Bionanotechnology application of polypeptides in a hair color product: self-assembly enables expression, processing, and functionality.

Bionanotechnology aims to impart new properties to materials from unique functionalities present in biomolecules. However, the promise of bionanotechnology has not materialized beyond the biomedical field due in large part to issues of scalability, purity, and cost of manufacturing. In this work we demonstrate an approach to co-engineer production and system functionality into a single polypeptide. We designed a system to anchor particles onto hair via a multifunctional polypeptide composed of two domains, one with affinity to hair and the other capable of strong interactions with the particle surface. These strong interactions, exemplified by resistance to anionic surfactants, stem from the ability to self-assemble into higher order structures, which were observed by atomic force microscopy. At the same time, the controlled solubility properties of the particle binding domain permit the scalable production in Escherichia coli via inclusion bodies and cost effective purification. We believe this is a significant advance toward the development of bionanotechnology for industrial applications.

High yield recombinant silk-like protein production in transgenic plants through protein targeting.

DP1B is a synthetic analogue of spider dragline silk protein. It can be spun to form silk fiber. Previously, it had been expressed in transgenic plants, showing the general feasibility of the plant-based DP1B production. However, success of such a plant-based platform requires a great increase of DP1B productivity in plant cells to reduce production cost. This report describes a protein targeting approach to accumulate DP1B in apoplast, ER lumen, and vacuole in Arabidopsis cells, by utilizing appropriate combinations of sporamin-targeting determinant peptides and ER retention peptide. The approach has dramatically enhanced DP1B accumulation, resulting in high production yield. The accumulation can be as high as 8.5 and 6.7% total soluble protein in leaf tissue by targeting to apoplast and ER lumen, respectively, or as high as 18 and 8.2% total soluble protein in seeds by targeting to ER lumen and vacuole, respectively. However, the vacuole targeting in leaves and the apoplast targeting in seeds have failed to accumulate full length DP1B molecules or any DP1B at all, respectively, suggesting that they may not be suitable for applications in leaf tissues and seeds. Data in this study recommend a combination of seed-specific expression and ER-targeting as one of the best strategies for yield enhancement of plant-based DP1B production.

Effects of electrospinning and solution casting protocols on the secondary structure of a genetically engineered dragline spider silk analogue investigated via Fourier transform Raman spectroscopy.

Micrometer and submicrometer diameter fibers of recombinant dragline spider silk analogues, synthesized via protein engineering strategies, have been electrospun from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and compared with cast films via Raman spectroscopy in order to assess changes in protein conformation that may result from the electrospinning process. Although the solvent casting process was shown to result in predominantly beta-sheet conformation similar to that observed in the bulk, the electrospinning process causes a major change in conformation from beta-sheet to alpha-helix. A possible mechanism involving electric field-induced stabilization of alpha-helical segments in HFIP solution during the electrospinning process is discussed.

Molecular analysis of Dehalococcoides 16S ribosomal DNA from chloroethene-contaminated sites throughout North America and Europe.

The environmental distribution of Dehalococcoides group organisms and their association with chloroethene-contaminated sites were examined. Samples from 24 chloroethene-dechlorinating sites scattered throughout North America and Europe were tested for the presence of members of the Dehalococcoides group by using a PCR assay developed to detect Dehalococcoides 16S rRNA gene (rDNA) sequences. Sequences identified by sequence analysis as sequences of members of the Dehalococcoides group were detected at 21 sites. Full dechlorination of chloroethenes to ethene occurred at these sites. Dehalococcoides sequences were not detected in samples from three sites at which partial dechlorination of chloroethenes occurred, where dechlorination appeared to stop at 1,2-cis-dichloroethene. Phylogenetic analysis of the 16S rDNA amplicons confirmed that Dehalococcoides sequences formed a unique 16S rDNA group. These 16S rDNA sequences were divided into three subgroups based on specific base substitution patterns in variable regions 2 and 6 of the Dehalococcoides 16S rDNA sequence. Analyses also demonstrated that specific base substitution patterns were signature patterns. The specific base substitutions distinguished the three sequence subgroups phylogenetically. These results demonstrated that members of the Dehalococcoides group are widely distributed in nature and can be found in a variety of geological formations and in different climatic zones. Furthermore, the association of these organisms with full dechlorination of chloroethenes suggests that they are promising candidates for engineered bioremediation and may be important contributors to natural attenuation of chloroethenes.