Part:BBa_K4905002
Elastin-like Polypeptide (VPGIG)[60] (VPGAG)3(VPGGG)2[40]
Elastin-like polypeptides (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 a transition temperature (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(Despanie et al., 2016).
The general structure of polymeric ELPs is (VPGXG)n, where the monomeric unit is Val-Pro-Gly-X-Gly, and the "X" denotes a variable amino acid that can have consequences on the general properties of the ELP, such as the transition temperature (Tt). Specifically, the hydrophilicity or hydrophobicity and the presence or absence of a charge on the guest residue play a great role in determining the Tt. Also, the solubilization of the guest residue can affect the Tt. The "n" denotes the number of monomeric units that comprise the polymer(Christensen et al., 2023). In general, these polymers are linear below the Tt, but aggregate into spherical clumps above the Tt.(Hassouneh et al., 2010)
The TU-Eindhoven 2023 team used this part in a composite part for the formation of a hydrogel in e.coli. The repeating sequence of this part is (VPGAG)40 and (VPGIG)60 which creates a hydrophilic and a hydrophobic part. It has been codon optimized for expression in e.coli bl21 cells.
Characterization:
We ran an electrophoresis gel with the plasmid, digested with enzymes AcuI and BglI. The band of A[40]I[60] is shown in slot 2. A 60 kb ladder (L) was used. Slots 1 and 3 were used for different parts. The bands formed as expected. Sequencing samples were also prepared and these results showed that we had made the DNA that we wanted.
I[60]-A[60]: AcuI + BglI
A[40]-I[60]: BseRI + BglI
A[60]-I[60]: BseRI + BglI
This digested DNA was used for the development of further composite parts.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 910
Illegal NgoMIV site found at 1087
Illegal NgoMIV site found at 1177
Illegal NgoMIV site found at 1357 - 1000COMPATIBLE WITH RFC[1000]
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