Genetically controlled syntheses of novel polymeric materials

Research directed toward the biological production of novel polymeric materials is presented, in which synthetic genes, acting as templates for polymer production, enforce tight control over all aspects of chain microstructure. Synthetic genes encoding polypeptides of sequences 1 and 2 were constructed through the enzymatic ligation of small, repetitive portions of chemically synthesized DNA.$$\eqalignno{&\rm\lbrack (Ala{-}Gly)\sb3{-}Pro{-}Glu{-}Gly\rbrack\sb{n}&{\bf 1}\cr&\rm\lbrack(Ala{-}Gly)\sb3{-}Asn{-}Gly\rbrack\sb{n}&{\bf 2}\cr}$$Empirical rules relating protein sequence and structure predicted that materials with these sequences should adopt chain folded structures in the solid state, with folding initiated at the periodic proline, glutamic acid, or asparagine residues. Tandemly repeated fragments were constructed through use of nonpalindromic BanI restriction sites at the 5$\sp\prime$ and 3$\sp\prime$ termini. Multimerized fragments were cloned into the unique BanI site of p937.51, and isolated from recombinant vectors by digestion with BamHI. These fragments were inserted into the unique BamHI site of pET3-b, a powerful T7-based bacterial expression vector. Recombinant plasmids containing the inserts in the correct orientation were used to transform E. coli strain BL21(DE3) pLysS. This vector-host system places the synthetic gene under the control of the viral T7 promoter. The requisite T7 RNA polymerase gene is integrated into the bacterial genome under lacUV5 control, and can be induced by the addition of IPTG. Four proteins of sequence 1 with n varying from 10 to 54 and of sequence 2 with n equal to 16 were produced. One protein containing 54 repeats of sequence 1 was chosen for further analysis. High levels of expression were obtained using a natural translational start from bacteriophage T7. A simple purification procedure has been developed, typically yielding 8-10 mg of highly purified protein per liter of culture. Non-repetitive N- and C-termini were removed by treatment with cyanogen bromide. Structural analysis using FTIR and wide angle x-ray scattering on purified samples failed to indicate the predicted chain folded lamellar structures, indicating that the materials adopted amorphous structures in the solid state