GENETICALLY DIRECTED SYNTHESES OF NEW POLYMERIC MATERIALS - EXPRESSION OF ARTIFICIAL GENES ENCODING PROTEINS WITH REPEATING (ALAGLY)3PROGLUGLY ELEMENTS

The goals of this work were to develop reliable methods for the bacterial expression of artificial repetitive polypeptides and to examine the connection between primary sequence and solid-state conformation in such materials. DNA fragments encoding variable numbers of repeating (AlaGly)3ProGluGly elements (1) were constructed via chemical synthesis and cloning of short oligonucleotides followed by self-ligation. The resulting multimer population was size fractionated and cloned into an adapter plasmid that provides methionine residues at positions flanking the repetitive sequences. DNA multimers modified in this way, and encoding 10, 18, 28, or 54 repeats of sequence 1, were transferred to an expression vector that features a T7 phage promoter, and protein expression in Escherichia coli was monitored via incorporation of [H-3]glycine. Purification of the target proteins was accomplished by selective precipitation with ammonium sulfate and subsequently with dilute acetic acid, followed by ion-exchange chromatography on DEAE-Sephadex. Typical yields of purified protein were ca. 10 mg/L of fermentation medium. The structure of the longest chain length variant was confirmed by amino acid compositional analysis, N-terminal protein sequencing (through 58 residues), matrix-assisted laser desorption mass spectrometry, H-1 and C-13 NMR spectrometry, cyanogen bromide cleavage, and combustion analysis. This polymer can be cast into coherent, optically clear films and displays a reversible glass transition at 170-degrees-C. Thermal decomposition starts at ca. 250-degrees-C and results ultimately in the loss of 70% of sample weight. Evidence from differential scanning calorimetry, X-ray scattering, and Fourier transform infrared spectroscopy suggests that this polymer and its CNBr cleavage product form amorphous glasses at room temperature.