Biosynthesis of an elastin-mimetic polypeptide with two different chemical functional groups within the repetitive elastin fragment.
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 beta-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.
Junger
Andreas
A
TU München, Lehrstuhl für Makromolekulare Stoffe, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Kaufmann
Doris
D
Scheibel
Thomas
T
Weberskirch
Ralf
R
D001483:Base Sequence; D040761:Biomimetic Materials / Q000737:chemistry; D004549:Elastin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
C033223:polyhistidine; D006639:Histidine; D004549:Elastin