Difference between revisions of "Part:BBa K5398030"
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<partinfo>BBa_K5398030 short</partinfo>
 
<partinfo>BBa_K5398030 short</partinfo>
  
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In the Mfp5-TRn4 fusion proteins, Mfp5 is derived from the mussel foot proteins, whose tyrosine residues are oxidized by tyrosinase into dopamine, which primarily forms &#960;-&#960; bonds and hydrogen bonds with the surface materials, allowing them to adhere to various materials. TRn4 is a protein obtained by repeating the sequence from squid ring teeth proteins four times, and the &#946;-sheet on its structure can connect with the &#946;-sheet on the structure of highly repetitive squid ring teeth proteins through hydrogen bonds. Tyrosine on Mfp5 generates dopa when tyrosinase is present, which makes Mfp5-TRn4 fusion protein adhere to the surface materials.  
+
In the Mfp5-TRn4 fusion proteins, Mfp5 is derived from the mussel foot proteins, whose tyrosine residues are oxidized by tyrosinase into dopa, which primarily forms &#960;-&#960; bonds and hydrogen bonds with the surface materials, allowing them to adhere to various materials. TRn4 is a protein obtained by repeating the sequence from squid ring teeth proteins four times, and the &#946;-sheet on its structure can connect with the &#946;-sheet on the structure of highly repetitive squid ring teeth proteins through hydrogen bonds. Tyrosine on Mfp5 generates dopa when tyrosinase is present, which makes Mfp5-TRn4 fusion protein adhere to the surface materials.  
  
 
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<center><img src="https://static.igem.wiki/teams/5398/trn4-mfp5/pet-pc-sumo-8-8-map.webp"with="1000" height="" width="500" height=""/></center>
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<center><img src="https://static.igem.wiki/teams/5398/trn4-mfp5/pet-pc-sumo-trn4-mfp5-map-white.webp"with="1000" height="" width="500" height=""/></center>
 
</html>
 
</html>
  
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<p style="text-align: center!important;"><b>Fig. 1 The plasmid map of TRn4-mfp5.
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<p style="text-align: center!important;"><b>Fig. 1 | The plasmid map of TRn4-mfp5.
 
</b></p>
 
</b></p>
  
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===Characterization===
 
===Characterization===
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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.
+
In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. 2)—pET-28a(+), pET-SUMO, and pET-21a(+)—and tried different strategies for TRn4-mfp5 protein production and purification.
  
  
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<body>
 
<body>
 
     <div class="module">
 
     <div class="module">
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/three-plasmid-trn4mfp5.webp" width="700" height="auto" alt="Protein purification">
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/three-plasmid-map-white.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>
+
         <p><b>Fig. 2 | Three different vectors used in protein expression.</b></p>
 
         <p><b>a.</b> The plasmid map of pET-28a(+)-His-SUMO-TRn4-mfp5;
 
         <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;
 
         <b>b.</b> The plasmid map of pET-SUMO-TRn4-mfp5;
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====Protein Expression====
 
====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 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>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 h, we found that the protein expressed better under the 16°C for 20 h condition, as indicated by the stronger bands in Fig. 3. 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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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/16-37-lb-pet21a-new.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. 3 | Comparison of fusion protein expression in different temperature using vector pET-21a(+).</b></p>
 
         <p>
 
         <p>
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Lanes 1-6 (LB 37°C 4 h):
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Lane 1: Protein ladder; Lanes 2-7 (LB 37°C 4 h): Lane 2: Total liquid (IPTG); Lane 3: Supernatant
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1. Protein ladder;
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(IPTG); Lane 4: Precipitate (IPTG); Lane 5: Total liquid; Lane 6: Supernatant; Lane 7: Precipitate;
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2. total liquid (+IPTG);
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Lanes 8-13 (TB 16°C 20 h): Lane 8: Total liquid (IPTG); Lane 9: Supernatant (IPTG); Lane 10:
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3. supernatant (+IPTG);
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Precipitate (IPTG); Lane 11: Total liquid; Lane 12: Supernatant; Lane 13: Precipitate; Lane 14:
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4. precipitate (+IPTG);
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Protein ladder.</p>
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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). </p>
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     </div>
 
     </div>
 
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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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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/tb-lb-prt21a-new.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>
+
         <p><b>Fig. 4 | Comparison of fusion protein expression in LB and TB media using vector pET-21a(+).</b></p>
 
         <p>
 
         <p>
  
−
1. Protein ladder; 
+
Lanes 1-6 (LB 16°C 20 h): Lane 1: Total liquid (IPTG); Lane 2: Supernatant (IPTG); Lane 3:
−
Lanes 2-7 (LB 16°C 20 h):  
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Precipitate (IPTG); Lane 4: Total liquid; Lane 5: Supernatant; Lane 6: Precipitate; Lane 7: Protein
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2. total liquid (+IPTG);
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ladder; Lanes 8-13 (TB 16°C 20 h): Lane 8: Total liquid (IPTG); Lane 9: Supernatant (IPTG); Lane
−
3. supernatant (+IPTG);
+
10: Precipitate (IPTG); Lane 11: Total liquid; Lane 12: Supernatant; Lane 13: Precipitate.  
−
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). 
+
 
</p>
 
</p>
 
     </div>
 
     </div>
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     <div class="module">
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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">
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/rostta-bl21-de3-trn4-mfp-new.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>
+
         <p><b>Fig. 5 | Comparison of fusion protein expression in <i>E. coli</i> strains BL21(DE3) and Rosetta.</b></p>
 
         <p>
 
         <p>
−
1. Protein ladder;
+
Lane 1: Protein ladder; Lanes 2-4 (BL21(DE3) LB 37℃ 4 h): Lane 2: Total liquid (IPTG);
−
Lanes 2-4 (BL21(DE3) LB 37℃ 4h)
+
Lane 3: Supernatant (IPTG); Lane 4: Precipitate (IPTG); Lanes 5-7 (Rosetta LB 37℃ 4 h) Lane 5:
−
2. total liquid (+IPTG);
+
Total liquid (IPTG); Lane 6: Supernatant (IPTG); Lane 7: Precipitate (IPTG)
−
3. supernatant (+IPTG);
+
−
4. precipitate (+IPTG);  
+
−
Lanes 5-7 (Rosetta LB 37℃ 4h)
+
−
5. total liquid (+IPTG);
+
−
6. supernatant (+IPTG);
+
−
7. precipitate (+IPTG). 
+
 
</p>
 
</p>
 
     </div>
 
     </div>
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====Protein Purification====
 
====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.</p>
 
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 2-4, 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>As shown in Figures 3-5, 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>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 2 to 5, indicating successful expression of the target protein, with a particularly strong band in the supernatant after denaturation (Fig. 5, lane 7). After purification, the target protein was mainly found in the 150 mM and 300 mM imidazole elution fractions.</p>
+
<p>The target protein bands were present in lanes 2 to 5, indicating successful expression of the target protein, with a particularly strong band in the supernatant after denaturation (Fig. 6, 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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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/purification-trn4-mfp5.webp" width="700" height="auto" alt="Protein purification">
+
         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/purification-trn4-mfp5-new.webp" width="700" height="auto" alt="Protein purification">
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         <p><b>Fig. 5 | SDS-PAGE of purified fusion protein TRn4-mfp5(35.4 kDa) uses vector pET-SUMO.</b></p>
+
         <p><b>Fig. 6 | SDS-PAGE of purified fusion protein TRn4-mfp5(35.4 kDa) using vector pET-SUMO.</b></p>
 
         <p>
 
         <p>
−
Lane 1: Protein - Binding buffer;
+
Lane 1: Protein-Binding buffer; Lane 2: 20 mM imidazole and 8 M urea elution; Lane 3: 50 mM
−
Lane 2: 20 mM imidazole and 8 M urea elution;
+
imidazole and 8 M urea elution; Lane 4: 150 mM imidazole and 8 M urea elution; Lane 5: 300 mM
−
Lane 3: 50 mM imidazole and 8 M urea elution;
+
imidazole and 8 M urea elution; Lane 6: 500 mM imidazole and 8 M urea elution; Lane 7:
−
Lane 4: 150 mM imidazole and 8 M urea elution;
+
Supernatant; Lane 8: Impurities; Lane 9: Protein ladder.
−
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.
+
 
</p>
 
</p>
 
     </div>
 
     </div>
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     <div class="module">
 
     <div class="module">
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/wb-all-final.webp" width="700" height="auto" alt="Protein purification">
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/wb-new.webp" width="700" height="auto" alt="Protein purification">
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         <p><b>Fig. 6 | Western Blot of purified fusion protein TRn4-mfp5(35.4 kDa) uses vector pET-SUMO.</b></p>
+
         <p><b>Fig. 7 | Western Blot of purified fusion protein TRn4-mfp5(35.4 kDa) using vector pET-SUMO.</b></p>
 
         <p>
 
         <p>
−
<p><b>a.</b> Western blot of the pre-expressed protein;<b>b.</b> Western blot after column purification of the supernatant following denaturation.
+
<b>a.</b> Western blot of the pre-expressed protein. Lane 1: Total liquid (IPTG); Lane 2:
 +
Supernatant (IPTG); Lane 3: Precipitate (IPTG), <b>b.</b> Western blot 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.
 
</p>
 
</p>
 
     </div>
 
     </div>
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====Adhesive test====
 
====Adhesive test====
−
We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 7). The final yield was about 25 mg/L bacterial culture.  
+
We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 8). The final yield was about 25 mg/L bacterial culture.  
 
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/protein-freeze-actual-picture-new.webp" width="700" height="auto" alt="Protein purification">
 
         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/protein-freeze-actual-picture-new.webp" width="700" height="auto" alt="Protein purification">
−
         <p><b>Fig. 7 | The protein sample freeze-dried by a lyophilizer.</b></p>
+
         <p><b>Fig. 8 | The protein sample freeze-dried by a lyophilizer.</b></p>
 
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</body>
 
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</html>
  
−
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.
+
Next, we dissolved protein samples in Buffer A (10 mL 20 mM Tris pH = 8) to reach  0.3 mg/mL, and conduct adhesive ability tests on the fusion protein(Fig. 9). We applied 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 successfully adhered.
  
 
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         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/part-fig10.webp" width="700" height="auto" alt="Protein purification">
 
         <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/part-fig10.webp" width="700" height="auto" alt="Protein purification">
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         <p><b>Fig. 8 | Adhesive ability test of fusion protein on plastic surface</b></p>
+
         <p><b>Fig. 9 | Adhesive ability test of fusion protein on plastic surface.</b></p>
 
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<b>Viscosity Calculations</b>
 
<b>Viscosity Calculations</b>
  
−
<p>Surface Area Calculation:</p>
+
<p><b>Surface Area Calculation:</b></p>  
−
<p>The surface area of a 10 µl pipette tip with an inner diameter of 3.7 mm is calculated as:</p>
+
<p>The surface area for the annular region of the pipette tip is calculated as:</p>  
−
<div class="calculation">
+
<p>
−
    <p>S = π × r² = π × (0.185 cm)² = 0.1075 cm²</p>
+
<strong>S</strong> = π × (r<sub>outer</sub><sup>2</sup> - r<sub>inner</sub>  
−
</div>
+
<sup>2</sup>)
−
 
+
</p>
−
<p>Force Calculation:</p>
+
<p>
−
<p>The total mass is (5.951 + 0.448 × 3) grams, and the force is:</p>
+
Where: <br>r<sub>outer</sub> = 3 mm = 0.3 cm <br>
−
<div class="calculation">
+
r<sub>inner</sub> = 1.85 mm = 0.185 cm
−
    <p>F = 7.295 g × 9.8 N/kg = 0.07149 N</p>
+
</p>
−
</div>
+
<p>
−
 
+
Substituting these values, we get:
−
<p>Adhesive Force Calculation:</p>
+
</p>
−
<p>The adhesive force produced by the protein is:</p>
+
<p>
−
<div class="calculation">
+
<strong>S</strong> = π × (0.3<sup>2</sup> - 0.185<sup>2</sup>) = π × (0.09 - 0.034225) =  
−
    <p>P = F / S = 0.07149 N / 0.1075 cm² = 0.665 N/cm² ≈ 6.65 KPa</p>
+
π × 0.055775 ≈ 0.1753 cm<sup>2</sup>  
−
</div>
+
</p>  
−
 
+
<p><b>Force Calculation:</b></p>  
−
<p>Adhesive Force per Milligram of Protein:</p>
+
<p>The total force is calculated as:</p>  
−
<p>The adhesive force per milligram of protein is:</p>
+
<p>  
−
<div class="calculation">
+
<strong>F</strong> = (5.951 + 0.448 × 15) g × 9.8 N/kg = 12.671 g × 9.8 N/kg ≈ 0.12418 N  
−
    <p>P' = P / m = 6.65 KPa / 1 mg = 6.65 KPa/mg</p>
+
</p>  
 +
<p><b>Adhesive Force Calculation:</b></p>  
 +
<p>The adhesive force produced by the protein is:</p>  
 +
<p>  
 +
<strong>P</strong> = <strong>F</strong> / <strong>S</strong> = 0.12418 N / 0.1753
 +
cm<sup>2</sup> ≈ 0.708 N/cm<sup>2</sup> = 7.08 kPa
 +
</p>  
 +
<p><b>Adhesive Force per Milligram of Protein:</b></p>  
 +
<p>The adhesive force per milligram of protein is:</p>  
 +
<p>  
 +
<strong>P'</strong> = <strong>P</strong> / m = 7.08 kPa / 1 mg = 7.08 kPa/mg  
 +
</p>
 +
<p>
 +
This demonstrates that the protein exhibits significant adhesive capability, with an adhesive force of 7.08 kPa per milligram of protein, which is enough for our daily production and usage.
 +
</p>
 
</div>
 
</div>
  

Latest revision as of 13:37, 2 October 2024


A fusion protein that adheres to the surface of dentate ring protein and substrate of squid.

In the Mfp5-TRn4 fusion proteins, Mfp5 is derived from the mussel foot proteins, whose tyrosine residues are oxidized by tyrosinase into dopa, which primarily forms π-π bonds and hydrogen bonds with the surface materials, allowing them to adhere to various materials. TRn4 is a protein obtained by repeating the sequence from squid ring teeth proteins four times, and the β-sheet on its structure can connect with the β-sheet on the structure of highly repetitive squid ring teeth proteins through hydrogen bonds. Tyrosine on Mfp5 generates dopa when tyrosinase is present, which makes Mfp5-TRn4 fusion protein adhere to the surface materials.

Fig. 1 | The plasmid map of TRn4-mfp5.

pET-SUMO-TRn4-mfp5

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.


Characterization

In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. 2)—pET-28a(+), pET-SUMO, and pET-21a(+)—and tried different strategies for TRn4-mfp5 protein production and purification.


Protein purification

Fig. 2 | 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. 3. This suggests that lower temperature incubation may enhance protein solubility and proper folding, resulting in improved yield.

Protein purification

Fig. 3 | 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.
Protein purification

Fig. 4 | 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.
Protein purification

Fig. 5 | 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 3-5, 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 2 to 5, indicating successful expression of the target protein, with a particularly strong band in the supernatant after denaturation (Fig. 6, lane 7). After purification, the target protein was mainly found in the 150 mM and 300 mM imidazole elution fractions.

Protein purification

Fig. 6 | 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 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. 7 | Western Blot of purified fusion protein TRn4-mfp5(35.4 kDa) using vector pET-SUMO.

a. Western blot of the pre-expressed protein. Lane 1: Total liquid (IPTG); Lane 2: Supernatant (IPTG); Lane 3: Precipitate (IPTG), b. Western blot 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. 8). The final yield was about 25 mg/L bacterial culture.

Protein purification

Fig. 8 | 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 conduct adhesive ability tests on the fusion protein(Fig. 9). We applied 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 successfully adhered.

Protein purification

Fig. 9 | Adhesive ability test of fusion protein on plastic surface.

Viscosity Calculations Viscosity Calculations

Surface Area Calculation:

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

S = π × (router2 - rinner 2)

Where:
router = 3 mm = 0.3 cm
rinner = 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, which is enough for our daily production and usage.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1035
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 189
    Illegal XhoI site found at 1018
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

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.