From 2014.igem.org

Latest revision as of 03:13, 18 October 2014

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Consensus sequences:

In the literature we found out that the natural elastin has some repetitions, in particular the sequence VPGXG seems to be repeated. X is V, L, or A, and it represents 9% of the whole polypeptide. This particular sequence were translated with codon optimization for E.coli to produce a (VPGXG)x protein. Elastin properties were checked to see if they are still existing after cloning into bacteria to produce the recombinant protein named elastin like polypeptide (ELP). We studied the original peptide sequences of these proteins to bring out some consensus sequences. Then it revealed a UCST and LCST (see on the purification results) that gives ELP a transition temperature with interesting particularities (see on the characterization results), it is more soluble when chilled and on the contrary it precipitates at higher temperature.

On the same way we search others elastic proteins and find spider silks and resilin.

For the resilin we use the exon 1 from Drosophila melanogaster2. We identify a repetitive pattern, PSXXYGAP. So we blast it to know if the pattern were conserved into different species or specific to Drosophila.

We have taken XX randomly and we brought out conserved pattern so we used it to do our resilin like polypeptide with the following pattern: (RLP) MW-[(PSSSYGAP)(PSNSYGAP)(PSTSYGAP)(PVAYGAP)]3

The literature delivers some mechanical properties concerning native resilin as high resilience (<90%) that allows the protein to recover its initial folding after a high tensile stress. Thus it can be an interesting characteristic to show and use, so we wanted to test it on our RLP.

Keep following our idea we do the same on spider silks. We previously identified consensus sequences, GPGQQ, GPGGY and GPGGX. Later we find in literature some leads that confirm our choice of consensus sequences. So we designed our SLP synthetic protein as MW-[(GPGGV)2(GPGQQ)(GPGGY)]5.

We first wanted to synthesize our SLP gene ourselves : You can see the strategy here

References:

1- Doreen M. Floss1, Kai Schallau2, Stefan Rose-John. ELASTIN-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends in Biotechnology Vol.28 No.1 (http://www.ncbi.nlm.nih.gov/pubmed/19897265)

2- David H. Ardell , Svend Olav Andersen. TENTATIVE identification of a resilin gene in Drosophila melanogaster. Insect Biochemistry and Molecular Biology 31 (2001) 965–970 (http://www.ncbi.nlm.nih.gov/pubmed/11483432)

3- Christopher M. Elvin, Andrew G. Carr, Mickey G. Huson. SYNTHESIS and properties of crosslinked recombinantpro-resilin. Vol 437|13 October 2005|doi:10.1038/nature04085 (http://www.ncbi.nlm.nih.gov/pubmed/16222249)

4- Michael B. Hinman, Justin A. Jones and Randolph V. Lewis. SYNTHETIC spider silk: a modular fiber. TIBTECH SEPTEMBER 2000 (Vol. 18) (http://www.ncbi.nlm.nih.gov/pubmed/10942961)