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_K1317002).

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 solubilize at lower temperature. If it is used in a protein mixture (a lysate for example) after a few cycles of thermal cycling (from 4°C to 50°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 and purification of the polymer for downstream applications

Strain

The protein ELP has been produced in BLR strain of E.coli (F–ompT hsdSB(rB– mB–) gal dcm Δ(srl-recA)306::Tn10)

BLR is more suited for the production of ELP, it is a BL21 derivative in which plasmids containing repetitive sequences are stabilized.

Culture

Culture was performed in LB-glucose (ELP60) or LB-glycerol (ELP20, 40). After inoculation from a preculture the transformed BLR strain was incubated at 37°C in 1L of appropriate LB medium. Induction was performed when the OD reached 0.8 with 0.8mM IPTG (typically after 2.75h for VPGXG20, 5.25h for VPGXG40 and 1.25h for VPGXG60). Temperature was lowered at 25°C. Culture is stopped 21h after induction

Link to the registry of Standard Parts: [http://parts.igem.org/Part:BBa_K1317003 http://parts.igem.org/Part:BBa_K1317003]

References

[1] 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)

[2] 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

[3] 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

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

[5] Trabbic-Carlson et al. (2004), Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. Protein Science, 13: 3274–3284. doi: 10.1110/ps.04931604

[6] K. Trabbic‐Carlson et al. Effect of protein fusion on the transition temperature of an environmentally responsive elastin‐like polypeptide: a role for surface hydrophobicity? Protein Engineering, Design and Selection (2004) 17 (1): 57-66. doi: 10.1093/protein/gzh006

Team:Bordeaux/Parts/BBa K1317003

From 2014.igem.org

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