Engineering the extracellular matrix: a novel approach to polymeric biomaterials. I. Control of the physical properties of artificial protein matrices designed to support adhesion of vascular endothelial cells.
Methods of genetic engineering were applied to the design and biosynthesis of three extracellular matrix protein analogues constructed from identical elastin- and fibronectin-derived repeating units but characterized by different molecular weights in the range of 14,000 to 59,000. Expression levels were enhanced by the serendipitous choice of an N-terminal fusion sequence such that gram-scale syntheses were achieved for each protein. Purification protocols were developed that resulted in proteins of high purity and correct sequence, as determined by amino acid analysis, NMR spectroscopy, and lower critical solution temperature (LCST). Glutaraldehyde was shown to insolubilize the otherwise soluble proteins in a concentration-dependent manner. Tensile moduli of cross-linked protein films were measured and found to be inversely related to the molecular weights of the engineered proteins, which in each case corresponds to the theoretical molecular weight between cross-links. At the highest cross-link density (lowest molecular weight) the elastic modulus was similar to that of native elastin.
Welsh
E R
ER
Department of Polymer Science and Engineering, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Tirrell
D A
DA
D000595:Amino Acid Sequence; D001672:Biocompatible Materials; D015153:Blotting, Western; D002448:Cell Adhesion; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology*; D005109:Extracellular Matrix / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013718:Tensile Strength
D001672:Biocompatible Materials; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins