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Applications of BBa_K5398003

In order to obtain proteins with self-healing properties, we used pET-29a(+)-cmRNA(TRn5) to desiged a circular mRNA (cmRNA). We tried different strategies for TRn protein production and purification and tested its function.

Characterization

Protein expression

The synthetic plasmid pET-29a(+)-cmRNA(TRn5) was transformed into E.coli BL21 (DE3) and recombinant proteins were expressed using LB medium (Fig. 1).

Fig. 1 | The plasmid map of pET-29a(+)-cmRNA(TRn5).

Optimization of incubation temperature

Aim: To determine which incubation temperature is better for protein expression using mRNA circularization.

Methods: The cells were inoculated in LB media at 37℃ for 5 h, 23℃ for 16 h and 16℃ for 20 h respectively. The cultures were induced with 1 mM IPTG and the proteins were expressed. An SDS-gel was used to assess the results.

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 (Fig. 2).

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 (Fig. 4), we found there were few differences among them. This showed the strategy employing cmRNA to express TRn had a low requirement.

Fig. 2 | 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

Aim: To determine which IPTG concentration is better for protein expression using mRNA circularization.

Methods: The cells were inoculated in LB media at 37℃ for 5 h. The cultures were induced with 0.5 mM and 1 mM IPTG and the proteins were expressed. An SDS-gel was used to assess the results.

Results: From the SDS-PAGE (Fig. 3), we found that the TRn expression level at two IPTG concentration (0.5 mM and 1 mM) had little difference and the protreins also formed a ladder on the gel.

Fig. 3 | SDS-PAGE of expression products of cmRNA induced with different IPTG concentration.

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)

Methods: The cells were induced with 0.5 mM IPTG and inoculated in LB media at 37℃ for 5 h. The cultures were centrifugated to get the supernatant and pellet. Next, the following steps were used:

Results: From the SDS-PAGE (Fig. 4), we found that the TRn expression level was too low to verify by SDS-PAGE. 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. 4 | 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: 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 imidazoles.

Protein purification using a new protocol

Aim: To purify the protein using a new protocol containing 5% acetic acid.

Methods: The cells were induced with 0.5 mM IPTG and inoculated in LB media at 37℃ for 5 h. The cultures were centrifugated to get the supernatant and pellet. Next, the following steps were used:

Results: From the SDS-PAGE (Fig. 5), we found that the TRn dissolved in 5% acetic acid still presented a ladder on the gel. And due to unpredictable and intermittent translation, the bands of TRn were a little shallow to recognize.

Fig. 5 | 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.

Self-healing test

We obtained protein samples of TRn by freezedrying 24 h. The final yield was about 187.2 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. 6 | 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 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. 9). 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 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. In brief, all of these findings indicate that the cmRNA is a good strategy to produce highly repetitive squid ring teeth proteins with excellent self-healing.

Fig. 7 | Self-healing of TRn protein films after puncture damage.

Model

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.

Simulation of the individual hydrogen bond network

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. We abstracted β-sheet as a point in a grid and set a connection between two points as their interaction through hydrogen bonds. We perturbed the system 20 times and regenerated a new hydrogen bond network. We calculated the point-line ratio for each of the 20 hydrogen bond networks formed, and took the average value as an evaluation index for the stability of the protein under this number of translations. The figure below shows the changes and result analysis of the intermolecular hydrogen bond network formed by proteins under different translation times.

Fig. 8 | 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. 9 | Results of intermolecular hydrogen bond network.

From Fig. 9, we can see that as the number of TRn repetitions increases, the point-line ratio of the hydrogen bond network 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 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.

Simulation of the overall hydrogen bond network

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. We abstracted the protein molecule into a sphere, abstracted β-sheet as a point and put them in a three-dimensional "box". Then we placed four spheres in the space and placed several evenly distributed points in the sphere according to the number of β-sheet. The remaining rules mainly followed the simulation of the individual hydrogen bond network above. The following is a simulation display and result analysis:

Fig. 10 | 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. 11 | Results of overall hydrogen bond network.

As can be seen from Fig. 11, 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 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 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. We directly used the total energy of the system to reflect the stability of the network. The results were as follows:

Fig. 12 | Unit energy of different TRn.

From the results, 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.

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