Difference between revisions of "Part:BBa K4905010"
 
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<h2>General applications</h2>
 
<h2>General applications</h2>
 
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ELPs are protein polymers derived from human tropoelastin. One of their key features is that they exhibit a phase separation that is often reversible whereby samples remain soluble below Tt but form coacervates above Tt. They have many possible applications in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators<sup>3</sup>.
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ELPs are protein polymers derived from human tropoelastin. One of their key features is that they exhibit a phase separation that is often reversible whereby samples remain soluble below Tt but form coacervates above Tt. They have many possible applications in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators<sup>2</sup>.
 
</p>
 
</p>
  
 
<h2>Construct design</h2>
 
<h2>Construct design</h2>
 
<p>
 
<p>
−
The construct consists of ELPs and FKBP12. In general, ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature-dependent. They are made from a repeating VPGXG sequence, with X replaced by specific amino acids. This results in specific properties of the ELPs, especially related to the transition temperature Tt at which the ELPs will interact with each other on the hydrophobic sites<sup>2</sup>. When the temperature is below Tt, the water molecules surrounding the hydrophobic parts will go into the bulk water phase which gains the solvent entropy. This makes it possible to form interactions with other ELP molecules<sup>3</sup>.  
+
The construct consists of ELPs and FKBP12. In general, ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature-dependent. They are made from a repeating VPGXG sequence, with X replaced by specific amino acids. This results in specific properties of the ELPs, especially related to the transition temperature Tt at which the ELPs will interact with each other on the hydrophobic sites<sup>1</sup>. When the temperature is below Tt, the water molecules surrounding the hydrophobic parts will go into the bulk water phase which gains the solvent entropy. This makes it possible to form interactions with other ELP molecules<sup>2</sup>.  
 
</p>
 
</p>
 
<p>
 
<p>
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This construct has a hydrophilic region in the middle (A[120]) and a hydrophobic region on each side of it (I[60]). On the ends, FKBP12 domains are located for stronger interactions between the ELPs. FKBP12 can form a complex with Rapamycin and the FKBP-rapamycin binding (FRB) domain. This mechanism is normally used in the mTOR pathway inside cells, which regulates cellular proliferation, protein synthesis, differentiation and survival, and lipid metabolism. In the case of a hydrogel, the addition of rapamycin to a cell with these ELPs and ELPs with FRB at the ends can induce crosslinking between them to form a network (bron inobe en yandong liu). These stronger interactions make them useful in the formation of a hydrogel.
+
This construct has a hydrophilic region in the middle (A[120]) and a hydrophobic region on each side of it (I[60]). On the ends, FKBP12 domains are located for stronger interactions between the ELPs. FKBP12 can form a complex with Rapamycin and the FKBP-rapamycin binding (FRB) domain. This mechanism is normally used in the mTOR pathway inside cells, which regulates cellular proliferation, protein synthesis, differentiation and survival, and lipid metabolism. In the case of a hydrogel, the addition of rapamycin to a cell with these ELPs and ELPs with FRB at the ends can induce crosslinking between them to form a network<sup>3,4</sup>. These stronger interactions make them useful in the formation of a hydrogel.
 
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As soon as the hydrogel is made inside <i>E.coli</i> BL21 cells, the cells are prevented from dividing. However, the cells remain functional. So they can still be used to express therapeutic agents, like Interleukin 10 in the TU Eindhoven 2023 teams project.  
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This parts needs to be combined with part <a style="color:#F6B227" href="https://parts.igem.org/Part:BBa_K4905014"> BBa_K4905014</a> that contains the FRB domain.  
 
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<h1>References</h1>
 
<h1>References</h1>
 
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1. Christensen, T., Hassouneh, W., Trabbic-Carlson, K., & Chilkoti, A. (2023). Predicting Transition Temperatures of Elastin-Like Polypeptide Fusion Proteins. https://doi.org/10.1021/bm400167h
 
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2. Despanie, J., Dhandhukia, J. P., Hamm-Alvarez, S. F., & MacKay, J. A. (2016). Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines. Journal of Controlled Release, 240, 93–108. https://doi.org/10.1016/j.jconrel.2015.11.010
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3. Inobe, T., & Nukina, N. (2016). Rapamycin-induced oligomer formation system of FRB–FKBP fusion proteins. Journal of Bioscience and Bioengineering, 122(1), 40–46. https://doi.org/10.1016/J.JBIOSC.2015.12.004
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<p>
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4. Liu, Y., Yang, F., Zou, S., & Qu, L. (2019). Rapamycin: A bacteria-derived immunosuppressant that has anti-atherosclerotic effects and its clinical application. Frontiers in Pharmacology, 9(JAN). https://doi.org/10.3389/FPHAR.2018.01520
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Latest revision as of 09:49, 6 October 2023


Elastin-Like Polypeptide Triblock with FKBP12

Information

This part is made up of the basic parts: two times Elastin-Like Polypeptide (ELP) sequence A[60]I[60] (BBa_K4905001) and two times FKBP12 (BBa_K3610022). This results in the sequence FKBP12-I[60]-A[120]-I[60]-FKBP12. With A[5] the sequence (VPGAG[3]VPGGG[2]), since there are five VPGXG repeats, and I the sequence (VPGIG). The numbers indicate the number of repeats of these sequences. This construct was used by the TU Eindhoven 2023 team to form a hydrogel outside as well as inside E.coli BL21 cells. A schematic overview of this is shown in figure 1.

Figure 1 | Schematic overview of the sequence of this construct. (VPGAG[3]VPGGG[2]) is from now on referred to as A[5] and VPGIG is referred to as I.

General applications

ELPs are protein polymers derived from human tropoelastin. One of their key features is that they exhibit a phase separation that is often reversible whereby samples remain soluble below Tt but form coacervates above Tt. They have many possible applications in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators2.

Construct design

The construct consists of ELPs and FKBP12. In general, ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature-dependent. They are made from a repeating VPGXG sequence, with X replaced by specific amino acids. This results in specific properties of the ELPs, especially related to the transition temperature Tt at which the ELPs will interact with each other on the hydrophobic sites1. When the temperature is below Tt, the water molecules surrounding the hydrophobic parts will go into the bulk water phase which gains the solvent entropy. This makes it possible to form interactions with other ELP molecules2.

This construct has a hydrophilic region in the middle (A[120]) and a hydrophobic region on each side of it (I[60]). On the ends, FKBP12 domains are located for stronger interactions between the ELPs. FKBP12 can form a complex with Rapamycin and the FKBP-rapamycin binding (FRB) domain. This mechanism is normally used in the mTOR pathway inside cells, which regulates cellular proliferation, protein synthesis, differentiation and survival, and lipid metabolism. In the case of a hydrogel, the addition of rapamycin to a cell with these ELPs and ELPs with FRB at the ends can induce crosslinking between them to form a network3,4. These stronger interactions make them useful in the formation of a hydrogel.

This parts needs to be combined with part BBa_K4905014 that contains the FRB domain.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2222
    Illegal EcoRI site found at 4148
    Illegal XbaI site found at 339
    Illegal XbaI site found at 2265
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2222
    Illegal EcoRI site found at 4148
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2222
    Illegal EcoRI site found at 4148
    Illegal XhoI site found at 2239
    Illegal XhoI site found at 4165
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2222
    Illegal EcoRI site found at 4148
    Illegal XbaI site found at 339
    Illegal XbaI site found at 2265
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2222
    Illegal EcoRI site found at 4148
    Illegal XbaI site found at 339
    Illegal XbaI site found at 2265
    Illegal NgoMIV site found at 396
    Illegal NgoMIV site found at 576
    Illegal NgoMIV site found at 666
    Illegal NgoMIV site found at 846
    Illegal NgoMIV site found at 2322
    Illegal NgoMIV site found at 2502
    Illegal NgoMIV site found at 2592
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

References

1. Christensen, T., Hassouneh, W., Trabbic-Carlson, K., & Chilkoti, A. (2023). Predicting Transition Temperatures of Elastin-Like Polypeptide Fusion Proteins. https://doi.org/10.1021/bm400167h

2. Despanie, J., Dhandhukia, J. P., Hamm-Alvarez, S. F., & MacKay, J. A. (2016). Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines. Journal of Controlled Release, 240, 93–108. https://doi.org/10.1016/j.jconrel.2015.11.010

3. Inobe, T., & Nukina, N. (2016). Rapamycin-induced oligomer formation system of FRB–FKBP fusion proteins. Journal of Bioscience and Bioengineering, 122(1), 40–46. https://doi.org/10.1016/J.JBIOSC.2015.12.004

4. Liu, Y., Yang, F., Zou, S., & Qu, L. (2019). Rapamycin: A bacteria-derived immunosuppressant that has anti-atherosclerotic effects and its clinical application. Frontiers in Pharmacology, 9(JAN). https://doi.org/10.3389/FPHAR.2018.01520