Our goal was to develop self-healing materials produced by engineered bacteria. This material addressed the numerous challenges soft robots encountered during ocean exploration. To achieve this, we needed to accomplish three steps. First, we produced a stable self-healing material composed of squid-inspired biosynthetic proteins. Second, we created an adhesive protein that could connect the soft robot with the self-healing protein. Finally, we increased the Levodopa (L-DOPA) content in the adhesive protein and prevented its over-oxidation. Our experimental results confirmed that our designed method could effectively complete these tasks. Next, we presented the results of our experiments.

Fig. 1 | An efficient strategy of producing the tandem repeat polypeptides with n repetitions (TRn) derived from squid ring teeth proteins using circular mRNA (cmRNA).

The tandem repeat polypeptides with n repetitions (TRn) derived from squid ring teeth proteins show excellent healing properties due to their supramolecular β-sheet-stabilized networks. It's proved there exists a positive correlation between the number of repeat units and self-healing properties of our materials containing TRn. In order to obtain materials with better self-healing ability, we need to express TRn proteins with more repeat units, which might lead to excess burden of chassis and low yield finally. To solve these problems above, we utilized the td intron to design circular mRNAs (cmRNA). A self-cleaving RNA cyclase ribozyme was incorporated to form the cmRNAs, allowing ribosomes to repeatedly translate the sequence of interest and producing proteins with different repeat times. Thus, we could obtain materials composed of TRn with exceptional self-healing properties.

• We found an effective means of production and purification of TRn.
• We confirmed that materials composed of TRn had the properties of self-healing.
• We succeeded in producing squid-inspired biosynthetic proteins with various long tandem repeats by a strategy of mRNA circularization.
• We verified our materials with excellent self-healing ability from function tests and mathematical modeling.

Expression and optimization of TRn5 using linear mRNA

We first used TRn5 to produce our self-healing material and tested its function. From the result of TRn5 predicted using AlphaFold3 (Fig. 2), we found the TRn5 as we expected had β-sheet composed of five β-strands and were connected by flexible chains, which provided solid foundation for our next experiment.

Fig. 2 | TRn5 predicted by AlphaFold3.

Then, we constructed pET-29a(+)-TRn5 plasmid to express TRn5 (Fig. 3).

Fig. 3 | 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(+) vector (5170 bp).

According to the results of colony PCR and Sanger sequencing (Fig. 4), we concluded that the pET-29a(+)-TRn5 plasmids were constructed successfully.

Fig. 4 | Verification of recombinant plasmid 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.

Next, we expressed TRn5 in E.coli BL21(DE3) using LB medium. After incubation at 23℃ for 16 h and 37℃ for 5 h respectively, we found that most TRn5 (17.58 kDa) existed in precipitate 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.

Then, we denatured TRn5 with 8 M urea overnight and renatured it by dialysis, which proved great protein loss as shown in SDS-PAGE. As a result, when we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC), 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; Lane 7-11: samples eluted with 20, 50, 150, 300, 500 mM imidazoles.

In order to optimize the expression of TRn5, we conducted a comprehensive review of the existing literature, revealing that the presence of Histidine facilitates the effortless dissolution of TRn5 in 5% acetic acid. Consequently, we implemented a novel protocol for the purification of TRn5. Upon solubilization in 5% acetic acid, a distinct and clear band of TRn5 was 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 by freezedrying 24 h (Fig. 8). The final yield was about 150.4 mg/L bacterial culture. Next, we dissolved protein samples in 5% acetic acid to reach 20 mg/μL, cast them into square models and dried them at 70℃ for 3 h to obtain protein films.

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

To examine the property of self-healing, we punctured a TRn5 protein film to create a hole defect by a needle (Fig. 9a). After putting the punctured film at room temperature for 12 h, we clearly saw the hole defect healing (Fig. 9b). So it was proved that this kind of film made of TRn5 had self-healing properties. 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 films after puncture damage.

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

Expression and optimization of TRn using circular mRNA (cmRNA)

In order to improve the self-healing ability of our materials, we needed to produce squid-inspired biosynthetic proteins with various long tandem repeats. Thus, we synthesized pET-29a(+)-cmRNA(TRn5) plasmid (Fig. 10), transferred it into E.coli BL21(DE3) and express it.

Fig. 10 | The plasmid map of pET-29a(+)-cmRNA(TRn5).

Optimization of incubation temperature

To determine which incubation temperature is better for protein expression using mRNA circularization, we inoculated the cells at 37℃ for 5 h, 23℃ for 16 h and 16℃ for 20 h respectively.

From the SDS-PAGE (Fig. 11), we concluded these following results:

① Proteins formed a ladder on the gel

The TRn polypeptide was composed of repeating units with a size of 16 kDa, which was formed by the ribosome traveling one round along the cmRNA. Due to uncertainty of the round number that the ribosome traveled, TRn sample was a mixture of proteins with various sizes that formed a ladder on the gel. According to the protein marker, we supposed that the sizes of the proteins ranged from about 8 to 96 kDa, indicating that the ribosome could travel along the cmRNA at most 6 rounds.

② The strategy of cmRNA facilitated the solubility of TRn

It was proved that TRn is a sort of inclusion body protein expressed in E.coli from plenty of literature. In our SDS-PAGE results, though part of TRn in the precitate, a substantial portion of TRn existed in inclusion body protein supernatant, which indicated the strategy using cmRNA could improve protein solubility.

③ Incubation temperature barely influenced the TRn expression

From the SDS-PAGE of expression products of cmRNA at different incubation temperatures, we found there were few differences among them. This showed the strategy employing cmRNA to express TRn had a low requirement.

Fig. 11 | SDS-PAGE of expression products of cmRNA at different incubation temperatures.

a. SDS-PAGE of cmRNA expressed at 23℃. Lane 1: marker; lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells, respectively. b. 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.

Optimization of IPTG concentration

To determine which IPTG concentration is better for protein expression using mRNA circularization, we induced the cells with 0.5 mM and 1 mM IPTG respectively.

Then, we found that the TRn expression level at two IPTG concentrations (0.5 mM and 1 mM) had little difference and the proteins also formed a ladder on the gel (Fig. 12).

Fig. 12 | SDS-PAGE of expression products of cmRNA induced with different IPTG concentrations.

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 with 1 mM IPTG, respectively.

Protein purification by Immobilized Metal Affinity Chromatography (IMAC)

Then, we purified the proteins by IMAC (Immobilised Metal Affinity Chromatography) using Ni-NTA resin.

We found that the TRn expression level was too low to verify by SDS-PAGE (Fig. 13), just like the result of TRn5. On the one hand, we speculated this kind of protein is not suitable for IMAC to purify. On the other hand, we supposed the His tag on TRn could not function well because it was not at the C or N terminal of targeting proteins like others, which posed a challenge for protein purification.

Fig. 13 | SDS-PAGE of expression products of cmRNA purified by IMAC.

Lanes 1-6: induced cell samples at 16℃; Lane 1: sample after being bound to Ni-NTA resin; Lane 2-6: sample eluted with 20 mM Tris-HCl; Lanes 3-6: samples eluted with 50, 150, 300 and 500 mM imidazole; Lane 7: marker; Lanes 8-13, induced cell samples at 37℃; Lane 8: sample after being bound to Ni-NTA resin; Lane 9: sample eluted with 20 mM Tris-HCl; Lanes 10-13: samples eluted with 50, 150 and 300 mM imidazole.

Protein purification using a new protocol

Therefore, we used the protocol containing 5% acetic acid again. We found that the TRn dissolved in 5% acetic acid still presented a ladder on the gel (Fig. 14). And due to unpredictable and intermittent translation, the bands of TRn were a little shallow to recognize.

Fig. 14 | SDS-PAGE of expression products of cmRNA 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.

Functional testing

We used the same methods to obtain TRn protein films (Fig. 15).

Fig. 15 | The freeze-dried protein samples.

Obtaining our TRn protein films, we found that they were more dense than those composed TRn5 (BBa_K5398001) under a stereomicroscope. Subsequently, in order to test the property of self-healing materials composed of TRn5, we punctured a TRn5 protein film to create a hole defect by a needle. After putting the punctured film at room temperature, we clearly saw the hole defect healing at a quick pace (Fig. 16). Compared with the self-healing effciency of TRn5 protein film (12 h of healing), this self-healing efficiency of TRn produced by the strategy of mRNA circularization is far higher because nearly 90% healing were completed in 4 h. Meanwhile, it should be noticed that the hole defect of TRn protein film is much bigger than that of TRn5 protein film. In brief, all of these findings indicate that the cmRNA is a good strategy to produce the squid-inspired biosynthetic proteins with various long tandem repeats.

Fig. 16 | Self-healing capabilities of TRn protein films after puncture damage.

mathematical modeling

In order to test the stability of proteins formed by different translation times of cmRNA, we performed mathematical and biological simulations. In mathematical simulation, we described the stability of the protein through the hydrogen bond network formed between β-sheet.

First, we selected proteins which were translated 1, 2, 3, 4, 5 and 6 times respectively through circular mRNA to study the stability of the intermolecular hydrogen bond network. The figure below shows the changes and result analysis of the intermolecular hydrogen bond network formed by proteins under different translation times.

Fig. 17 | Intermolecular hydrogen bond networks in different proteins.

TRn with n=5, 10, 15, 20, 25 and 305 from top to bottom, left to right.

TRn with n=n=5, 10, 15, 20, 25 and 305 from top to bottom, left to right.

Fig. 18 | Results of intermolecular hydrogen bond network.

From Fig. 18, we can see that as the number of TRn 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. Networks with dense lines usually have higher stability and can more effectively resist external disturbances. In addition, such networks are not prone to deformation due to the dense distribution of their lines. When a line is broken, other surrounding lines can share its load and reduce the impact of the break on the overall structure. Therefore, in microscopic protein structures with self-healing function 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.

Subsequently, we introduced several proteins with the same number of translations and placed them in a three-dimensional space to simulate the overall hydrogen bond network. The following is a simulation display and result analysis:

Fig. 19 | 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. 20 | Results of overall hydrogen bond network.

As can be seen from Fig. 20, the point-to-line ratio of the hydrogen bond network between multiple protein molecules is generally lower than that of the hydrogen bond network within a protein, indicating that the hydrogen bond interactions between multiple proteins are more intensive and the connectivity of the formed network is stronger. This further confirms the more TRn is repeated, the more stable the hydrogen bond network between protein molecules, which makes the interacting protein molecules more resistant to deformation when facing external perturbations, and have higher self-healing ability and adaptability.

We then used GROMACS to perform molecular dynamics simulations and calculate the energy in the system, hoping to verify the validity of our mathematical modeling through molecular dynamics methods. The results were as follows:

Fig. 21 | Unit energy of different TRn.

From the results (Fig. 21), we can see that as the number of TRn repetitions increases, the energy of the system becomes lower and lower, so the protein becomes more stable, which is consistent with the results we obtained through mathematical modeling, and to a certain extent, it shows the accuracy of the mathematical modeling results.

More information about the project for which the part was created: SAMUS model (NAU-CHINA 2024).

In summary, we have succeeded in producing squid ring proteins with various long tandem repeats by a strategy of mRNA circularization. Simultaneously, we verified our materials with excellent self-healing ability from function tests and mathematical modeling.

To further enhance the self-healing and adhesive properties of the protein, we designed and constructed the TRn4-mfp5 fusion protein. TRn4 is derived from squid ring teeth protein, and its unique β-sheet structure allows it to form strong hydrogen bonds with other tandem repetitions (TRns) of the squid-inspired building block. Mfp5 is derived from mussel foot protein and rich in tyrosine residues, which can form strong adhesive forces through conversion to DOPA in moist environments. By fusing these two proteins, we aim to create a material with dual-sided adhesive properties.

• We successfully expressed and purified the TRn4-Mfp5 fusion protein in E. coli BL21(DE3), with a final yield of approximately 25 mg/L.
• The adhesive force of TRn4-mfp5 was calculated to be approximately 7.08 kPa/mg, demonstrating the fusion protein's significant adhesive potential.
• We integrated a tyrosinase oxidation system, which oxidized tyrosine residues in Mfp5 to L-DOPA.

Successful expression of TRn4-Mfp5 fusion protein:

We first designed and produced the TRn4-mfp5 fusion protein and tested its adhesive function. From the AlphaFold3 prediction of TRn4-Mfp5 (Fig. 22), we found that, as expected, TRn4 formed β-sheets containg four β-strands, and the Mfp5 domain remained independent and unaffected by TRn4, providing a solid foundation for our subsequent experiments.

Fig. 22 | TRn4-mfp5 predicted by AlphaFold3.

In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. 23)—pET-28a(+), pET-SUMO, and pET-21a(+)—and tried different strategies for TRn4-mfp5 protein production and purification.

Fig. 23 | Three different vectors used in protein expression.

a. The plasmid map of pET-28a(+)-His-SUMO-TRn4-mfp5; b. The plasmid map of pET-SUMO-TRn4-mfp5; c. The plasmid map of pET-21a(+)-TRn4-mfp5.

We expressed the protein in E. coli BL21(DE3) using LB medium. After incubation at 16°C for 20 h or at 37°C for 4 h, we found that the protein expressed better under the 16°C for 20 h condition, as indicated by the stronger bands in Fig. 24. This suggests that lower temperature incubation may enhance protein solubility and proper folding, resulting in yield increase.

Fig. 24 | Comparison of fusion protein expression in different temperature using vector pET-21a(+).

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.

Since there was some discrepancy in the target band size observed in the SDS-PAGE gel, and the bands were not very distinct, we also tried another medium in an attempt to increase the expression level of the fusion protein. We additionally used TB medium and compared its expression efficiency with that of LB medium. 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, although the difference was not significant.

Fig. 25 | 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.

We compared protein expression between the E. coli BL21(DE3) and E. coli Rosetta strains. Rosetta, derived from BL21, includes a compatible chloramphenicol-resistant plasmid that provides tRNA genes for six rare codons (AUA, AGG, AGA, CUA, CCC, GGA) which are often underrepresented in E. coli . This modification is designed to overcome expression limitations when eukaryotic genes, which frequently use these rare codons, are expressed in a prokaryotic system. We used the pET-SUMO vector for expression.

While Rosetta is optimized to address these rare codon issues and can be advantageous when expressing eukaryotic proteins with high rare codon usage, our results showed that protein expression levels were higher in the BL21(DE3) strain. This discrepancy could be due to several factors. One possibility is that our target protein does not contain a sufficient number of rare codons to significantly hinder translation in BL21(DE3). Additionally, the extra plasmid load in Rosetta could impose a metabolic burden, reducing its overall protein production efficiency. As a result, in cases where rare codon usage is not a critical factor, BL21(DE3) might provide a more efficient platform for protein expression.

The results indicate that the protein expression level in the BL21(DE3) strain is higher compared to that in the Rosetta strain.

Fig. 26 | Comparison of fusion protein expression in E. coli strains 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)

After considering both expression efficiency and practical experimental constraints, we decided to express the fusion protein at 37°C for 4 h in LB medium using the pET-SUMO-TRn4-mfp5 plasmid.

As shown in Fig. 24-26, the target protein was present in the pellet after cell lysis. Therefore, we denatured the pellet of the fusion protein TRn4-mfp5 with 8M urea overnight and renatured it through dialysis. This process resulted in some protein loss, as confirmed by SDS-PAGE analysis.

Consequently, we proceeded to purify the fusion protein TRn4-mfp5 using a Ni-NTA Gravity Column.

The target protein bands were present in lanes 4-7, indicating successful expression of the target protein, with a particularly strong band in the supernatant after denaturation (Fig. 27, Lane 7). After purification, the target protein was mainly found in the 150 mM and 300 mM imidazole elution fractions.

Fig. 27 | SDS-PAGE of purified fusion protein TRn4-mfp5(35.4 kDa) uses vector pET-SUMO.

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 further confirm the expression of TRn4-mfp5, we performed a Western blot, which provided a clear and definitive conclusion, verifying the successful expression of the TRn4-mfp5 protein under the conditions mentioned above.

Fig. 28 | Western blotting(WB) of purified fusion protein TRn4-mfp5(35.4 kDa) uses vector pET-SUMO.

a. Western blotting(WB) of the pre-expressed protein. Lane 1: Total liquid (IPTG); Lane 2: Supernatant (IPTG); Lane 3: Precipitate (IPTG), b. Western blotting(WB) 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.

Overall, We constructed Part BBa_K5398020, the plasmid pET-SUMO-TRn4-mfp5, which includes a His-tag and SUMO-tag. The expression of the TRn4-mfp5 fusion protein was induced in E. coli BL21(DE3) using IPTG. The protein was predominantly found in the supernatant, as confirmed through SDS-PAGE and Western blotting(WB). We used Ni-NTA Gravity Column to purify the TRn4-mfp5 protein, followed by dialysis for further experiments.

Adhesive Analysis:

We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 29). We finally got about 25 mg protein from 1 L bacterial culture.

Fig. 29 | The protein sample freeze-dried by a lyophilizer.

Next, we dissolved protein samples in Buffer A (10 mL 20 mM Tris pH = 8.0) 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.

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

Video 1 | NAU-CHINA: TRn4-mfp5 adhesive ability test (2024)

To quantify the adhesive strength of the fusion protein, we calculated the adhesive force using the following parameters

Fig. 32 | Synthesis scheme of L-DOPA and further oxidized product L-dopaquinone.

Through extensive literature review, we found that tyrosinase TyrVs can oxidize tyrosine in TRn4-mfp5 to L-DOPA, thereby enhancing its adhesive properties. However, TyrVs may also over-oxidize L-DOPA to dopaquinone, which diminishes the adhesive effect. Fortunately, mussel foot protein type 6 (Mfp6) can effectively prevent over-oxidation. Based on this finding, we designed a tyrosinase oxidation system that co-expressed TyrVs and Mfp6, aiming to control the oxidation process and ensure that tyrosine is oxidized to L-DOPA instead of dopaquinone.

• We confirmed that TyrVs can oxidize the majority of tyrosine to L-DOPA, with a small portion of L-DOPA further oxidized to dopaquinone.
• We also confirmed that Mfp6 can effectively reduce dopaquinone back to L-DOPA.

Successful expression and activity assay of TyrVs

We constructed the pET-SUMO-TyrVs(BBa_K5398610) and transformed it into E. coli BL21(DE3).

Fig. 33 | 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.

Then, the engineered bacteria were further incubated at 16°C for 20 h. Through SDS-PAGE (Fig. 34) and western blotting(WB)(Fig. 35), we found that SUMO-TyrVs (52.2 kDa) was primarily present in the supernatant, indicating that it was expressed in a soluble form.

Fig. 34 | Expression of recombinant TyrVs in E. coliBL21(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.

Fig. 35 | Western blotting(WB) analysis recombinant TyrVs in E. coli BL21(DE3) with pET-SUMO-TyrVs.

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

We used a Hypur T Ni-NTA 6FF (His-Tag) Prepacked Chromatographic Column, 1 mL for immobilized metal affinity chromatography (IMAC) purification of the supernatant.

Fig. 36 | 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 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. 37 | 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 (K_m) and maximum velocity (V_max) 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 (K_m) and maximum velocity (V_max) were 8787 μmol/L and 0.86 μmol·L^-1·s^-1, respectively.

Fig. 38 | 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.

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 L-DOPA quantity changes is similar, and the yield fluctuation is small, our reaction system has strong environmental adaptability and stability.

Fig. 39 | Mathematical modeling Analysis of TyrVs Enzymatic Reaction.

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.

Successful expression and activity assay of Mfp6

We constructed pET-28a(+)-Mfp6, which includes a His-tag and SUMO-tag. Then, we added 10 μM IPTG to induce the expression of TyrVs in E. coli BL21(DE3). Through SDS-PAGE and western blotting(WB), we found that TyrVs existed in the pellet (Fig. 40).

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

a.The plasmid map of pET-28a(+)-Mfp6. b.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. c.Western blot of pET-28a(+)-Mfp6(28 kDa). Lane 1: Mfp6-Whole Cell Lysate (IPTG); Lane 2: Mfp6-Supernatant (IPTG); Lane 3: Mfp6-Pellet-PBS (IPTG).

Therefore, we dialyzed the extracted SUMO-Mfp6 with acetic acid overnight. 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.

Fig. 41 | SDS-PAGE of pET-28a(+)-Mfp6(28 kDa).

Lane 1: Protein Marker; Lane2: Extraction Buffer; Lane 3: Supernatant; Lane 4: Elution Buffer (50 mM Imidazole); Lane 5: Elution Buffer (100 mM Imidazole); Lane 6: Elution Buffer (250 mM Imidazole); Lane 7: Elution Buffer (500 mM Imidazole).

After that, we adopted two strategies to test the activity of Mfp6. One was testing 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. 42). 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. 42 | Result of activity analysis.

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

The other was testing the enzyme activity of Mfp6 at different reaction time. We found the OD₄₇₅ in the experimental group gradually decreased, while those of the control group remained virtually unchanged (Fig. 43). This indicates that Mfp6 progressively reduced dopaquinone back to dopamine as the reaction progressed.

Fig. 43 | Mfp6 activity analysis at different reaction time.

Co-expression of TyrVs and Mfp6

Furthermore, we co-transformed pET-SUMO-TyrVs and pET-28a(+)-Mfp6 into E. coli BL21(DE3) and induced expression with 10 µM IPTG, followed by cultivation. In the end, we used SDS-PAGE and western blotting (WB) for verification, but the results did not meet our expectations.