Design, biosynthesis and characterization of spider dragline silk analogue: Self-assembled polypeptide block copolymer that undergoes structural rearrangement

The design and synthesis of artificial protein polymer materials is an attractive area that has potential applications in structural biology, materials science, and biomedical engineering. Unlike polymers produced by conventional chemical synthetic processes, genetically engineered protein polymers can be designed and produced with high precision in molecular weight, sequence, and structure.; Among the natural protein polymers that could be used as templates for artificial protein design, spider dragline silk protein has aroused much interest for its unusual combination of high tensile and compressive strength that is unmatched in synthetic fibers. The spider dragline silk is a semicrystalline material made of amorphous flexible chains reinforced by strong crystals. The crystalline domain is made of hydrophobic alanine-rich sequences. The arrangement of theses segments into hydrogen-bonded β-sheets provides mechanical strength through the formation of crystalline cross-links between protein chains. The amorphous matrix is formed from oligopeptide chains rich in glycine which comprise approximately 70% of the dragline silk protein, and are believe to provide the elasticity of the silk fiber.; We chose the sequence of the spider dragline silk protein as a model for the design of a polypeptide block-copolymer material. This protein polymer is comprised of alternating repeats of a 16 residue alanine-rich oligopeptide crystalline domain and a 30 residue glycine-rich oligopeptide amorphous domain. The sequence of the crystalline block was based on a self-complementary amphiphilic oligopeptide (AEAEAKAK)₂, which self-assembles under ambient conditions into extremely stable crystallites, fibrils, and membranes. The sequence of the amorphous domain consisted of six (GPGQQ) repeats, which was derived from dragline silk fibroin. The monomer sequence design of the protein polymer was as below: GPGQQ 6GAEAEA KAK2AGS ; The DNA cassettes encoding the individual domains were synthesized independently and joined enzymatically to create the DNA monomer encoding the above sequence. A DNA cassette multimerization strategy was used to isolate synthetic multimer genes encoding multiple repeats of the consensus sequence of the target polypeptide block-copolymer. A 9-mer gene was chosen to be introduced into pET-19b and expressed in E. coli strain BL21(DE3). Expression of the target gene from pET-19b which has a N-terminal decahistidine tag allowed the fusion protein to be purified to homogeneity under native conditions with immobilized metal affinity chromatography. The decahistidine tag was removed by cyanogen bromide cleavage, liberating the target protein polymer from the fusion protein. The degree of multimerization was confirmed by MALDI-TOF MS. The composition and N-terminal sequence of the polypeptide were validated by total amino acid compositional analysis and automated Edman degradation sequencing.; An optically transparent membrane was formed spontaneously upon concentration of aqueous solutions of the silk analogue. FTIR of the membrane exhibited absorptions that were consistent with presence of a β-sheet structure. High resolution SEM of the membrane suggested a morphology in which β-sheet crystallites were embedded in a matrix of amorphous polypeptide. However, the silk analogue demonstrated an β-helix structure before the formation of membrane. NMR and attenuated total reflection FTIR spectra suggested that the (AEAAEAKAK)₂ segment of the protein polymer underwent a conformational transition from α-helix to β-sheet upon self-assembly from solution into the membrane. Since the final β-sheet content of the membrane was sensitive...