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−	This section encodes the TRn4-mfp5 fusion protein, which combines the adhesive properties of mfp5 from the mussel foot protein family with the unique functionality of the four tandem repeats of the squid-inspired building block (TRn4). In our project, we utilized this protein as a 'dual-sided adhesive' and examined its adhesive ability through various production and purification strategies.The dual-sided adhesion of the TRn4-mfp5 fusion protein leverages the strengths of both mfp5 and TRn4. Mfp5, through the oxidation of tyrosine into dopamine, forms strong π-π stacking interactions and hydrogen bonds with a wide range of surfaces. On the other side, TRn4's β-sheet structures enable strong hydrogen bonding between protein strands, which could adhere to other TRn proteins, providing robust structural integrity. Together, these two mechanisms allow the fusion protein to adhere effectively to different surfaces simultaneously.	+	This section encodes the TRn4-mfp5 fusion protein, which combines the adhesive properties of mfp5 from the mussel foot protein family with the unique functionality of the four tandem repeats of the squid-inspired building block (TRn4). In our project, we utilized this protein as a 'dual-sided adhesive' and examined its adhesive ability through various production and purification strategies. The dual-sided adhesion of the TRn4-mfp5 fusion protein leverages the strengths of both mfp5 and TRn4. Mfp5, through the oxidation of tyrosine into dopamine, forms strong π-π stacking interactions and hydrogen bonds with a wide range of surfaces. On the other side, TRn4's β-sheet structures enable strong hydrogen bonding between protein strands, which can adhere to other TRn proteins, providing robust structural integrity. Together, these two mechanisms allow the fusion protein to adhere effectively to different surfaces simultaneously.
	===Usage and Biology===		===Usage and Biology===

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Revision as of 06:34, 29 September 2024

This section encodes the TRn4-mfp5 fusion protein, which combines the adhesive properties of mfp5 from the mussel foot protein family with the unique functionality of the four tandem repeats of the squid-inspired building block (TRn4). In our project, we utilized this protein as a 'dual-sided adhesive' and examined its adhesive ability through various production and purification strategies. The dual-sided adhesion of the TRn4-mfp5 fusion protein leverages the strengths of both mfp5 and TRn4. Mfp5, through the oxidation of tyrosine into dopamine, forms strong π-π stacking interactions and hydrogen bonds with a wide range of surfaces. On the other side, TRn4's β-sheet structures enable strong hydrogen bonding between protein strands, which can adhere to other TRn proteins, providing robust structural integrity. Together, these two mechanisms allow the fusion protein to adhere effectively to different surfaces simultaneously.

Usage and Biology

The TRn4-mfp5 fusion protein combines two proteins: mfp5 from Mytilus foot proteins and TRn4 from squid ring teeth proteins. Mfp5 is derived from Mytilus, known for their ability to adhere to different materials' surfaces. This adhesion is primarily driven by the tyrosine residues in mfp5, which, upon oxidation by tyrosinase, are converted into dopamine. Dopamine forms π-π stacking interactions and hydrogen bonds with various substrates, including metal, glass, and polymer surfaces. This ability allows mfp5 to provide the bioadhesive strength that is crucial for surface attachment in various environments. TRn4 consists of a four-time repeated sequence derived from squid ring teeth proteins. The structural strength of squid ring teeth is attributed to the formation of β-sheets, which allow hydrogen bonding between protein strands. In TRn4, this repetitive sequence enables robust structural integrity, as the β-sheets can bond with other β-sheet structures.

The fusion of mfp5 and TRn4 creates a unique protein that leverages the adhesive capabilities of mfp5 and the function of TRn4. When exposed to tyrosinase, the mfp5 portion generates dopamine, allowing the fusion protein to adhere to various materials through strong molecular interactions. The β-sheets in TRn4 allow hydrogen bonding between TRn4 and other repeated squid ring teeth protein. This fusion protein has the potential for applications in surface coatings, and bio-inspired materials that require both strong adhesion and mechanical stability. The combination of mfp5’s versatile binding properties and TRn4’s structural offers an innovative solution for challenges in areas such as marine technology, biomedical adhesives, and sustainable material development.

We utilized AlphaFold to predict the structure of the TRn4-Mfp-5 fusion protein. After selecting the most accurate model, we aligned the predicted structures of TRn4 and Mfp-5 with the fusion protein and found consistent results (Fig. 2). This confirms that our design preserves the structural integrity and functionality of both components. Additionally, the molecular dynamics simulation showed that the overall conformation remained stable throughout, providing further confidence in the robustness of the fusion protein.

Characterization

In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. 3)—pET-28a(+), pET SUMO, and pET-21a(+)—and tried different strategies for TRn4-mfp5 protein production and purification.

Protein Expression

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

Since there was some discrepancy in the target band size observed in the protein gel image, 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. We compared protein expression between the BL21(DE3) and Rosetta E. coli 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) that are lacking in E. coli. This modification is intended to address expression limitations associated with the high usage frequency of these rare codons in eukaryotic genes. We used the pET SUMO vector for expression. The results indicate that the protein expression level in the BL21(DE3) strain is higher compared to that in the Rosetta strain.

Protein Purification

After testing various expression conditions, the optimal condition was found to be 16°C for 20 hours in LB medium using the pET SUMO-TRn4-mfp5 plasmid. However, due to practical constraints, we ultimately expressed the fusion protein under 37°C for 4 hours in LB medium using the pET SUMO-TRn4-mfp5 plasmid.

As shown in Figures 4-6, 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 to 7, indicating successful expression of the target protein, with a particularly strong band in the supernatant after denaturation (Fig 7, lane 7). After purification, the target protein was mainly found in the 150 mM and 300 mM imidazole elution fractions.

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.

WB的图片

Protein Purification

We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 9). The final yield was about 25 mg/L bacterial culture.

Next, we dissolved protein samples in Buffer A (10 mL 20 mM Tris pH8) to reach 0.3 mg/mL, and conduct adhesive ability tests on the fusion protein(Fig. 10). 200 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 8 hours, the pipette tip successfully adhered.

Reference

[1] Jung H., Pena-Francesch A., Saadat A, et al. Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins[J]. PNAS, 2016, 113(23), 6478–6483.

[2] Zhang C, Wu B, Zhou Y, et al. Mussel-inspired hydrogels: from design principles to promising applications[J]. Chem Soc Rev, 2020, 49(3605): 3605-3637.