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| <p>Inspired by the autocatalytic formation of the isopeptide bond between a specific Lys and an Asp residue in Streptococcus pyogenes (Spy) fibronectin-binding protein FbaB, researchers split its autocatalytic domain, CnaB2, and obtained two peptides which they named SpyTag and SpyCatcher. The resulting peptides are able to form isopeptide bonds with each other spontaneously. SpyTag, SpyCatcher and their stable cross-linking products provided us with an ideal tool to manipulate the protein polymer network<sup>1</sup>.</p> | | <p>Inspired by the autocatalytic formation of the isopeptide bond between a specific Lys and an Asp residue in Streptococcus pyogenes (Spy) fibronectin-binding protein FbaB, researchers split its autocatalytic domain, CnaB2, and obtained two peptides which they named SpyTag and SpyCatcher. The resulting peptides are able to form isopeptide bonds with each other spontaneously. SpyTag, SpyCatcher and their stable cross-linking products provided us with an ideal tool to manipulate the protein polymer network<sup>1</sup>.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/c/cd/F1.large.jpg" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/5/51/T--Peking--image_crosslinking_fig1.jpg" style="width:100%;" alt=""/> |
| <figcaption style="text-align:justify"> | | <figcaption style="text-align:justify"> |
| Fig. 1. Spontaneous intermolecular amide bond formation by SpyTag. (A) Amide bond formation between Lys and Asp side chains. (B) Key residues for amide bond formation in CnaB2 shown in stick format, based on PDB 2X5P. (C) Cartoon of SpyTag construction. Streptococcus pyogenes (Spy) CnaB2 was dissected into a large N-terminal fragment (SpyCatcher, left) and a small C-terminal fragment (SpyTag, right).For sake of brevity, SpyTag and SpyCatcher shall from now on be abbreviated as A and B, respectively. | | Fig. 1. Spontaneous intermolecular amide bond formation by SpyTag. (A) Amide bond formation between Lys and Asp side chains. (B) Key residues for amide bond formation in CnaB2 shown in stick format, based on PDB 2X5P. (C) Cartoon of SpyTag construction. Streptococcus pyogenes (Spy) CnaB2 was dissected into a large N-terminal fragment (SpyCatcher, left) and a small C-terminal fragment (SpyTag, right).For sake of brevity, SpyTag and SpyCatcher shall from now on be abbreviated as A and B, respectively. |
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| <p>The SpyTag / SpyCatcher System could be applied in the field via polymerization to obtain the “Spy Crosslinking Network”. The functional domains provided by SpyTag and SpyCatcher were linked via an elastin like protein (ELP), yielding the new fusion protein monomers able to undergo three-dimensional polymerization (TDP). This kind of polymerization could yield hyper-branched products and polymer network at low concentrations, and gels with a high degree of cross-linkage at high concentrations<sup>2,3</sup>.</p> | | <p>The SpyTag / SpyCatcher System could be applied in the field via polymerization to obtain the “Spy Crosslinking Network”. The functional domains provided by SpyTag and SpyCatcher were linked via an elastin like protein (ELP), yielding the new fusion protein monomers able to undergo three-dimensional polymerization (TDP). This kind of polymerization could yield hyper-branched products and polymer network at low concentrations, and gels with a high degree of cross-linkage at high concentrations<sup>2,3</sup>.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/3/38/%E7%AC%AC%E4%BA%8C%E5%BC%A0%E5%9B%BE.png" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/0/00/T--Peking--image_crosslinking_fig2.png" style="width:100%;" alt=""/> |
| <figcaption> | | <figcaption> |
| Fig. 2. The Spy Crosslinking Network. (A) Monomers containing multiple functional groups, SpyTag or SpyCatcher, react with each other both in situ and in vitro. (B) Schematic illustration of the products formed by mixing protein precursors 3A and 2B. Their mixing at high concentration (w.t. >=7.5%) leads to the formation of a covalently cross-linked gel. | | Fig. 2. The Spy Crosslinking Network. (A) Monomers containing multiple functional groups, SpyTag or SpyCatcher, react with each other both in situ and in vitro. (B) Schematic illustration of the products formed by mixing protein precursors 3A and 2B. Their mixing at high concentration (w.t. >=7.5%) leads to the formation of a covalently cross-linked gel. |
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| <p>To answer these questions, we have analyzed the crosslinking ability of the fundamental monomers 3A, 3A-SUP, 3A-mSA and 3B in detail. They were first diluted to a certain concentration, and were made to react at equimolar concentrations of A and B. The monomers containing A modules were contacted with 3B at 25°C and pH=7.3 for 2 hours.</p> | | <p>To answer these questions, we have analyzed the crosslinking ability of the fundamental monomers 3A, 3A-SUP, 3A-mSA and 3B in detail. They were first diluted to a certain concentration, and were made to react at equimolar concentrations of A and B. The monomers containing A modules were contacted with 3B at 25°C and pH=7.3 for 2 hours.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/c/cd/%E8%83%B6%E5%9B%BE.png" style="width:100%;" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/f/f0/T--Peking--image_crosslinking_fig4.png" style="width:100%;" alt=""/> |
| <figcaption> | | <figcaption> |
| Fig. 4. Exploration of the polymerization ability of the 3A-SUP/3A-mSA with 3B. “3A-SUP” stands for “Triple SpyTag-SUP”, “3A-mSA” for “Triple SpyTag-mSA”, and “3B” is the abbreviation of “Triple SpyCatcher”. (A) SDS-PAGE of basic experiment, which illustrates the basic cross-linking ability of 3A-SUP/3A-mSA and 3B. Lane 1, Marker; lane 2, 3A-SUP; lane 3, 3A-mSA; lane 4, 3A-SUP+3A-mSA; lane5, 3B; lane 6, 3A-SUP+3B; lane 7, 3A-mSA+3B. (Molecular Weight: 3A-SUP, 21.4kDa; 3A-mSA, 25.4kDa; 3B, 62.0kDa.) (B) The OD value of the lane 5 of oligomers produced by the mix of 3A-SUP and 3B. Peaks illustrate the monomers and the possible products: ① 3A-SUP (21.4kDa); ② 3B (62.0kDa); ③ 1x 3A-SUP+1x 3B (83.4kDa); ④ 3x 3A-SUP+1x 3B (126.2kDa); ⑤ 2x 3A-SUP+2x 3B (166.8kDa); ⑥ 4x 3A-SUP+4x 3B (333.6kDa). (C) The OD value of the lane 7 of oligomers produced by the mix of 3A-mSA and 3B. Peaks illustrate the monomers and the possible products: ① 3A-mSA (25.4kDa); ② 3B (62kDa); ③ 1x 3A-mSA+1x 3B (87.4kDa); ④ 1x 3A-mSA+2x 3B (149.4kDa); ⑤ 2x 3A-mSA+2x 3B (174.4kDa); ⑥ 3x 3A-mSA+4x 3B (324.2kDa). | | Fig. 4. Exploration of the polymerization ability of the 3A-SUP/3A-mSA with 3B. “3A-SUP” stands for “Triple SpyTag-SUP”, “3A-mSA” for “Triple SpyTag-mSA”, and “3B” is the abbreviation of “Triple SpyCatcher”. (A) SDS-PAGE of basic experiment, which illustrates the basic cross-linking ability of 3A-SUP/3A-mSA and 3B. Lane 1, Marker; lane 2, 3A-SUP; lane 3, 3A-mSA; lane 4, 3A-SUP+3A-mSA; lane5, 3B; lane 6, 3A-SUP+3B; lane 7, 3A-mSA+3B. (Molecular Weight: 3A-SUP, 21.4kDa; 3A-mSA, 25.4kDa; 3B, 62.0kDa.) (B) The OD value of the lane 5 of oligomers produced by the mix of 3A-SUP and 3B. Peaks illustrate the monomers and the possible products: ① 3A-SUP (21.4kDa); ② 3B (62.0kDa); ③ 1x 3A-SUP+1x 3B (83.4kDa); ④ 3x 3A-SUP+1x 3B (126.2kDa); ⑤ 2x 3A-SUP+2x 3B (166.8kDa); ⑥ 4x 3A-SUP+4x 3B (333.6kDa). (C) The OD value of the lane 7 of oligomers produced by the mix of 3A-mSA and 3B. Peaks illustrate the monomers and the possible products: ① 3A-mSA (25.4kDa); ② 3B (62kDa); ③ 1x 3A-mSA+1x 3B (87.4kDa); ④ 1x 3A-mSA+2x 3B (149.4kDa); ⑤ 2x 3A-mSA+2x 3B (174.4kDa); ⑥ 3x 3A-mSA+4x 3B (324.2kDa). |
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| <figure> | | <figure> |
− | <p style="text-align:center;"><img style="width: 90% ;" src=" https://static.igem.org/mediawiki/2016/4/4b/T--Peking--images_crosslinking_fig6.png" alt=""/></p> | + | <p style="text-align:center;"><img style="width: 80% ;" src="https://static.igem.org/mediawiki/2016/4/4b/T--Peking--images_crosslinking_fig6.png" alt=""/></p> |
| <figcaption style="text-align:left;"> | | <figcaption style="text-align:left;"> |
| Fig. 6. pH Gradient Experiment. (A) The reactive extent for 3B at different pH. Experiments were repeated three times and error bars were added. (B) The mass distribution of oligomers at different time at pH7.3 (70kDa-450kDa). </figcaption> | | Fig. 6. pH Gradient Experiment. (A) The reactive extent for 3B at different pH. Experiments were repeated three times and error bars were added. (B) The mass distribution of oligomers at different time at pH7.3 (70kDa-450kDa). </figcaption> |
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| <p>The second part of the gradient experiment is concerned with temperature. According to common environmental temperatures, three typical values, 16°C, 25°C, 37°C were introduced in this experiment. Similar to the pH Gradient Experiment, the monomers 3A-SUP and 3B were first dissolved in TBS at pH=7.3, after which they were contacted at the temperatures from the mentioned gradient. The reactions were conducted at pH=7.3 for 2 hours. The subsequent steps were the same as described for the pH gradient experiment.</p> | | <p>The second part of the gradient experiment is concerned with temperature. According to common environmental temperatures, three typical values, 16°C, 25°C, 37°C were introduced in this experiment. Similar to the pH Gradient Experiment, the monomers 3A-SUP and 3B were first dissolved in TBS at pH=7.3, after which they were contacted at the temperatures from the mentioned gradient. The reactions were conducted at pH=7.3 for 2 hours. The subsequent steps were the same as described for the pH gradient experiment.</p> |
| <figure> | | <figure> |
− | <p style="text-align:center;"><img style="width: 90% ;" src="https://static.igem.org/mediawiki/2016/3/3c/Figure_7_peking_igem_2016.png" alt=""/></p> | + | <p style="text-align:center;"><img style="width: 70% ;" src="https://static.igem.org/mediawiki/2016/0/0b/T--Peking--image_crosslinking_fig7.png" alt=""/></p> |
| <figcaption style="text-align:left;"> | | <figcaption style="text-align:left;"> |
| Fig. 7. Temperature Gradient Experiment. (A) The reactive extent for 3B at different temperature. Experiments were repeated three times and error bars were added. (B) The mass distribution of oligomers at different time at 16℃ (70kDa-400kDa). </figcaption> | | Fig. 7. Temperature Gradient Experiment. (A) The reactive extent for 3B at different temperature. Experiments were repeated three times and error bars were added. (B) The mass distribution of oligomers at different time at 16℃ (70kDa-400kDa). </figcaption> |
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| <p>The third part is concerned with optimal monomer concentrations. At low concentrations, the polymers would cross-link and not polymerize, but oligomerize. According to the basics of polymer science, the differences between these two processes could be described according to crosslinking density, which is large in the former, and rather small in the latter case. From this point of view, if we hope to increase the mass, strength and contact area of polymers, we have to mix the monomers at high concentrations. Thus, understanding the relationship between the monomer concentrations and the variation of mass distribution of polymers is quite essential.</p> | | <p>The third part is concerned with optimal monomer concentrations. At low concentrations, the polymers would cross-link and not polymerize, but oligomerize. According to the basics of polymer science, the differences between these two processes could be described according to crosslinking density, which is large in the former, and rather small in the latter case. From this point of view, if we hope to increase the mass, strength and contact area of polymers, we have to mix the monomers at high concentrations. Thus, understanding the relationship between the monomer concentrations and the variation of mass distribution of polymers is quite essential.</p> |
| <figure> | | <figure> |
− | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/3/34/Figure_8_peking_igem_2016_.png" alt=""/> | + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/9/9c/T--Peking--images_crosslinking_fig81.png" alt=""/> |
| + | </br> |
| + | <img class="featurette-image" src="https://static.igem.org/mediawiki/2016/4/41/T--Peking--images_crosslinking_fig82.png" alt=""/> |
| <figcaption> | | <figcaption> |
| Fig. 8. The mass distribution of oligomers of concentration gradient experiment at different concentration. (A) SDS-PAGE of concentration gradient experiment, which clearly showed the change of mass distribution according to the change of concentration. Lane 1, Marker; lane 2, 3A-SUP; lane 3, 3A-mSA; lane 4, 3A-SUP+3A-mSA; lane5, 3B; (1mg/mL) lane 6, 3A-SUP+3B; lane 7, 3A-mSA+3B; lane 8, 3A-SUP+3A-mSA+3B; (5mg/mL) lane 9, 3A-SUP+3B; lane 10, 3A-mSA+3B; lane 11, 3A-SUP+3A-mSA+3B; (10mg/mL) lane 12, 3A-SUP+3B; lane 13, 3A-SUP+3A-mSA+3B. (Molecular Weight: 3A-SUP, 21.4kDa; 3A-mSA, 25.4kDa; 3B, 62.0kDa.) The mass of samples in lanes was equal to each other. (B) The OD value of the lanes 2, 5, 6, 9, 12 of the oligomers produced by the mix of 3A-SUP and 3B. Peaks illustrate the monomers and the possible products, ① 3A-SUP (21.4kDa); ② 3B (62kDa); ③ 1x 3A-SUP+1x 3B (83.4kDa); ④ 3x 3A-SUP+1x 3B (126.2kDa); ⑤ 2x 3A-SUP+2x 3B (166.8kDa); ⑥ 4x 3A-SUP+4x 3B (333.6kDa); ⑦ oligomers larger than 750kDa (~6x 3A-sup+10x 3B or 9x 3A-SUP+9x 3B). | | Fig. 8. The mass distribution of oligomers of concentration gradient experiment at different concentration. (A) SDS-PAGE of concentration gradient experiment, which clearly showed the change of mass distribution according to the change of concentration. Lane 1, Marker; lane 2, 3A-SUP; lane 3, 3A-mSA; lane 4, 3A-SUP+3A-mSA; lane5, 3B; (1mg/mL) lane 6, 3A-SUP+3B; lane 7, 3A-mSA+3B; lane 8, 3A-SUP+3A-mSA+3B; (5mg/mL) lane 9, 3A-SUP+3B; lane 10, 3A-mSA+3B; lane 11, 3A-SUP+3A-mSA+3B; (10mg/mL) lane 12, 3A-SUP+3B; lane 13, 3A-SUP+3A-mSA+3B. (Molecular Weight: 3A-SUP, 21.4kDa; 3A-mSA, 25.4kDa; 3B, 62.0kDa.) The mass of samples in lanes was equal to each other. (B) The OD value of the lanes 2, 5, 6, 9, 12 of the oligomers produced by the mix of 3A-SUP and 3B. Peaks illustrate the monomers and the possible products, ① 3A-SUP (21.4kDa); ② 3B (62kDa); ③ 1x 3A-SUP+1x 3B (83.4kDa); ④ 3x 3A-SUP+1x 3B (126.2kDa); ⑤ 2x 3A-SUP+2x 3B (166.8kDa); ⑥ 4x 3A-SUP+4x 3B (333.6kDa); ⑦ oligomers larger than 750kDa (~6x 3A-sup+10x 3B or 9x 3A-SUP+9x 3B). |
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| <p>The crosslinking experiments showed that monomers containing SpyTag domains could react with triple SpyCatcher (3B) monomers, and that the crosslinking reaction may be favored at appropriately low pH (in the experiments, pH=6.3) and appropriately high temperature (in the experiments, T=25℃). Crosslinking leads to oligomerization at low monomer concentrations, and produces highly-crosslinked polymers with larger masses at high monomer concentrations. To this end, we have determined the optimal conditions for polymerization, which consisted of appropriately low pH, appropriately high temperature and comparatively higher monomer concentrations. The findings were in accordance with the universal principles of polyester condensation, as expected.</p> | | <p>The crosslinking experiments showed that monomers containing SpyTag domains could react with triple SpyCatcher (3B) monomers, and that the crosslinking reaction may be favored at appropriately low pH (in the experiments, pH=6.3) and appropriately high temperature (in the experiments, T=25℃). Crosslinking leads to oligomerization at low monomer concentrations, and produces highly-crosslinked polymers with larger masses at high monomer concentrations. To this end, we have determined the optimal conditions for polymerization, which consisted of appropriately low pH, appropriately high temperature and comparatively higher monomer concentrations. The findings were in accordance with the universal principles of polyester condensation, as expected.</p> |
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