Part:BBa_K4905009
Elastin-Like Polypeptide Triblock with Leucine Zippers
Information
This part is made up of the basic parts: two times Leucine zipper Z2 (BBa_K4905005), Elastin-Like Polypeptide (ELP) sequence A[60]I[60] (BBa_K4905001), and ELP sequence A[40]I[60] (BBa_K4905002). This results in the sequence Z2-I[60]-A[100]-I[60]-Z2. 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 modulators3.
Construct design
The construct consists of ELPs and two different leucine zippers that have affinity for each other. 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 sites2. 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 molecules3.
As shown in figure 2, this construct has a hydrophilic region in the middle (A[100]) and a hydrophobic region on each side of it (I[60]). On the ends the leucine zipper Z2 is located for stronger interactions between the ELPs. Leucine zippers consist of a repeating unit that forms an alpha helix. Two leucine zippers together form ion pairs between the helices, which causes association1. These stronger and reversible interactions make them useful in the formation of a hydrogel at a specific Tt. In the end, the hydrogel is formed with electrostatic and hydrophobic interactions between the ELPs.
Figure 2 | Schematic representation of the composite part, an ELP with leucine zippers on the ends.
As soon as the hydrogel is made inside E.coli 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1982
Illegal XbaI site found at 99 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1982
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1982
Illegal XhoI site found at 1999 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1982
Illegal XbaI site found at 99 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1982
Illegal XbaI site found at 99
Illegal NgoMIV site found at 156
Illegal NgoMIV site found at 336
Illegal NgoMIV site found at 426
Illegal NgoMIV site found at 606 - 1000COMPATIBLE WITH RFC[1000]
Results
References
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