Difference between revisions of "Part:BBa K5398030"
(→Adhesive test) |
|||
(8 intermediate revisions by 2 users not shown) | |||
Line 3: | Line 3: | ||
<partinfo>BBa_K5398030 short</partinfo> | <partinfo>BBa_K5398030 short</partinfo> | ||
− | In the Mfp5-TRn4 fusion proteins, Mfp5 is derived from the mussel foot proteins, whose tyrosine residues are oxidized by tyrosinase into | + | 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. |
<html> | <html> | ||
− | <center><img src="https://static.igem.wiki/teams/5398/trn4-mfp5/pet-pc-sumo- | + | <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> | ||
− | <p style="text-align: center!important;"><b>Fig. 1 The plasmid map of TRn4-mfp5. | + | <p style="text-align: center!important;"><b>Fig. 1 | The plasmid map of TRn4-mfp5. |
</b></p> | </b></p> | ||
Line 17: | Line 17: | ||
===Characterization=== | ===Characterization=== | ||
− | In order to obtain proteins, test suitable expression conditions, and evaluate the function of TRn4-mfp5, we chose three different expression vectors (Fig. | + | 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. |
Line 37: | Line 37: | ||
<body> | <body> | ||
<div class="module"> | <div class="module"> | ||
− | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/three-plasmid- | + | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/three-plasmid-map-white.webp" width="700" height="auto" alt="Protein purification"> |
− | <p><b>Fig. | + | <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; | ||
Line 47: | Line 47: | ||
====Protein Expression==== | ====Protein Expression==== | ||
− | <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. | + | <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. |
<html lang="zh"> | <html lang="zh"> | ||
<head> | <head> | ||
Line 65: | Line 65: | ||
<body> | <body> | ||
<div class="module"> | <div class="module"> | ||
− | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/16-37-lb-pet21a.webp" width="700" height="auto" alt="Protein purification"> | + | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/16-37-lb-pet21a-new.webp" width="700" height="auto" alt="Protein purification"> |
− | <p><b>Fig. | + | <p><b>Fig. 3 | Comparison of fusion protein expression in different temperature using vector pET-21a(+).</b></p> |
<p> | <p> | ||
− | + | 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; | |
− | 2 | + | 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: | |
− | 4 | + | Protein ladder.</p> |
− | 5 | + | |
− | 6 | + | |
− | 7 | + | |
− | Lanes 8-13 (TB 16°C 20 h): | + | |
− | 8 | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | 13 | + | |
− | + | ||
</div> | </div> | ||
</body> | </body> | ||
Line 107: | Line 96: | ||
<body> | <body> | ||
<div class="module"> | <div class="module"> | ||
− | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/tb-lb-prt21a.webp" width="700" height="auto" alt="Protein purification"> | + | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/tb-lb-prt21a-new.webp" width="700" height="auto" alt="Protein purification"> |
− | <p><b>Fig. | + | <p><b>Fig. 4 | Comparison of fusion protein expression in LB and TB media using vector pET-21a(+).</b></p> |
<p> | <p> | ||
− | 1 | + | 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. | |
− | + | ||
− | + | ||
− | 6 | + | |
− | + | ||
− | + | ||
− | Lanes 8-13 (TB 16°C 20 h): | + | |
− | 8 | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | 13. | + | |
− | + | ||
</p> | </p> | ||
</div> | </div> | ||
Line 154: | Line 130: | ||
<body> | <body> | ||
<div class="module"> | <div class="module"> | ||
− | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/rostta-bl21-de3-trn4- | + | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/rostta-bl21-de3-trn4-mfp-new.webp" width="700" height="auto" alt="Protein purification"> |
− | <p><b>Fig. | + | <p><b>Fig. 5 | Comparison of fusion protein expression in <i>E. coli</i> strains BL21(DE3) and Rosetta.</b></p> |
<p> | <p> | ||
− | 1 | + | 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℃ | + | Lane 3: Supernatant (IPTG); Lane 4: Precipitate (IPTG); Lanes 5-7 (Rosetta LB 37℃ 4 h) Lane 5: |
− | 2 | + | Total liquid (IPTG); Lane 6: Supernatant (IPTG); Lane 7: Precipitate (IPTG) |
− | 3 | + | |
− | 4 | + | |
− | Lanes 5-7 (Rosetta LB 37℃ | + | |
− | 5 | + | |
− | 6 | + | |
− | 7 | + | |
</p> | </p> | ||
</div> | </div> | ||
Line 176: | Line 146: | ||
====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 | + | <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. | + | <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> |
<html lang="zh"> | <html lang="zh"> | ||
Line 197: | Line 167: | ||
<body> | <body> | ||
<div class="module"> | <div class="module"> | ||
− | <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"> |
− | <p><b>Fig. | + | <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> | ||
Line 233: | Line 198: | ||
<body> | <body> | ||
<div class="module"> | <div class="module"> | ||
− | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/wb- | + | <img src="https://static.igem.wiki/teams/5398/trn4-mfp5/wb-new.webp" width="700" height="auto" alt="Protein purification"> |
− | <p><b>Fig. | + | <p><b>Fig. 7 | Western Blot of purified fusion protein TRn4-mfp5(35.4 kDa) using vector pET-SUMO.</b></p> |
<p> | <p> | ||
− | + | <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> | ||
Line 243: | Line 213: | ||
====Adhesive test==== | ====Adhesive test==== | ||
− | We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. | + | We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 8). The final yield was about 25 mg/L bacterial culture. |
<html lang="zh"> | <html lang="zh"> | ||
<head> | <head> | ||
Line 262: | Line 232: | ||
<div class="module"> | <div class="module"> | ||
<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. | + | <p><b>Fig. 8 | The protein sample freeze-dried by a lyophilizer.</b></p> |
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> | ||
− | Next, we dissolved protein samples in Buffer A (10 mL 20 mM Tris | + | 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. |
<html lang="zh"> | <html lang="zh"> | ||
Line 287: | Line 257: | ||
<div class="module"> | <div class="module"> | ||
<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"> | ||
− | <p><b>Fig. | + | <p><b>Fig. 9 | Adhesive ability test of fusion protein on plastic surface.</b></p> |
</div> | </div> | ||
</body> | </body> | ||
Line 313: | Line 283: | ||
<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 | + | <p>The surface area for the annular region of the pipette tip is calculated as:</p> |
− | < | + | <p> |
− | + | <strong>S</strong> = π × (r<sub>outer</sub><sup>2</sup> - r<sub>inner</sub> | |
− | </ | + | <sup>2</sup>) |
− | + | </p> | |
− | <p>Force Calculation:</p> | + | <p> |
− | <p>The total | + | Where: <br>r<sub>outer</sub> = 3 mm = 0.3 cm <br> |
− | < | + | r<sub>inner</sub> = 1.85 mm = 0.185 cm |
− | + | </p> | |
− | < | + | <p> |
− | + | Substituting these values, we get: | |
− | < | + | </p> |
− | <p>The adhesive force produced by the protein is:</p> | + | <p> |
− | < | + | <strong>S</strong> = π × (0.3<sup>2</sup> - 0.185<sup>2</sup>) = π × (0.09 - 0.034225) = |
− | + | π × 0.055775 ≈ 0.1753 cm<sup>2</sup> | |
− | </ | + | </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> |
− | < | + | <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><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.
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.
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.
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.
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. 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.
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.
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.
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.
Fig. 9 | Adhesive ability test of fusion protein on plastic surface.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1035
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 189
Illegal XhoI site found at 1018 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.