The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

Background Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.This work was mainly supported by ERA Biotech (www.erabiotech.com). Additional support was supplied by grant SGR 2009/703 funded by the Generalitat de Catalunya (www10.gencat.net) and grants CDS2007/00036 of Consolider Ingenio program and TRA 2009/0124 of TRACE program funded by Ministerio de Ciencia e Inovación (MICINN, www.micinn.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Actin-based spring in horseshoe crab sperm

The conversion of chemical energy into mechanical forces that powers cell movements is a ubiquitous theme across biology. Besides molecular motors such as kinesin-microtubule and actin-myosin complexes, biological springs and ratchets can also store and release energy to rectify motion. The acrosome reaction of horseshoe crab sperm is a simple example of a biological spring where a 60μm-long crystalline bundle of actin filaments, tightly cross-linked by actin bundling protein scruin, straightens from a coiled conformation and extends from the cell to penetrate an egg in about five seconds. To identify the basis and mechanism for this movement, we examine the possible sources of chemical and mechanical energy and show that the stored elastic energy alone is sufficient to drive the reaction. We also provide an estimate of the maximum force generated during the uncoiling by stalling the bundle using an agarose gel to show the reaction produces enough force to penetrate the egg

Dual targeting of xylanase to chloroplasts and peroxisomes as a means to increase protein accumulation in plant cells

One of the limiting factors in the production of recombinant proteins in transgenic plants is the low level of protein accumulation. A strategy was investigated for a high level of protein accumulation in plant cells. A fungal xylanase encoded by XYLII of Trichoderma reesei was chosen as the model protein because xylanases have a high potential for applications in environment-related technologies. Xylanase was expressed in the cytosol or targeted either to chloroplasts or peroxisomes alone, or to both organelles simultaneously. When xylanase was targeted to both chloroplasts and peroxisomes simultaneously the amount of xylanase accumulated was 160% of that in chloroplasts alone and 240% of that in peroxisomes alone although the transcript levels were similar among these constructs. The growth stage of the transgenic plants also affected the total amount of xylanase; the highest level of accumulation occurred at the time of flowering. This study provides genetic and biochemical data demonstrating that a high level of protein accumulation in transgenic plants can be obtained by targeting a protein to both chloroplasts and peroxisomes at the same time.X114870sciescopu

New Random Copolymer Acceptors Enable Additive-Free Processing of 10.1% Efficient All-Polymer Solar Cells with Near-Unity Internal Quantum Efficiency

Finding effective molecular design strategies to optimize the active layer blend morphology is among the long-standing challenges in developing efficient all-polymer solar cells (all-PSCs). Here we show that new biselenophene/selenophene-linked naphthalene diimide random copolymer acceptors BSSx (x = 10, 20, 50) facilitate the achievement of high-performance all-PSCs without the use of any solution processing additive. Blends of BSS10 with donor polymer PBDB-T combined 10.1% power conversion efficiency with 97% internal quantum efficiency and 0.59 eV optical band gap energy loss (Eloss). BSS10- and BSS20-based devices have the best combination of high external quantum efficiency (>85%) and small Eloss (<0.6 eV) among all-PSCs yet reported. The results demonstrate that the blend morphology, charge carrier mobilities, and photovoltaic properties of all-PSCs could be rationally optimized by means of a synthetic variablethe random copolymer composition