Producing Human Therapeutic Proteins in Plastids

Plastid transformation technology is set to become a major player in the production of human therapeutic proteins. Protein expression levels that can be achieved in plant plastids are hundreds of times greater than the expression levels generally obtained via nuclear transformation. Plastids can produce human proteins that are properly folded and are biologically active. Effective protein purification strategies and strategies that can achieve inducible plastid gene expression are being developed within the system. Plastid transformation technology has been extended to edible plant species, which could minimize down-stream processing costs and raises the possibility of “edible protein therapies”. The system is limited by the fact that plastid-produced proteins are not glycosylated and that, at the moment, it can be difficult to predict protein stability within the plastid. The high level of protein expression that can be obtained in plastids could make it possible to produce high-value therapeutic proteins in plants on a scale that could be accommodated in contained glasshouse facilities and still be economically viable. Growing plastid-transformed plants under contained conditions, and coupled with the level of bio-safety conferred by maternal inheritance of plastid transgenes, would address many of the social and environmental concerns relating to plant based production of human therapeutic proteins

Gene Expression in Gut Symbiotic Organ of Stinkbug Affected by Extracellular Bacterial Symbiont

<div><p>The bean bug <i>Riptortus pedestris</i> possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus <i>Burkholderia</i>. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from <i>Burkholderia</i>-infected and uninfected <i>R. pedestris</i>. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations.</p></div

Synthesis and Biological Activity of Tetrameric Ribitol Phosphate Fragments of Staphylococcus aureus Wall Teichoic Acid

A systematically designed and synthesized ribitol phosphate (RboP) oligomer using a series of building blocks, which make up the wall teichoic acid (WTA) of S. aureus, is presented. Based on the use of a solution-phase phosphodiester synthesis, a library of ribitol phosphate tetramers, decorated with d-alanine and <i>N</i>-acetylglucosamine (GlcNAc), were generated. The synthesized RboP tetramers showed increased cytokine levels in mice in a subcutaneous air pouch model

WTA immunization reduces MW2 CA-MRSA infection in WT mice while no difference is seen in the absence of MBL.

<p>(2A) Abscess formation. Mice immunized with PBS control or WTA were infected 20 days after the last immunization. Abscess formation was examined on day 10 following systemic infection with MW2 CA-MRSA as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069739#s2" target="_blank">Materials and Methods</a>. Abscess formation is expressed as numbers of mice with abscess and total mice in each group. * indicates p<0.0001 against all other groups (Likelihood Ratio). (2B) Bacterial load in the kidney. Bacterial titers were measured in homogenates of two combined kidneys and are expressed as cfu/g of kidneys in a box plot. Numbers of mice used are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069739#pone-0069739-g002" target="_blank">figure 2A</a>. * and ** indicates p<0.05 and p<0.001, respectively compared to WT immunized with PBS control (Nonparametric comparisons for each pair by Wilcoxon methods).</p

Abscess formation in systemic MRSA infection.

*<p>indicates p<0.0001 (Likelihood ratio) compared to WT in MW2 CA-MRSA infection.</p

Serum from WTA-immunized mice inhibits bacterial growth in whole blood assays <i>ex vivo</i>.

<p>Two MRSA strains, COL HA-MRSA (4A and 4B) and MW2 CA-MRSA, (4C and 4D) were used. Bacteria were incubated with whole blood from MBL KO (4A and 4C) or WT mice (4B and 4D) with serum from MBL KO mice immunized with either PBS control or WTA, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069739#s2" target="_blank">Materials and Methods</a>. Bacterial titers are expressed as mean <b>±</b> SD in each group of four samples. Each sample was measured in duplicate and the average measurement was used for statistical analysis. Representative results from two repeated experiments are shown. *, **, and *** indicate p<0.05, 0.001, and 0.0001 (Student's t-test), respectively.</p

Multiple sequence alignment.

<p>Sequence homologues of Lpg0393 were identified by HHpred, and its non-redundant close relatives were aligned to a set of 10 distant Vps9 domains in the PF02204 of the Pfam database. Jalview was used for rendering. The secondary structure of Rabex-5 (PDB entry: 2OT3) and the predicted secondary structure of Lpg0393 are shown at the bottom of the alignment. Two key residues for the GEF activity of Rabex-5 and AtVps9a are coloured white on red. The aligned sequences are represented by a name of species. <i>Legionella shakespearei</i> (<i>Ls</i>; UPI000377E818), <i>Legionella longbeachae</i> (<i>Ll</i>; D3HLE8), <i>Legionella drancourtii</i> LLAP 12 (<i>Ld</i>; G9ET11), <i>Legionella tunisiensis</i> (<i>Lt</i>; UPI0002ECA82A), <i>Fluoribacter dumoffii</i> (<i>Fd</i>; UPI00026C7A1A), <i>Albugo laibachii</i> Nc14 (<i>Al</i>; F0WZN8), <i>Danio rerio</i> (<i>Dr</i>; F1QIZ1), <i>Entamoeba dispar</i> (<i>Ed</i>; entry code = B0EDB3), <i>Entamoeba histolytica</i> (<i>Eh</i>; C4LYL6), <i>Micromonas</i> sp. (<i>Ms</i>; C1E5E9), <i>Nematostella vectensis</i> (<i>Nv</i>; A7SBH6), and <i>Trichomonas vaginalis</i> (<i>Tv</i>; A2DH58).</p

Lpg0393 has a Vps9-like domain.

<p>(A) Structural comparison with the catalytic core of Rabex-5. Four residues important for the GEF activity of Rabex-5 and corresponding Lpg0393 residues are shown in sticks (<i>left</i>) and highlighted (<i>right</i>). Asp313 and Tyr354 are identically conserved in Lpg0393, while Pro317 and Glu351 are substituted by unrelated amino acids. (B) Topological comparison with the catalytic core of Rabex-5. The domain organization is reversed, and the topology of the helical bundle domain is reversed in the two proteins. (C) Structure-based sequence alignment. The N-terminal domain of Lpg0393 and the Vps9 domains of Rabex-5 and <i>Arabidopsis</i> Vps9a are aligned. The secondary structural elements of Lpg0393 and Rabex-5 are shown at the top and bottom of the alignment, respectively. The four highlighted residues in <i>A</i> are indicated by arrows.</p

Lpg0393 weakly localizes to the Golgi.

<p>Confocal images of the two indicated cells are shown that transiently expressed YFP-tagged full-length Lpg0393 and CFP-tagged β-1,4-galactosyltransferase (a Golgi marker), Rab5b(Q79L) or Rab5b(S34N). The scale bars indicate 10 μm. (A) Lpg0393 is dispersed throughout the cytosol with noticeable enrichment on the Golgi. (B) Lpg0393 colocalizes closely with Rab5b(S34N). (C) Lpg0393 colocalizes partially with Rab5b(Q79L), by with a similar overall pattern as observed in <i>A</i> and <i>B</i>. The right panels in <i>A</i>-<i>C</i> show fluorescence intensities from sections indicated by the red arrows. Discrepancies in the fluorescence intensity are notable in C.</p

Overall structure of Lpg0393Δ17.

<p>(A) Ribbon drawings in two orientations. The dotted line indicates a disordered segment in the crystal structure. (B) Two Lpg0393Δ17 molecules (Chain A and Chain B) forming a crystallographic dimer. The experimental SAD map together with the final refined model is shown for a region that contains a methionine residue. (C) Gel filtration analysis. Lpg0393Δ17 (5 mg/ml) was eluted from a HiLoad 26/60 Superdex 75 column at a rate of 1.5 ml/min with 30 mM TrisHCl buffer (pH 8.0) containing 100 mM NaCl and 3 mM dithiothreitol. The size marker proteins were bovine serum albumin (67 kDa), ovalbumin (43 kDa), yellow fluorescent protein (27 kDa) and chymotrypsinogen A (25 kDa).</p