Fig. 21)

Fig. 21 | Comparison of fusion protein expression in LB and TB media use vector pET-21a(+).

Lanes 1-6 (LB 16°C 20 h): Lane 1: Total liquid (IPTG); Lane 2: Supernatant (IPTG); Lane 3: Precipitate (IPTG); Lane 4: Total liquid; Lane 5: Supernatant; Lane 6: Precipitate; Lane 7: Protein ladder; Lanes 8-13 (TB 16°C 20 h): Lane 8: Total liquid (IPTG); Lane 9: Supernatant (IPTG); Lane 10: Precipitate (IPTG); Lane 11: Total liquid; Lane 12: Supernatant; Lane 13: Precipitate.

In Fig. 21, We found that the bands in the TB medium were indeed thicker than those in the LB medium, indicating a slight increase in expression levels.

However, the theoretical size of the fusion protein TRn4-mfp5 on the pET-21a(+) vector is 21.2 kDa, but the bands in Fig. 20-21 did not appear in the expected position.

Based on the result above, we have doubted the expression results of the TRn4-mfp5 fusion protein using the pET-21a(+) vector. This discrepancy could be due to unsuitable expression vectors or conditions, or it may be caused by structural interference between the TRn4 and Mfp5 domains, leading to an incorrect protein size.

The fusion protein plays a crucial role in our material. It comprises TRn4, Mfp5, and a GS linker (which links the terminal proteins). However, the structure data for these proteins are currently absent in common protein databases such as RCSB, UniProt, and NCBI.

Consequently, we must predict their structures separately to validate their properties. Fortunately, we can collect possible structural characteristics of TRn4 and Mfp5 from the literature, which will guide our predictions and improve the accuracy of the modeled structures, allowing us to prioritize predictions for these two proteins.

The fusion protein plays a crucial role in our material. It comprises TRn4, Mfp5, and a GS linker (amino acid sequence: GGGGSGGGGSGGGGS, which links the terminal proteins).

However, the structure data for these proteins are currently absent in common protein databases such as RCSB, UniProt, and NCBI. Consequently, we predicted their structures separately to validate their properties. Fortunately, we can collect possible structural characteristics of TRn4 and Mfp5 from the literature, which will guide our predictions and improve the accuracy of the modeled structures, allowing us to prioritize predictions for these two proteins.

Given the limitations of SWISS-MODEL in predicting the structure of larger proteins, we chose to use AlphaFold for predicting the structure of this fusion protein.

To further validate our design, we conducted molecular dynamics simulations, based on the AlphaFold prediction results (Fig. 22), the results of which are shown in Fig. 23. These simulations provided critical insights into the protein's stability, as well as potential dynamic behaviors and conformational changes under various environmental conditions.

To assess whether there was a trend in the root mean square deviation (RMSD) graph, we applied the Cox-Stuart trend test during the 2-3 ns stage of the simulation. This non-parametric test helped determine whether the data exhibited an upward or downward trend over time, providing key analysis to confirm the stability and overall suitability of the fusion protein for real-world applications.

Fig. 22 | Fusion protein predicted by AlphaFold

Fig. 23 | Molecular dynamics simulation for fusion protein.

From the molecular dynamics simulation of the fusion protein, we observed that the overall conformation remained relatively stable throughout the simulation, with no significant structural dissociation or major deformations.

Thus, one of our hypothesis that the fusion protein's unexpectd expression was due to structural interference has been resolved, as the model confirmed that the TRn4 and Mfp5 domains do not interfere with each other.

So we tried to change expression vectors to address the problem with protein expression.

Several studies suggest that using the Rosetta strain may be advantageous, as it provides tRNA genes for rare codons that are often underrepresented in E. coli. This strain is specifically engineered to enhance the expression of eukaryotic genes in prokaryotic systems. We anticipated that utilizing Rosetta would significantly improve protein yield and overall expression efficiency.

Studies have shown that a SUMO tag can enhance protein solubility and reduce the formation of inclusion bodies.

We decided to incorporate a SUMO tag into the plasmid to enhance protein solubility. To achieve this, we planned to construct the plasmid using pET-SUMO (Fig. 24) not only in BL21(DE3) but also in Rosetta strains, which provides tRNA for rare codons, to improve both the expression and quality of the TRn4-mfp5 protein. Additionally, the expression conditions were optimized to further increase protein yield and solubility.

Fig. 24 | The plasmid map of pET-SUMO-TRn4-mfp5.

We expressed the proteins under the optimized conditions of induction at 37°C for 4 h using 0.5 mM IPTG. After lysis, the target protein was primarily found in the pellet, which we denatured with 8 M urea for 12 h. The fusion protein was then purified using a Ni-NTA Gravity Column.

Test

The target protein was successfully expressed in both BL21(DE3) and Rosetta E. coli strains. Although the electrophoresis bands showed slight deviations from the theoretical molecular weight, they remained within an acceptable range, likely due to potential modifications. In Fig. 25, we compared the expression efficiency between the two strains.

Fig. 25 | Comparison of fusion protein expression in E. coli BL21(DE3) and Rosetta.

Lane 1: Protein ladder; Lanes 2-4 (BL21(DE3) LB 37℃ 4 h): Lane 2: Total liquid (IPTG); Lane 3: Supernatant (IPTG); Lane 4: Precipitate (IPTG); Lanes 5-7 (Rosetta LB 37℃ 4 h) Lane 5: Total liquid (IPTG); Lane 6: Supernatant (IPTG); Lane 7: Precipitate (IPTG)

Through SDS-PAGE analysis of the purified protein by using a Ni-NTA Gravity Column, we observed the target protein in lanes 4 to 7, with a particularly strong band in the supernatant after denaturation (Fig. 26, Lane 7), confirming successful expression.

Fig. 26 | Comparison of fusion protein expression in E. coli BL21(DE3) and Rosetta.

Lane 1: Protein-Binding buffer; Lane 2: 20 mM imidazole and 8 M urea elution; Lane 3: 50 mM imidazole and 8 M urea elution; Lane 4: 150 mM imidazole and 8 M urea elution; Lane 5: 300 mM imidazole and 8 M urea elution; Lane 6: 500 mM imidazole and 8 M urea elution; Lane 7: Supernatant; Lane 8: Impurities; Lane 9: Protein ladder.

To precisely confirm the expression of TRn4-mfp5, we performed a western blotting analysis, which provided clear and definitive results, verifying the successful expression of the TRn4-mfp5 protein under the aforementioned conditions (Fig. 27).

Fig. 27 | Western blotting of purified fusion protein TRn4-mfp5(35.4 kDa) expressed by vector pET-SUMO.

a. Western blotting of the pre-expressed protein. Lane 1: Total liquid (IPTG); Lane 2: Supernatant (IPTG); Lane 3: Precipitate (IPTG), b. Western blotting after column purification of the supernatant following denaturation. Lane 1: Supernatant; Lane 2: 20 mM imidazole and 8 M urea elution; Lane 3: 50 mM imidazole and 8 M urea elution; Lane 4: 150 mM imidazole and 8 M urea elution; Lane 5: 300 mM imidazole and 8 M urea elution; Lane 6: 500 mM imidazole and 8 M urea elution.

We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 28). We finally got about 25 mg protein from 1 L bacterial culture.

Fig. 28 | The protein sample freeze-dried by a lyophilizer.

The above addressed the expression issues of the fusion protein TRn4-mfp5. In the following steps, we planned to conduct experiments co-expressing it with tyrosinase to verify that the tyrosine residues in mfp5 need to be converted into L-DOPA, as highlighted in our design, for it to exhibit adhesive properties.

We aimed to ensure that the tyrosine residues in the Mfp5 portion of TRn4-mfp5 can be catalyzed by tyrosinase to form L-DOPA, thereby enabling its adhesive functionality.

To achieve the co-expression of Mfp5 and tyrosinase, we designed a system utilizing two separate plasmids. Tyrosinase was cloned into the pET-SUMO vector to ensure proper folding and solubility, while the fusion protein (TRn4-mfp5) was inserted into the pET-28a(+) vector. This strategic design aims to enable the simultaneous expression of both proteins in the same host cell, allowing for the efficient oxidation of tyrosine residues in Mfp5 during the expression process, thus enhancing its adhesive properties.

Both plasmids were co-transformed into BL21(DE3) cells for simultaneous expression. This was chosen to make the tyrosine residues in Mfp5 oxidized into L-DOPA during the expression process, thereby enhancing its adhesive properties.

To confirm the successful co-expression of tyrosinase and the fusion protein (TRn4-mfp5), we performed a western blotting analysis using an anti-His antibody. Since the fusion protein (TRn4-mfp5) was tagged with a His-tag, this allowed us to specifically detect its expression. The results showed a clear band at the expected molecular weight of TRn4-mfp5, confirming that the fusion protein was successfully expressed. Additionally, a band corresponding to tyrosinase was also detected, suggesting that the tyrosinase may have been expressed with some affinity for the His-tag, likely due to interactions between the proteins during the expression process. The WB image below shows these results.

Fig. 29 | Western blotting analysis of tyrosinase and TRn4-mfp5 co-expression.

Lane 1: total liquid (IPTG); Lane 2: supernatant (IPTG); Lane 3: precipitate (IPTG).

We dissolved protein samples in Buffer A (10 mL 20 mM Tris pH = 8) to reach 0.3 mg/mL, and conduct adhesive ability tests on the fusion protein(Fig. 30). 20 μL of the protein solution was applied, and the pipette tip was placed on a plastic Petri dish lid. After incubation at 37°C for 4 h, the pipette tip successfully adhered.

We found that when tyrosinase is added, the tyrosine residues in Mfp5 are successfully converted to L-DOPA, allowing the fusion protein to adhere effectively. However, without the addition of tyrosinase, the fusion protein fails to exhibit adhesive properties.

Fig. 30 | Adhesive ability test of fusion protein on plastic surface.

Surface Area Calculation:

The surface area for the annular region of the pipette tip is calculated as:

S = π × (r_outer² - r_inner²)

Where:
r_outer = 3 mm = 0.3 cm
r_inner = 1.85 mm = 0.185 cm

Substituting these values, we get:

S = π × (0.3² - 0.185²) = π × (0.09 - 0.034225) = π × 0.055775 ≈ 0.1753 cm²

The total force is calculated as:

F = (5.951 + 0.448 × 15) g × 9.8 N/kg = 12.671 g × 9.8 N/kg ≈ 0.12418 N

The adhesive force produced by the protein is:

P = F / S = 0.12418 N / 0.1753 cm² ≈ 0.708 N/cm² = 7.08 kPa

The adhesive force per milligram of protein is:

P' = P / m = 7.08 kPa / 1 mg = 7.08 kPa/mg

From this experiment, we learned that tyrosinase successfully oxidized the tyrosine residues in Mfp5, which is crucial for enhancing its adhesive properties. We also recognized the potential issue of L-DOPA being further oxidized into dopaquinone, which could negatively impact the adhesive performance of Mfp5. To address this, we planned to add Mfp6 in the co-expression system, as it has the ability to reduce DOPA-quinone back to DOPA, maintaining the adhesive functionality of Mfp5.

Tyrosinase can oxidize tyrosine residues in TRn4-mfp5 to L-DOPA, thereby imparting adhesive properties. After reviewing relevant literature, we selected the tyrosinase TyrVs from Verrucomicrobium spinosum, which exhibits high expression efficiency in heterologous hosts and possesses high monophenolase activity. It effectively converts L-tyrosine into L-DOPA, with greater specificity for L-tyrosine and lower consumption of L-DOPA compared to other bacterial tyrosinases. Based on the reasons above, we chose to express the tyrosinase TyrVs in E. coli.

Fig. 31 | Synthesis scheme of L-DOPA and further oxidized product L-dopaquinone.

We first synthesized the plasmid pETDuet-1-TyrVs-mfp6 and transformed it into E. coli BL21(DE3).

Fig. 32 | Expression plasmids of the co-expression system to obtain in vivo-modified TRn4-mfp5.

We tested the parts we constructed through Sanger sequencing. The results showed that we had successfully constructed the plasmid. The E. coli BL21(DE3) carrying the pETDuet-1-TyrVs-mfp6 were grown at 16°C for 20 h, and a separate batch was grown at 24°C for 20 h in Luria-Bertani (LB) medium.

After protein extraction, different proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (Fig. 33). The results revealed that the expression levels of TyrVs (38 kDa) were low.

Fig. 33 | Expression of recombinant TyrVs in E. coli BL21(DE3) with pETDuet-1-TyrVs-mfp6.

a. SDS-PAGE analysis for the expression of TyrVs cultured for 20 h at 16℃. 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.b. SDS-PAGE analysis for the expression of TyrVs cultured for 20 h at 24℃. 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 considered that the low protein expression might be due to the weak strength of the promoter in the pETDuet-1 vector, and certain sequences in the vector may also affect the expression of TyrVs.

We cloned TyrVs into other high-copy vectors with a strong promoter and further reduced the protein expression temperature in an attempt to obtain soluble TyrVs.

We cloned TyrVs into pET-29a(+) (Fig. 34)and pET-SUMO (Fig. 35). We tested the parts we constructed through Sanger sequencing. The results showed that we had successfully constructed plasmids.

Fig. 34 | Plasmid pET-29a(+)-TyrVs construction results.

a. Expression plasmids of TyrVs. b. PCR results of pET-29a(+)-TyrVs. Lane 1: Marker; Lanes 2,3: Vector; Lanes 4,5: Gene.

Fig. 35 | Plasmid pET-SUMO-TyrVs construction results.

a. Expression plasmids of TyrVs. b.PCR results of pET-SUMO-TyrVs. Lane 1: Marker; Lanes 2,3: Vector; Lanes 4,5: Gene.

The engineered bacteria were further incubated at 16°C for 20 h and 37℃ for 6 h.

After protein extraction, different proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The Coomassie Brilliant Blue staining results showed whether the cultivation temperature is 16℃ or 37 ℃, E. coli BL21(DE3) carrying pET-29a(+) did not exhibit significant expression of TyrVs.

Fig. 36 | Expression of recombinant TyrVs in E. coli BL21(DE3) with pET-29a(+)-TyrVs.

a. SDS-PAGE analysis for the expression of TyrVs cultured for 20 h at 16℃. 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. b. SDS-PAGE analysis for the expression of TyrVs cultured for 6 h at 37℃. Lane 1: marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively.

The E. coli BL21(DE3) carrying pET-SUMO-TyrVs cultured at 16°C were successfully expressed SUMO-TyrVs (52.2 kDa) and it was primarily present in the supernatant, indicating that it was expressed in a soluble form.

Fig. 37 | Expression of recombinant TyrVs in E. coli BL21(DE3) with pET-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 further confirmed the expression of SUMO-TyrVs through western blotting (WB).

Fig. 38 | Western blotting (WB) analysis of recombinant TyrVs in E. coliBL21 (DE3) with pET-SUMO-TyrVs.

Lanes 1-3: whole-cell lysate, pellet and supernatant from induced cells with 0.5 mM IPTG respectively.

Subsequently, we conducted enzymatic activity tests and determined that the K_m and V_max for the catalysis of tyrosine to L-DOPA are: 456.8 μmol/L and 0.31 μmol·L^-1·s^-1 ; the K_m and V_max for the conversion of L-DOPA to dopaquinone are: 8787 μmol/L and 0.86 μmol·L^-1·s^-1.

Through the aforementioned methods and attempts, we successfully achieved the soluble expression of tyrosinase and tested its enzymatic activity, laying the foundation for the modification of TRn4-mfp5.

Unfortunately, we found that a portion of L-DOPA is excessively oxidized to L-dopaquinone, which reduces the adhesion ability of TRn4-mfp5. Through an extensive literature review, we found that mussel foot protein 6 (Mfp6) is particularly rich in cysteine, a sulfur-containing amino acid that can reduce the oxidized form of dopaquinone back to L-DOPA. Therefore, we decided to express the Mfp6 protein to address the issue of excessive oxidation.

We added the Mfp6 protein into the tyrosinase catalytic system and explored its reducing capacity. Furthermore, we learned that the Mfp6 protein is sensitive to high concentrations of IPTG. So, we adjusted the final IPTG concentration to 10 μM during the experimental process.

We cloned Mfp6 into the pET-28a(+) vector, constructed the plasmid pET-28a(+)-Mfp6 and selected E. coli BL21(DE3) as the host to express the Mfp6 protein individually.

Fig. 39 | The results of Plasmid amplification and reconstruction.

a.The plasmid map of pET-28a(+)-Mfp6. b.1 % agarose gel electrophoresis of the PCR amplified Mfp6 and pET-28a(+) vector. Lane 1: 5000bp DNA Marker；Lanes 2,3: the PCR amplified Mfp6(363 bp); Lanes 4,5: the PCR amplified pET-28a(+) vector(5725 bp).

Then, we dialyzed the extracted SUMO-Mfp6 with acetic acid at 16℃ for 12 h. Finally, we used a Hypur T Ni-NTA 6FF (His-Tag) Prepacked Chromatographic Column, 1mL for immobilized metal affinity chromatography (IMAC) purification of the sample and dialyzed it with acetic acid again.

Through SDS-PAGE and western blotting(WB), we found that TyrVs existed in the pellet.

Fig. 40 | Expression of pET-28a(+)-Mfp6(28 kDa).

a.SDS-PAGE of pET-28a(+)-Mfp6(28 kDa). Lane 1: Protein Marker; Lane 2: Mfp6-Whole Cell Lysate (IPTG); Lane 3: Mfp6-Supernatant (IPTG); Lane 4: Mfp6-Pellet-PBS (IPTG); Lane 5: Mfp6-Pellet-Extraction Buffer (IPTG); Lane 6: Mfp6-Whole Cell Lysate-1; Lane 7: Mfp6-Supernatant-1; Lane 8: Mfp6-Pellet-PBS-1; Lane 9: Mfp6-Pellet-Extraction Buffer-1; Lane 10: Mfp6-Whole Cell Lysate-2; Lane 11: Mfp6-Supernatant-2; Lane 12: Mfp6-Pellet-PBS-2; Lane 13: Mfp6-Pellet-Extraction Buffer-2. b.Western blotting(WB) of pET-28a(+)-Mfp6(28 kDa). Lane 1: Mfp6-Whole Cell Lysate (IPTG); Lane 2: Mfp6-Supernatant (IPTG); Lane 3: Mfp6-Pellet-PBS (IPTG).

After obtaining active Mfp6 protein through denaturation and renaturation methods, we adopted two strategies to test the activity of it.

The first strategy: We tested the activity of Mfp6 at different concentrations. We found the OD₄₇₅ in the experimental group were consistently lower than those in the control group across all tested concentrations. Besides, an increasingly obvious colour occurs in experimental group compared to control group along with the increase of concentration (Fig. 41). This suggests that Mfp6 indeed facilitated the reduction of some dopaquinone back to L-DOPA, with the reduction effect being more significant at higher substrate concentrations.

Fig. 41 | Result of activity analysis.

a.Mfp6 activity analysis at different substrate concentrations. b.Mfp6 activity analysis on a 96-Well Plate.

The second strategy: We tested the activity of Mfp6 at different reaction time. We knew the OD₄₇₅ in the experimental group gradually decreased, while those of the control group remained virtually unchanged (Fig. 42). This indicates that Mfp6 progressively reduced dopaquinone back to L-DOPA as the reaction progressed.

Fig. 42 | Mfp6 activity analysis at different reaction time.

In summary, although the experimental process was very arduous, we ultimately verified the feasibility of this project in the experimental environment. In the future, we will continue to optimize this system and strive to expand it to other application areas as soon as possible.