Part:BBa_K5398020

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. Contents - 1 Usage and Biology - 2 Characterization

 - 2.1 Cloning strategy and results
 - 2.2 Protein expression
 - 2.3 Self-healing test

- 3 Reference

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.

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.<p> <p>Additionally, the molecular dynamics simulation showed that the overall conformation remained stable throughout, providing further confidence in the robustness of the fusion protein.<p>

Fig. 2 Fusion protein TRn4-Mfp5 predicted by AlphaFold.

AlphaFold-predicted structure of the TRn4-Mfp-5 fusion protein. TRn4 (pink) and Mfp-5 (blue) are connected by a GS linker (green), showing structural integrity.

Characterization

<p>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.

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 hours and then 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.

Fig. 4 Fusion proteins expressed in different temperatures use vector pET-21a(+).

Lanes 1-6 (LB 37°C 4 h): 1. Protein ladder 2. pET21a total liquid (+IPTG) 3. pET21a supernatant (+IPTG) 4. pET21a precipitate (+IPTG) 5. pET21a total liquid (-IPTG) 6. pET21a supernatant (-IPTG) 7. pET21a precipitate (-IPTG) Lanes 8-13 (TB 16°C 20 h): 8. Protein ladder 9. pET21a total liquid (+IPTG) 10. pET21a supernatant (+IPTG) 11. pET21a precipitate (+IPTG) 12. pET21a total liquid (-IPTG) 13. pET21a supernatant (-IPTG) 14. pET21a precipitate (-IPTG)

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.

Fig. 5 Comparison of fusion protein expression in LB and TB media use vector pET-21a(+).

Lanes 1-6 (LB 16°C 20 h): 1. Protein ladder 2. pET21a total liquid (+IPTG) 3. pET21a supernatant (+IPTG) 4. pET21a precipitate (+IPTG) 5. pET21a total liquid (-IPTG) 6. pET21a supernatant (-IPTG) 7. pET21a precipitate (-IPTG) Lanes 8-13 (TB 16°C 20 h): 8. Protein ladder 9. pET21a total liquid (+IPTG) 10. pET21a supernatant (+IPTG) 11. pET21a precipitate (+IPTG) 12. pET21a total liquid (-IPTG) 13. pET21a supernatant (-IPTG) 14. pET21a precipitate (-IPTG)

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.