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

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. This protein was meant as a control group for the tested part BBa_K4905006 .

**Figure 2 |** Schematic representation of the composite part, an ELP with leucine zippers on the ends.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1982
Illegal XbaI site found at 99
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1982
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1982
Illegal XhoI site found at 1999
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1982
Illegal XbaI site found at 99
25
INCOMPATIBLE 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
1000
COMPATIBLE WITH RFC[1000]

Results

References

[1] Alber, T. (1992). Structure of the leucine zipper. Current Opinion in Genetics and Development, 2, 205–210

[2] 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