Difference between revisions of "Part:BBa K5398020:Experience"

(→‎Protein Expression)
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In order to obtain proteins with adhesive properties, we used the pET SUMO vector to express TRn4-mfp5 ( BBa_K5398020) ). We tried different strategies for TRn4-mfp5 protein production and purification and tested its function.
 
In order to obtain proteins with adhesive properties, we used the pET SUMO vector to express TRn4-mfp5 ( BBa_K5398020) ). We tried different strategies for TRn4-mfp5 protein production and purification and tested its function.
  
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===Characterization===
+
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.
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<p>In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. 1)—pET-28a(+), pET SUMO, and pET-21a(+)—and tried different strategies for TRn4-mfp5 protein production and purification.</p>
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 +
===Usage and Biology===
 +
<p>The TRn4-mfp5 fusion protein combines two proteins: mfp5 from Mytilus foot proteins and TRn4 from squid ring teeth proteins. Mfp5 is derived from <i>Mytilus </i>, 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. </p>
 +
 
 +
<p>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. </p>
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        <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/fig1.webp" width="700" height="auto" alt="Protein purification">
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        <p><b>Fig. 1 | Expected usage of the fusion protein TRn4-Mfp5.</b></p>
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        <p><b>a.</b>  Schematic diagram of the TRn4-Mfp5 fusion protein structure;
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        <b>b.</b> Expression and usage of TRn and TRn4-Mfp5 fusion proteins.</p>
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<p>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.
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Additionally, the molecular dynamics simulation showed that the overall conformation remained stable throughout, providing further confidence in the robustness of the fusion protein.
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/plasmid-map-3.webp" width="700" height="auto" alt="Protein purification">
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/model.webp" width="700" height="auto" alt="Protein purification">
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         <p><b>Fig. 1 | Three different vectors used in protein expression.</b></p>
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         <p><b>Fig. 2 | Fusion protein TRn4-Mfp5 predicted by AlphaFold.</b></p>
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         <p><b>a.</b> The plasmid map of pET-28a(+)-His-SUMO-TRn4-mfp5;
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         <p>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.</p>
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        <b>b.</b> The plasmid map of pET SUMO-TRn4-mfp5;
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        <b>c.</b> The plasmid map of pET-21a(+)-TRn4-mfp5.</p>
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====Protein Expression====
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<p>We expressed the protein in <i>E. coli</i> BL21 (DE3) using LB medium. After incubation at 16°C for 20 h and then 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. 2. This suggests that lower temperature incubation may enhance protein solubility and proper folding, resulting in improved yield.</p>
+
===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.</p>
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        <p><b>Fig. 3 | Three different vectors used in protein expression.</b></p>
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        <p><b>a.</b> The plasmid map of pET-28a(+)-His-SUMO-TRn4-mfp5;
 +
        <b>b.</b> The plasmid map of pET SUMO-TRn4-mfp5;
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        <b>c.</b> The plasmid map of pET-21a(+)-TRn4-mfp5.</p>
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====Protein Expression====
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<p>We expressed the protein in <i>E. coli </i> BL21(DE3) using LB medium. After incubation at 16°C for 20 h or at 37°C for 4 hours, 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.
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/16-37-lb-pet21a.webp" width="700" height="auto" alt="Protein purification">
 
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         <p><b>Fig. 2 | Comparison of fusion protein expression in different temperature use vector pET-21a(+).</b></p>
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         <p><b>Fig. 4 | Comparison of fusion protein expression in different temperature use vector pET-21a(+).</b></p>
 
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Lanes 1-6 (LB 37°C 4 h):   
 
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6. supernatant (-IPTG);   
 
6. supernatant (-IPTG);   
 
7. precipitate (-IPTG);   
 
7. precipitate (-IPTG);   
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Lanes 8-13 (TB 16°C 20 h):  
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Lanes 8-13 (TB 16°C 20 h):
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8. Protein ladder;   
 
8. Protein ladder;   
 
9. total liquid (+IPTG);   
 
9. total liquid (+IPTG);   
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<p>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.
+
 
 +
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.
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/tb-lb-prt21a.webp" width="700" height="auto" alt="Protein purification">
 
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         <p><b>Fig. 3 | Comparison of fusion protein expression in LB and TB media use vector pET-21a(+).</b></p>
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         <p><b>Fig. 5 | Comparison of fusion protein expression in LB and TB media use vector pET-21a(+).</b></p>
 
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Lanes 1-6 (LB 16°C 20 h): 
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1. Protein ladder;   
 
1. Protein ladder;   
 +
Lanes 2-7 (LB 16°C 20 h):
 
2. total liquid (+IPTG);   
 
2. total liquid (+IPTG);   
 
3. supernatant (+IPTG);   
 
3. supernatant (+IPTG);   
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We compared protein expression between the BL21(DE3) and Rosetta <i>E. coli</i> 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 <i>E. coli<i/>. 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.
+
We compared protein expression between the BL21(DE3) and Rosetta <i>E. coli </i> 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 <i>E. coli </i>. 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.<br>
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The results indicate that the protein expression level in the BL21(DE3) strain is higher compared to that in the Rosetta strain.
+
 
 +
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.<br>
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 +
The results indicate that the protein expression level in the BL21(DE3) strain is higher compared to that in the Rosetta strain.<br>
 
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/rostta-bl21-de3-trn4-mfp5png.webp" width="700" height="auto" alt="Protein purification">
 
         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/rostta-bl21-de3-trn4-mfp5png.webp" width="700" height="auto" alt="Protein purification">
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         <p><b>Fig. 4 | Comparison of fusion protein expression in <i>E. coli</i> strains BL21(DE3) and Rosetta.</b></p>
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         <p><b>Fig. 6 | Comparison of fusion protein expression in <i>E. coli</i> strains BL21(DE3) and Rosetta.</b></p>
 
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1. Protein ladder
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1. Protein ladder;
 
Lanes 2-4 (BL21(DE3) LB 37℃ 4h)
 
Lanes 2-4 (BL21(DE3) LB 37℃ 4h)
 
2. total liquid (+IPTG);   
 
2. total liquid (+IPTG);   
 
3. supernatant (+IPTG);   
 
3. supernatant (+IPTG);   
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4. precipitate (+IPTG);
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4. precipitate (+IPTG);  
 
Lanes 5-7 (Rosetta LB 37℃ 4h)
 
Lanes 5-7 (Rosetta LB 37℃ 4h)
 
5. total liquid (+IPTG);   
 
5. total liquid (+IPTG);   
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====Protein Purification====
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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.</p>
 +
<p>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. </p>
 +
<p>Consequently, we proceeded to purify the fusion protein TRn4-mfp5 using a Ni-NTA Gravity Column.</p>
 +
<p>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.</p>
  
 
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         <p><b>Fig. 4 | SDS-PAGE of purified fusion protein TRn4-mfp5 (35.4 kDa) using vector pET SUMO.</b></p>
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         <p><b>Fig. 7 | SDS-PAGE of purified fusion protein TRn4-mfp5(35.4 kDa) uses vector pET-SUMO.</b></p>
 
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            Lane 1: Protein - Binding buffer;
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Lane 1: Protein - Binding buffer;
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            Lane 2: 20 mM imidazole and 8 M urea elution;
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Lane 2: 20 mM imidazole and 8 M urea elution;
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            Lane 3: 50 mM imidazole and 8 M urea elution;
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Lane 3: 50 mM imidazole and 8 M urea elution;
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            Lane 4: 150 mM imidazole and 8 M urea elution;
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Lane 4: 150 mM imidazole and 8 M urea elution;
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            Lane 5: 300 mM imidazole and 8 M urea elution;
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Lane 5: 300 mM imidazole and 8 M urea elution;
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            Lane 6: 500 mM imidazole and 8 M urea elution;
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Lane 6: 500 mM imidazole and 8 M urea elution;
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            Lane 7: Supernatant;
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Lane 7: Supernatant;
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            Lane 8: Impurities;
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Lane 8: Impurities;
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            Lane 9: Protein ladder.
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Lane 9: Protein ladder.
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====Protein Purification====
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<p>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.</p>
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We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 6). The final yield was about 25 mg/L bacterial culture.
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         <p><b>Fig. 6 | The protein sample freeze-dried by a lyophilizer.</b></p>
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         <p><b>Fig. 8 | Western Blot of purified fusion protein TRn4-mfp5(35.4 kDa) uses vector pET-SUMO.</b></p>
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 +
<p><b>a.</b> Western blot of the pre-expressed protein;<b>b.</b>  Western blot after column purification of the supernatant following denaturation.
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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. 7). 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.
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====Adhesive test====
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We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 9). The final yield was about 25 mg/L bacterial culture.
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            text-align: center; /* 内容居中 */
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        <p><b>Fig. 9 | The protein sample freeze-dried by a lyophilizer.</b></p>
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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 h, the pipette tip successfully adhered.
  
 
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         <p><b>Fig. 7 | Adhesive Ability Test of Fusion Protein on Plastic Surface</b></p>
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         <p><b>Fig. 10 | Adhesive ability test of fusion protein on plastic surface</b></p>
 
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<b>Viscosity Calculations</b>
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<p>More information about the project for which the part was created:<a href="https://2024.igem.wiki/nau-china/description"> SAMUS (NAU-CHINA 2024).</a></p>
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<p>Surface Area Calculation:</p>
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<p>The surface area of a 10 µl pipette tip with an inner diameter of 3.7 mm is calculated as:</p>
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    <p>S = π × r² = π × (0.185 cm)² = 0.1075 cm²</p>
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<p>Force Calculation:</p>
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<p>The total mass is (5.951 + 0.448 × 3) grams, and the force is:</p>
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    <p>F = 7.295 g × 9.8 N/kg = 0.07149 N</p>
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<p>Adhesive Force Calculation:</p>
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<p>The adhesive force produced by the protein is:</p>
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    <p>P = F / S = 0.07149 N / 0.1075 cm² = 0.665 N/cm² ≈ 6.65 KPa</p>
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<p>Adhesive Force per Milligram of Protein:</p>
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<p>The adhesive force per milligram of protein is:</p>
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    <p>P' = P / m = 6.65 KPa / 1 mg = 6.65 KPa/mg</p>
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===Reference ===
 
===Reference ===
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<p>[1] Jung, H., Pena-Francesch, A., Saadat, A., et al. Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins [J]. <i>PNAS</i>, 2016, 113(23): 6478–6483.</p>
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<p>[1] Jung H., Pena-Francesch A., Saadat A, et al. Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins[J]. <i>PNAS</i>, 2016, 113(23), 6478–6483.</p>
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<p>[2] Zhang, C., Wu, B., Zhou, Y., et al. Mussel-inspired hydrogels: from design principles to promising applications [J]. <i>Chem Soc Rev</i>, 2020, 49: 3605-3637.</p>
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<p>[2] Zhang C, Wu B, Zhou Y, et al. Mussel-inspired hydrogels: from design principles to promising applications[J]. <i>Chem Soc Rev</i>, 2020, 49(3605): 3605-3637.</p>
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Revision as of 03:42, 30 September 2024


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K5398020

In order to obtain proteins with adhesive properties, we used the pET SUMO vector to express TRn4-mfp5 ( BBa_K5398020) ). We tried different strategies for TRn4-mfp5 protein production and purification and tested its function.

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.

Protein purification

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.

Protein purification

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

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 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 h or at 37°C for 4 hours, 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.

Protein purification

Fig. 4 | Comparison of fusion protein expression in different temperature use vector pET-21a(+).

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

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.
Protein purification

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

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

Fig. 6 | Comparison of fusion protein expression in E. coli strains BL21(DE3) and Rosetta.

1. Protein ladder; Lanes 2-4 (BL21(DE3) LB 37℃ 4h) 2. total liquid (+IPTG); 3. supernatant (+IPTG); 4. precipitate (+IPTG); Lanes 5-7 (Rosetta LB 37℃ 4h) 5. total liquid (+IPTG); 6. supernatant (+IPTG); 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 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.

Protein purification

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

Protein purification

Fig. 8 | Western Blot of purified fusion protein TRn4-mfp5(35.4 kDa) uses vector pET-SUMO.

a. Western blot of the pre-expressed protein;b. Western blot after column purification of the supernatant following denaturation.

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.

Protein purification

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

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 h, the pipette tip successfully adhered.

Protein purification

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

Viscosity Calculations Viscosity Calculations

Surface Area Calculation:

The surface area of a 10 µl pipette tip with an inner diameter of 3.7 mm is calculated as:

S = π × r² = π × (0.185 cm)² = 0.1075 cm²

Force Calculation:

The total mass is (5.951 + 0.448 × 3) grams, and the force is:

F = 7.295 g × 9.8 N/kg = 0.07149 N

Adhesive Force Calculation:

The adhesive force produced by the protein is:

P = F / S = 0.07149 N / 0.1075 cm² = 0.665 N/cm² ≈ 6.65 KPa

Adhesive Force per Milligram of Protein:

The adhesive force per milligram of protein is:

P' = P / m = 6.65 KPa / 1 mg = 6.65 KPa/mg

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.