Part:BBa_K1317003

CDS for Elastin like polypeptide (ELP)

This part is made with a consensus of the natural elastin sequence. Elastin is a protein involved in maintaining shape and elasticity of several tissues like skin. It has a high elasticity and resilience as a polymer.

In our project, the consensus sequence allows the use of a minimal pattern of the protein while keeping these particular properties. In an iGEM spirit the consensus itself can be used as a brick to vary length and nature of the polymers with our other biobricks (BBa_K1317001, BBa_K1317001).

The basic length of a polymer is 20 monomers. The coding sequence can be repeated easily using our improvement of the biobrick assembly system to get rid of the Stop Codon. After variation of the length different properties are attributed to the part. For example it can be used as a fusion tag to purify easily any fuse protein thanks to the thermal cycling purification.

ELP indeed have this particularity to precipitate at lower temperature. If it is used in a protein mixture (a lysate for example) after a few cycles of thermal cycling (from 50°C to 4°C), the pure fuse protein is obtained.

This part can be used as a purification tag for easy, quick and column-free purification or as coding sequence for a polymer usable to get fiber with good elasticity and resilience. The polymer is biocompatible, biodegradable and could be used as a tool for surgery or other health applications.

Usage and Biology

Production of the polymer

The protein ELP has been produced in BL21 strain of E.coli. After phase cycling purification it is possible to spin the protein into a wire, thanks to the wet-spinning method. The ELP can have different length regarding the number of copy of the gene. In these experiments, we used 40mer ELP (assemble of two gene copies).

Investigating the properties of the polymer

Polymer formation trial:

Fiber obtained by extruding ELP/Alginate in calcium chloride

In order to obtain polymer wires, we carried out severeal tests by mixing the ELP with different solvants. The following results have been observed:

• Sodium Sulfate (Na2So4) at 320 g/L => cottony aspect of the fiber, the structure is not stable

• Diethyl ether (C2H5)2O => No reaction because the drop is trapped. This solvent is not miscible with the polymer

• Acetone (CH3COCH3) => Formation of a white ring

• Heated Sodium Chloride (NaCl 5M) => Formation of a white cloud due to a lack of cohesiveness

• Calcium Chloride (CaCl2 15%) => 1.25% alginate mixture with ELP40 at 10mg/mL were extruded in the calcium chloride solution: a white-colored fiber is obtained.

This last trial has been performed with 0.75% alginate which seems to be the best experimental condition to get fibers from the ELP

Phase transition trial:

Phase transition of the ELP according to temperature

After getting the ELP40 fiber, it is necessary to wash it with hot water for extraction, the fiber could indeed dissolve at low temperature.

- Control remains transparent

- The aspect of the fiber changes at ambient temperature (from white to transparent)

Thanks to the mixture alginate + ELP40 (10 mg/L), we could observe the formation of the polymer and it can be deduced that:

- In presence of salt, product is losing cohesiveness

- With contact of hot water, the product is contracting

You can see the results on the phase transition on this video: https://www.youtube.com/watch?v=xoTQkoNoZu0

Wet-spinning and mechanical testing of fibers properties

The wet-spinning method has been used to create a wire out of the polymer.

Overview of the wet-sinning method

The mechanical traction machine

A traction machine allows to measure the resistance to rupture of a chosen material, in this case, the fiber obtained by wet-spinning. This experiment consists in placing a little stick of the material to be studied between the jaws of the traction machine. It will pull the stick until its rupture. The lenghtening and the applied force are recorded and then converted into distorsion and pressure data.

The mechanical testing allow us to state:

1. For the alginate
   • The experiment done with the alginate is reproducible while using the same conditions, but the fiber ends by breaking.
   • Alginate breaks faster because the experiment is carried out at high temperature
2. Comparison with carbon nanofibers
   • The elasticity is comparable with carbon nanofibers. Thus, ELP has characteristic properties of elasticity due to its polymeric nature.
   • The fibers are nevertheless fragile and break easily. Enhancing the resistance should be possible by using another part and fusing the proteins (BBa_K1317001, BBa_K1317002)

Another trial was carried out with two times more alginate. The result seems to be the same, the fiber breaks fast.

All data are available on the wiki page of this part:

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 312
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Doreen M. Floss et al. ELASTIN-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends in Biotechnology Vol.28 No.1 (PMID 19897265)

Dan W. Urry Entropic Elastic Processes in Protein Mechanisms. I. Elastic Structure Due to an Inverse Temperature Transition and Elasticity Due to Internal Chain Dynamics. Journal of Protein Chemistry, Vol. 7, No.. I, 1988

Dan W. Urry. Physical Chemistry of Biological Free Energy Transduction As Demonstrated by Elastic Protein-Based Polymers. J. Phys. Chem. B 1997, 101, 11007-11028

Dan E. Meyer and Ashutosh Chilkoti. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. NATURE BIOTECHNOLOGY VOL 17 NOVEMBER 1999