Biosynthesis of an Elastin-Mimetic Polypeptide with Two Different Chemical Functional Groups within the Repetitive Elastin Fragment
✍ Scribed by Andreas Junger; Doris Kaufmann; Thomas Scheibel; Ralf Weberskirch
- Publisher
- John Wiley and Sons
- Year
- 2005
- Tongue
- English
- Weight
- 246 KB
- Volume
- 5
- Category
- Article
- ISSN
- 1616-5187
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✦ Synopsis
Abstract
Summary: A new protein engineering strategy was utilized to synthesize an elastin‐mimetic polypeptide. The primary structure represents an elastic motif composed of thirty amino acids with one lysine and one glutamic acid per repeat unit EMM = (VPGVG VPGKG VGPVG VPGVG VPGEG VPGIG). The gene was constructed using a Seamless Cloning method by generating three DNA cassettes which all encoded the EMM repeat unit, but with different flanking restriction recognition sites. The DNA cassettes were assembled to yield a gene that could be directly cloned into the multiple cloning site of pBluescript® II SK+. The resulting gene (EMM)~7~ with approximately 650 base pairs in length was further cloned into the expression vector pET‐28b. Protein biosynthesis in E. coli strain BLR(DE3) resulted in the 21.5 kDa repeating polypeptide His~6~‐(EMM)~7~ yielding up to 50 mg · L^−1^ of cell culture. Secondary structure analysis by far UV circular dichroism revealed a minimum at 197 nm and a shoulder at 218 nm indicative for a random coil with some type II β‐turn conformation content. Lower critical solution temperature (LCST) behavior strongly depends on salt and polypeptide concentration. Importantly, first cross‐linking experiments indicate successful hydrogel formation with a surface structure reminiscent to natural elastin as visualized by SEM micrographs.
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