Recombinant exon-encoded resilins for elastomeric biomaterials.

Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediated di-tyrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative di-tyrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in vivo applications of resilin biomaterials.

Production of bioactive, post-translationally modified, heterotrimeric, human recombinant type-I collagen in transgenic tobacco.

Collagen's biocompatibility, biodegradability and low immunogenicity render it advantageous for extensive application in pharmaceutical or biotechnological disciplines. However, typical collagen extraction from animal or cadaver sources harbors risks including allergenicity and potential sample contamination with pathogens. In this work, two human genes encoding recombinant heterotrimeric collagen type I (rhCOL1) were successfully coexpressed in tobacco plants with the human prolyl-4-hydroxylase (P4H) and lysyl hydroxylase 3 (LH3) enzymes, responsible for key posttranslational modifications of collagen. Plants coexpressing all five vacuole-targeted proteins generated intact procollagen yields of approximately 2% of the extracted total soluble proteins. Plant-extracted rhCOL1 formed thermally stable triple helical structures and demonstrated biofunctionality similar to human tissue-derived collagen supporting binding and proliferation of adult peripheral blood-derived endothelial progenitor-like cells. Through a simple, safe and scalable method of rhCOL1 production and purification from tobacco plants, this work broadens the potential applications of human recombinant collagen in regenerative medicine.

Float and compress: honeycomb-like array of a highly stable protein scaffold.

Organizing nano-objects, proteins in particular, on surfaces is one of the primary goals of bio/chemical nanotechnology. A highly stable protein scaffold (6His-SP1) was organized into a hexagonal 2D array by a new, versatile method. The protein was expelled from solution into the air/water interface and compressed in a Langmuir trough into a closely packed monolayer without the use of phospholipids or other surfactants at the interface. The 2D arrays formed at the air/water interface were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM).

SP1 protein-based nanostructures and arrays.

Controlled formation of complex nanostructures is one of the main goals of nanoscience and nanotechnology. Stable Protein 1 (SP1) is a boiling-stable ring protein complex, 11 nm in diameter, which self-assembles from 12 identical monomers. SP1 can be utilized to form large ordered arrays; it can be easily modified by genetic engineering to produce various mutants; it is also capable of binding gold nanoparticles (GNPs) and thus forming protein-GNP chains made of alternating SP1s and GNPs. We report the formation and the protocols leading to the formation of those nanostructures and their characterization by transmission electron microscopy, atomic force microscopy, and electrostatic force microscopy. Further control over the GNP interdistances within the protein-GNP chains may lead to the formation of nanowires and structures that may be useful for nanoelectronics.

Aspen SP1, an exceptional thermal, protease and detergent-resistant self-assembled nano-particle.

Stable protein 1 (SP1) is a homo-oligomeric protein isolated from aspen (Populus tremula aspen) plants which forms a ring-shape dodecameric particle with a central cavity. The oligomeric form of SP1 is an exceptionally stable structure that is resistant to proteases (e.g., trypsin, V8, and proteinase K), high temperatures, organic solvents, and high levels of ionic detergent. Analytical ultra-centrifugation, chemical cross-linking, matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and transmission electron microscopy were used to further characterize the SP1 dodecamer. Introduction of a single cysteine at the N-terminus of SP1 enabled the formation of disulfide bridges within the SP1 dodecamer, concurrent with increased melting point. A six-histidine tag was introduced at the N-terminus of SP1 to generate 6HSP1, and the DeltaNSP1 mutant was generated by a deletion of amino acids 2-6 at the N-terminus. Both 6HSP1 and DeltaNSP1 maintained their ability to assemble a stable dodecamer. Remarkably, these SP1 homo-dodecamers were able to re-assemble into stable hetero-dodecamers following co-electro-elution from SDS-PAGE. The exceptional stability of the SP1-nano ring and its ability to self-assemble hetero-complexes paves the way to further research in utilizing this unique protein in nano-biotechnology.

The structural basis of the thermostability of SP1, a novel plant (Populus tremula) boiling stable protein.

We previously reported on a new boiling stable protein isolated from aspen plants (Populus tremula), which we named SP1. SP1 is a stress-related protein with no significant sequence homology to other stress-related proteins. It is a 108-amino-acid hydrophilic polypeptide with a molecular mass of 12.4 kDa (Wang, W. X., Pelah, D., Alergand, T., Shoseyov, O., and Altman, A. (2002) Plant Physiol. 130, 865-875) and is found in an oligomeric form. Preliminary electron microscopy studies and matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that SP1 is a dodecamer composed of two stacking hexamers. We performed a SDS-PAGE analysis, a differential scanning calorimetric study, and crystal structure determination to further characterize SP1. SDS-PAGE indicated a spontaneous assembly of SP1 to one stable oligomeric form, a dodecamer. Differential scanning calorimetric showed that SP1 has high thermostability i.e. Tm of 107 degrees C (at pH 7.8). The crystal structure of SP1 was initially determined to 2.4 A resolution by multi-wavelength anomalous dispersion method from a crystal belonging to the space group I422. The phases were extended to 1.8 A resolution using data from a different crystal form (P21). The final refined molecule includes 106 of the 108 residues and 132 water molecules (on average for each chain). The R-free is 20.1%. The crystal structure indicated that the SP1 molecule has a ferredoxin-like fold. Strong interactions between each two molecules create a stable dimer. Six dimers associate to form a ring-like-shaped dodecamer strongly resembling the particle visualized in the electron microscopy studies. No structural similarity was found between the crystal structure of SP1 and the crystal structure of other stress-related proteins such as small heat shock proteins, whose structure has been already determined. This structural study further supports our previous report that SP1 may represent a new family of stress-related proteins with high thermostability and oligomerization.

Crystallization and preliminary X-ray crystallographic analysis of SP1, a novel chaperone-like protein.

SP1 (108 amino acids) is a boiling-stable stress-responsive protein. It has no significant sequence homology to other stress-related proteins or to small heat-shock proteins (sHsps). SP1 activity is ATP-independent, similar to other small heat-shock proteins. Based on these features, it is expected that the structure-function relationship of SP1 will be unique. In this work, the crystallization and preliminary crystallographic data of native SP1 and its selenomethionine derivative are described. Recombinant SP1 and its selenomethionine derivative were expressed in Escherichia coli and used for crystallization experiments. SP1 crystals were grown from 0.1 M HEPES pH 7.5, 20% PEG 3K, 0.2 M NaCl. One to four single crystals appeared in each droplet within a few Days and grew to dimensions of about 0.5 x 0.5 x 0.8 mm after about two weeks. Diffraction studies of these crystals at low temperature indicated that they belong to space group I422, with unit-cell parameters a = 89, b = 89, c = 187 A. Efforts to crystallize the selenomethionine derivative of SP1 are in progress.