Large scale production of synthetic spider silk proteins in Escherichia coli.

Spider silk, which has remarkable mechanical properties, is a natural protein fiber produced by spiders. Spiders cannot be farmed because of their cannibalistic and territorial nature. Hence, large amounts of spider silk cannot be produced from spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk. Our group has produced synthetic spider silk proteins in E. coli to study structure/function and to produce biomaterials comparable to the silks produced by orb-weaving spiders. Here we give a detailed description of our cloning, expression, and purification methods of synthetic spider silk proteins ranging from ~30 to ~200 kDa. We have cloned the relevant genes of the spider Nephila clavipes and introduced them into bacteria to produce synthetic spider silk proteins using small and large-scale bioreactors. We have optimized the fermentation process, and we have developed protein purification methods as well. The purified proteins are spun into fibers and are used to make alternative materials like films and adhesives with various possible commercial applications.

Functional comparison of Deinococcus radiodurans Dps proteins suggests distinct in vivo roles.

Deinococcus radiodurans exhibits extreme resistance to DNA damage and is one of only few bacteria that encode two Dps (DNA protection during starvation) proteins. Dps-1 was shown previously to bind DNA with high affinity and to localize to the D. radiodurans nucleoid. A unique feature of Dps-2 is its predicted signal peptide. In the present paper, we report that Dps-2 assembly into a dodecamer requires the C-terminal extension and, whereas Dps-2 binds DNA with low affinity, it protects against degradation by reactive oxygen species. Consistent with a role for Dps-2 in oxidative stress responses, the Dps-2 promoter is up-regulated by oxidative stress, whereas the Dps-1 promoter is not. Although DAPI (4',6-diamidino-2-phenylindole) staining of Escherichia coli nucleoids shows that Dps-1 can compact genomic DNA, such nucleoid condensation is absent from cells expressing Dps-2. A fusion of EGFP (enhanced green fluorescent protein) to the Dps-2 signal peptide results in green fluorescence at the perimeter of D. radiodurans cells. The differential response of the Dps-1 and Dps-2 promoters to oxidative stress, the distinct cellular localization of the proteins and the differential ability of Dps-1 and Dps-2 to attenuate hydroxyl radical production suggest distinct functional roles; whereas Dps-1 may function in DNA metabolism, Dps-2 may protect against exogenously derived reactive oxygen species.

On the stoichiometry of Deinococcus radiodurans Dps-1 binding to duplex DNA.

DNA protection during starvation (Dps) proteins, dodecameric assemblies of four-helix bundle subunits, contribute to protection against reactive oxygen species. Deinococcus radiodurans, which is characterized by resistance to DNA damaging agents, encodes two Dps homologs, of which Dps-1 binds DNA with high affinity. DNA binding requires N-terminal extensions preceding the four-helix bundle core. Composed of six Dps-1 dimers, each capable of DNA binding by N-terminal extensions interacting in consecutive DNA major grooves, dodecameric Dps-1 would be predicted to feature six DNA binding sites. Using electrophoretic mobility shift assays and intrinsic tryptophan fluorescence, we show that dodecameric Dps-1 binds 22-bp DNA with a stoichiometry of 1:6, consistent with the existence of six DNA binding sites. The stoichiometry of Dps-1 binding to 26-bp DNA is 1:4, suggesting that two Dps-1 dodecamers can simultaneously occupy opposite faces of this DNA. Mutagenesis of an arginine (Arg132) on the surface of Dps-1 leads to a reduction in DNA binding. Altogether, our data suggest that duplex DNA lies along the dimer interface, interacting with Arg132 and the N-terminal α-helices, and they extend the hexagonal packing model for Dps-DNA assemblies by specifying the basis for occupancy of available DNA binding sites.

Regulation of adipogenesis by natural and synthetic REV-ERB ligands.

The nuclear hormone receptor, REV-ERB, plays an essential role in adipogenesis. Rev-erbalpha expression is induced in 3T3-L1 cells during adipogenesis, and overexpression of this receptor leads to expression of adipogenic genes. We recently demonstrated that the porphyrin heme functions as a ligand for REV-ERB, and binding of heme is required for the receptor's activity. We therefore hypothesized that REV-ERB ligands may play a role in regulation of adipogenesis. We detected an increase intracellular heme levels during 3T3-L1 adipogenesis that correlated with induction of aminolevulinic acid synthase 1 (Alas1) expression, the rate-limiting enzyme in heme biosynthesis. If the increase in Alas1 expression was blocked, adipogenesis was severely attenuated, indicating that induction of expression of Alas1 and the increase in heme synthesis is critical for differentiation. Inhibition of heme synthesis during adipogenesis leads to decreased recruitment of nuclear receptor corepressor to the promoter of a REV-ERB target gene, suggesting alteration of REV-ERB activity. Treatment of 3T3-L1 cells with a synthetic REV-ERB ligand, SR6452, resulted in induction of adipocyte differentiation to a similar extent as treatment with the peroxisomal proliferator-activated receptor-gamma agonist, rosiglitazone. Combination of SR6452 and rosiglitazone had an additive effect on stimulation of adipocyte differentiation. These results suggest that heme, functioning as a REV-ERB ligand, is an important signaling molecule for induction of adipogenesis. Moreover, synthetic small molecule ligands for REV-ERB are effective modulators of adipogenesis and may be useful for treatment of metabolic diseases.

The C-terminal domain of HU-related histone-like protein Hlp from Mycobacterium smegmatis mediates DNA end-joining.

Histone-like proteins (such as HU, H-NS, and Fis) participate in nucleoid organization and in DNA replication, recombination, and transcription. Cold shock and anoxia upregulates a homologue of HU (Hlp) in Mycobacterium smegmatis, the nonpathogenic model of Mycobacterium tuberculosis. We show using electrophoretic mobility shift assays that Hlp, which in addition to the HU fold has a basic C-terminal tail containing multiple PAKK and PAAK repeats, has very high affinity for DNA. The affinity of Hlp for 76 bp linear DNA is higher, K d = 0.037 +/- 0.001 nM, compared to an Hlp variant without the C-terminal repeats, K d = 2.5 +/- 0.1 nM and the isolated C-terminal repeat domain, K d = 0.8 +/- 0.2 nM, where K d in all cases reflects an aggregate affinity for the DNA probes, not the affinity for binding to a single site. Hlp lacking the entire C-terminal domain binds DNA only poorly. These data indicate that both Hlp domains contribute to high-affinity DNA binding. Hlp promotes DNA end-joining in the presence of T4 DNA ligase, and this property is mediated by the C-terminal repeats. At <100 nM concentration, Hlp represses transcription by T7 RNA polymerase in vitro whereas the individual N- and C-terminal domains do not, even when present together. Notably, while DNA end-joining can be achieved by the isolated C-terminal domain, transcriptional repression requires for both domains to be present on a single polypeptide. Given the low cellular concentration of Hlp, our data suggest that its primary functional role may be in DNA-dependent responses to environmental stress rather than in nucleoid organization.

The N-terminal extensions of Deinococcus radiodurans Dps-1 mediate DNA major groove interactions as well as assembly of the dodecamer.

Dps (DNA protection during starvation) proteins play an important role in the protection of prokaryotic macromolecules from damage by reactive oxygen species. Previous studies have suggested that the lysine-rich N-terminal tail of Dps proteins participates in DNA binding. In comparison with other Dps proteins, Dps-1 from Deinococcus radiodurans has an extended N terminus comprising 55 amino acids preceding the first helix of the 4-helix bundle monomer. In the crystal structure of Dps-1, the first approximately 30 N-terminal residues are invisible, and the remaining 25 residues form a loop that harbors a novel metal-binding site. We show here that deletion of the flexible N-terminal tail obliterates DNA/Dps-1 interaction. Surprisingly, deletion of the entire N terminus also abolishes dodecameric assembly of the protein. Retention of the N-terminal metal site is necessary for formation of the dodecamer, and metal binding at this site facilitates oligomerization of the protein. Electrophoretic mobility shift assays using DNA modified with specific major/minor groove reagents further show that Dps-1 interacts through the DNA major groove. DNA cyclization assays suggest that dodecameric Dps-1 does not wrap DNA about itself. A significant decrease in DNA binding affinity accompanies a reduction in duplex length from 22 to 18 bp, but only for dodecameric Dps-1. Our data further suggest that high affinity DNA binding depends on occupancy of the N-terminal metal site. Taken together, the mode of DNA interaction by dodecameric Dps-1 suggests interaction of two metal-anchored N-terminal tails in successive DNA major grooves, leading to DNA compaction by formation of stacked protein-DNA layers.

Crystal structure of Dps-1, a functionally distinct Dps protein from Deinococcus radiodurans.

DNA protection during starvation (Dps) proteins play an important role in protecting cellular macromolecules from damage by reactive oxygen species (ROS). Unlike most orthologs that protect DNA by a combination of DNA binding and prevention of hydroxyl radical formation by ferroxidation and sequestration of iron, Dps-1 from the radiation-resistant Deinococcus radiodurans fails to protect DNA from hydroxyl radical-mediated cleavage through a mechanism inferred to involve continuous release of iron from the protein core. To address the structural basis for this unusual release of Fe(2+), the crystal structure of D. radiodurans Dps-1 was determined to 2.0 Angstroms resolution. Two of four strong anomalous signals per protein subunit correspond to metal-binding sites within an iron-uptake channel and a ferroxidase site, common features related to the canonical functions of Dps homologs. Similar to Lactobacillus lactis Dps, a metal-binding site is found at the N-terminal region. Unlike other metal sites, this site is located at the base of an N-terminal coil on the outer surface of the dodecameric protein sphere and does not involve symmetric association of protein subunits. Intriguingly, a unique channel-like structure is seen featuring a fourth metal coordination site that results from 3-fold symmetrical association of protein subunits through alpha2 helices. The presence of this metal-binding site suggests that it may define an iron-exit channel responsible for the continuous release of iron from the protein core. This interpretation is supported by substitution of residues involved in this ion coordination and the observation that the resultant mutant protein exhibits significantly attenuated iron release. Therefore, we propose that D. radiodurans Dps-1 has a distinct iron-exit channel.

Efficient gene targeting in Drosophila with zinc-finger nucleases.

This report describes high-frequency germline gene targeting at two genomic loci in Drosophila melanogaster, y and ry. In the best case, nearly all induced parents produced mutant progeny; 25% of their offspring were new mutants and most of these were targeted gene replacements resulting from homologous recombination (HR) with a marked donor DNA. The procedure that generates these high frequencies relies on cleavage of the target by designed zinc-finger nucleases (ZFNs) and production of a linear donor in situ. Increased induction of ZFN expression led to higher frequencies of gene targeting, demonstrating the beneficial effect of activating the target. In the absence of a homologous donor DNA, ZFN cleavage led to the recovery of new mutants at three loci-y, ry and bw-through nonhomologous end joining (NHEJ) after cleavage. Because zinc fingers can be directed to a broad range of DNA sequences and targeting is very efficient, this approach promises to allow genetic manipulation of many different genes, even in cases where the mutant phenotype cannot be predicted.