Difference between revisions of "Part:BBa K5398006"

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	<p><b>Methods:</b> 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:		<p><b>Methods:</b> 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:
−	<li>Denature the supernatant with 8 mM urea overnight;</li>	+	<li>Denature the supernatant with 8 M urea overnight;</li>
	<li>Renature the detured solution by dialysis with 20 mM Tris-HCl buffer for 20 h (changing dialysate every 8 h);</li>		<li>Renature the detured solution by dialysis with 20 mM Tris-HCl buffer for 20 h (changing dialysate every 8 h);</li>
	<li>Purify proteins on a HisTrap Ni-NTA column with different concentrations of imidazole;</li>		<li>Purify proteins on a HisTrap Ni-NTA column with different concentrations of imidazole;</li>

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Revision as of 03:15, 30 September 2024

pET29a(+)-cmRNA

In order to obtain proteins with self-healing properties, we used the pET-29a(+) vector to express TRn5 ( BBa_K5398001) ). We tried different strategies for TRn5 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).

Optimization of incubation temperature

Aim: To determine which incubation temperature is beter 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 least 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.

Optimization of IPTG concentration

Aim: To determine which IPTG concentration is beter 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.

Protein purification by Immobilized Metal Affinity Chromatography (IMAC)

Aim: To purify the protein by IMAC (Immobilised Metal Affinity Chromatography) using Ni-NTA resin.

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:

Denature the supernatant with 8 M urea overnight;

Renature the detured solution by dialysis with 20 mM Tris-HCl buffer for 20 h (changing dialysate every 8 h);

Purify proteins on a HisTrap Ni-NTA column with different concentrations of imidazole;

Assess the results using SDS-PAGE.

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.

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:

Wash the pellets twice with 100 mL urea extraction buffer [100 mM Tris, pH 7.4, 5 mM EDTA, 2 M urea, 2% (vol/vol) Triton X-100] and centrifugat them to remove cell debris and other soluble proteins;

Wash the pellets with 100 mL washing buffer (100 mM Tris, pH 7.4, 5 mM EDTA) and centrifugat them to remove urea and TritonX-100.

Dissolve the pellets in 5% acetic acid.

Assess the results using SDS-PAGE.

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.

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.

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 was 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.

From Fig. 18, 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.

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:

As can be seen from Fig. 18, 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. We directly used the total energy of the system to reflect the stability of the network. The results were as follows:

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).

Reference

[1] LIU L, WANG P, ZHAO D, et al. Engineering Circularized mRNAs for the Production of Spider Silk Proteins[J]. Appl. Environ. Microbiol., 2022, 88(8): e00028-22.

[2] PERRIMAN R, ARES M. Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo[J]. RNA, 1998, 4(9): 1047-1054.

[3] LEE S O, XIE Q, FRIED S D. Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins[J]. ACS Cent. Sci., 2021, 7(10): 1736-1750.

[4] OBI P, CHEN Y G. The design and synthesis of circular RNAs[J]. Methods, 2021, 196: 85-103.

[5] GOMES R M O da S, SILVA K J G da, THEODORO R C. Group I introns: Structure, splicing and their applications in medical mycology[J]. Genet. Mol. Biol., 2024, 47: e20230228.

Sequence and Features