Part:BBa_K4905006
Elastin-Like Polypeptide Triblock with Leucine Zippers
Information
This part is made up of the basic parts: Leucine zipper Z1 (BBa_K4905004), Leucine zipper Z2 (BBa_K4905005), and two times Elastin-like polypeptide sequence A[60]I[60] (BBa_K4905001). This results in the sequence Z1-I[60]-A[120]-I[60]-Z2. With A the sequence (VPGAG(3)VPGGG(2)) 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 and VPGIG is referred to as I.
The construct consists of Elastin-like Polypeptides (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 sites [2]. 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 [3].
As shown in figure 2, 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 the leucine zippers Z1 and Z2 are 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 association [1]. 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.
Figure 2: Schematic representation of the composite part, an Elastin-Like Polypeptide 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 2023
Illegal EcoRI site found at 3949
Illegal XbaI site found at 140
Illegal XbaI site found at 2066 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2023
Illegal EcoRI site found at 3949
Illegal NheI site found at 4077 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2023
Illegal EcoRI site found at 3949
Illegal XhoI site found at 2040
Illegal XhoI site found at 3966 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2023
Illegal EcoRI site found at 3949
Illegal XbaI site found at 140
Illegal XbaI site found at 2066 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2023
Illegal EcoRI site found at 3949
Illegal XbaI site found at 140
Illegal XbaI site found at 2066
Illegal NgoMIV site found at 197
Illegal NgoMIV site found at 377
Illegal NgoMIV site found at 467
Illegal NgoMIV site found at 647
Illegal NgoMIV site found at 2123
Illegal NgoMIV site found at 2303
Illegal NgoMIV site found at 2393 - 1000COMPATIBLE WITH RFC[1000]
Results
Protein expression and purification
The protein has an expected molecular weight of 105.1 kDa Organic solvent extraction, ITC
SDS-page
- Z1-I60-A120-I60-Z2
- Z2-I60-A120-I60-Z2
- Z2-I60-A100-I60-Z2
Characterization
Massspec
Hydrogel formation
To see if a hydrogel could form, 10 wt% ELP was dissolved in cold MQ and left at room temperature to warm up and form a gel. This gel could be dissolved again when put at 4 °C overnight.
Figure 2 A ten percent hydrogel was formed inside of a mass spectrometry vial. ELP constructs were dissolved in MQ at 4 °C and left at room temperature as soon as they dissolved. Within minutes, a hydrogel started to form.
Transition temperature determination
To determine the transition temperature of the ELP constructs, different solutions of the proteins were made in PBS. Using a UV-VIS spectrometer, the absorbance of light at 350 and 600 nm was measured to find the temperature at which phase separation happens. First, the samples were heated up to find the temperature where the hydrogel forms. Later, the samples were cooled again to show their reversible behavior. Two transition temperatures can be seen, the first is where the hydrophobic parts aggregate and the hydrogel forms, the second transition temperature is where the hydrophilic blocks also collapse. Two different constructs are plotted together, Z1-I60-A120-I60-Z2 has complementary leucine zippers and Z2-I60-A120-I60-Z2 has two leucine zippers that are the same, so it acts as a control group. It can be seen that their transition temperatures differ. The transition temperature of Z2-I6-A120-I60-Z2 is about 17 °C and Z1-I60-A120-I60-Z2 has a transition temperature of around 20 °C. It can also be seen hat the Z1-I60-A120-I60-Z2 construct only exhibits one transition temperature, although two were expected, this might be because of the zippers already bringing the hydrophilic domains together which loses the second transition temperature.
Figure 3 Temperature dependent behaviour of the Z1-I60-A120-I60-Z2 and Z2-I60-A120-I60-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 350 nm. The temperature was varied with 0.5 °C per minute. The transition temperature of Z2-I60-A120-I60-Z2 is about 17 °C and Z1-A120-Z2 has a transition temperature of around 20 °C.
Figure 4 Temperature dependent behaviour of the Z1-I60-A120-I60-Z2 and Z2-I60-A120-I60-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 600 nm. The temperature was varied with 0.5 °C per minute. The transition temperature of Z2-I60-A120-I60-Z2 is about 17 °C and Z1-I60-A120-I60-Z2 has a transition temperature of around 20 °C.
Figure 5 Temperature dependent behaviour of the Z1-I60-A120-I60-Z2 and Z2-I60-A120-I60-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 350 nm. The temperature was varied with 2 °C per minute. The transition temperature of Z2-I60-A120-I60-Z2 is about 17 °C and Z1-A120-Z2 has a transition temperature of around 20 °C.
Figure 6 Temperature dependent behaviour of the Z1-I60-A120-I60-Z2 and Z2-I60-A120-I60-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 600 nm. The temperature was varied with 2 °C per minute. The transition temperature of Z2-I60-A120-I60-Z2 is about 17 °C and Z1-I60-A120-I60-Z2 has a transition temperature of around 20 °C.
Dye release from hydrogel
Inhibition of bacterial growth
To follow the growth inhibition of the bacteria because of the hydrogel, a calorimetric assay was conducted with CCK-8. This type of assay can be used to detect the concentration of live bacteria in a sample and relies on the reaction between CCK-8 and dehydrogenase, which results in the formation of orange-yellow formazan. The concentration of live bacteria is proportional to the absorbance value of formazan measured at 450 nm. According to literature, the OD600 is proportional to the number of bacteria in the sample, and the relation between the OD600 and OD450 measured in samples containing CCK-8 has been shown to have a linear relationship. Based on this information, a standard curve was made that relates the OD600 to the OD450. This curve was used in further experiments to determine the number of live (and over-time thus dividing) bacteria in each sample.
Figure 7 Standard curve relating the OD600 and OD450 of bacterial samples containing CCK-8. Imaging experiments
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 [3] 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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