Genetic engineering of designed fiber proteins to study structure/function relationships in fibrous proteins

Using spider silk and collagen as a model, we are investigating the role that various protein primary structural components play in fiber production. Spidroins, spider dragline silk protein components, are essentially characterized by an amino acid repeat containing a glycine rich motif (amorphous) followed by an alanine rich motif (crystalline-putatively responsible for fiber strength). I am testing the importance of alanine runs in these proteins and the role of this motif in the mechanical properties of the resulting fiber. To test the importance of alanine-rich motifs in the spidroin 1 proteins, I have engineered three types of spidroin 1-like genes containing sequences encoding for different amounts of alanine repeats in the protein (normal, low, and no alanine residues). I also have engineered three copolymer collagen-spidroin 1 genes using each of the three spidroin 1 synthetic genes. These copolymers were mimicked on existing natural block collagen-silk like protein copolymer found in the byssus thread of marine mussels. All of these constructs were introduced in yeast (Pichia pastoris) for protein production. High levels of protein production (intracellular and secretion) have been achieved in Pichia pastoris. I found that the presence of alanine motifs promotes stability in these recombinant proteins.; Large scale production of newly designed fiber proteins in higher plants will be investigated in seed storage protein producing plants such as legumes using a natural high-level seed specific promoter: the peanut stearoyl-ACP desaturase gene promoter. Stearoyl-ACP desaturase regulatory sequences as well as upstream A/T-rich region (putative enhancer) have been identified and isolated. The putative promoter (without A/T-rich region) was juxtaposed to a GUS reporter gene in a pBI121 derived vector and tested for activity. The construct was introduced in callus obtained from mature peanut seeds by particle bombardment. The GUS transient assay for this construct was positive meaning that the sequences isolated could promote gene transcription. Further studies will determine the putative enhancing role of the upstream A/T-rich region.