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Latest revision as of 09:05, 2 October 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 after proteins's production and purification strategies. The dual-sided adhesive fusion protein TRn4-mfp5 leverages the strengths of both mfp5 and TRn4. The tyrosine residues in mfp5, when oxidized into DOPA, can form 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 components: mfp5, derived from mussel foot proteins, and TRn4, an artificially synthesized protein inspired by squid 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 of mfp5, which, upon oxidation by tyrosinase, are converted into DOPA. DOPA 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 allows 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 DOPA, 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.

Fig. 1 | Expected usage of the fusion protein TRn4-mfp5.

a. Schematic diagram of the TRn4-mfp5 fusion protein structure; b. Expression and usage of TRn and TRn4-mfp5 fusion proteins.

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.

Fig. 2 | Fusion protein TRn4-mfp5 predicted by AlphaFold.

AlphaFold-predicted structure of the TRn4-mfp5 fusion protein. TRn4 (pink) and Mfp5 (blue) are connected by a GS linker (green), showing structural integrity.

Characterization

To obtain the TRn4-mfp5 protein, optimize its expression, and evaluate its function, we tested three different expression vectors—pET-28a(+), pET-SUMO, and pET-21a(+)—and employed various strategies for protein production and purification (Fig. 1). These vectors allowed us to explore suitable conditions for protein expression and purification, facilitating the subsequent functional evaluation of TRn4-mfp5.

Fig. 3 | 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.

Protein Expression

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. 4. This suggests that lower temperature incubation may enhance protein solubility and proper folding, resulting in improved yield.

Fig. 4 | 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. 5 | Comparison of fusion protein expression in LB and TB media using 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 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 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. 6 | 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)

Protein Purification

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 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 4-7, 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.

Fig. 7 | SDS-PAGE of purified fusion protein TRn4-mfp5(35.4 kDa) using 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 blotting, which provided a clear and definitive conclusion, verifying the successful expression of the TRn4-mfp5 protein under the conditions mentioned above.

Fig. 8 | Western blotting of purified fusion protein TRn4-mfp5(35.4 kDa) using 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.

Adhesive test

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

Fig. 9 | The protein sample freeze-dried by a lyophilizer.

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

Fig. 10 | Adhesive ability test of fusion protein on plastic surface.

Viscosity Calculations

Surface Area Calculation:

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

S = π × (r_outer² - r_inner ²)

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

Force Calculation:

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

Adhesive Force Calculation:

The adhesive force produced by the protein is:

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

Adhesive Force per Milligram of Protein:

The adhesive force per milligram of protein is:

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

This demonstrates that the protein exhibits significant adhesive capability, with an adhesive force of 7.08 kPa per milligram of protein.