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Most antibodies display very low brain exposure due to the blood-brain barrier (BBB) preventing their entry into brain parenchyma. Transferrin receptor (TfR) has been used previously to ferry antibodies to the brain by using different formats of bispecific constructs. Tetravalent bispecific tandem immunoglobulin Gs (IgGs) (TBTIs) containing two paratopes for both TfR and protofibrillar forms of amyloid-beta (Aβ) peptide were constructed and shown to display higher brain penetration than the parent anti-Aβ antibody. Additional structure-based mutations on the TfR paratopes further increased brain exposure, with maximal enhancement up to 13-fold in wild-type mice and an additional 4-5-fold in transgenic (Tg) mice harboring amyloid plaques, the main target of our amyloid antibody. Parenchymal target engagement of extracellular amyloid plaques was demonstrated using in vivo and ex vivo fluorescence imaging as well as histological methods. The best candidates were selected for a chronic study in an amyloid precursor protein (APP) Tg mouse model showing efficacy at reducing brain amyloid load at a lower dose than the corresponding monospecific antibody. TBTIs represent a promising format for enhancing IgG brain penetration using a symmetrical construct and keeping bivalency of the payload antibody., (© 2020 Sanofi.)

Background: Thapsigargin and nortrilobolide are sesquiterpene lactones found in the Mediterranean plant Thapsia garganica L. Thapsigargin is a potent inhibitor of the sarco/endoplasmic reticulum calcium ATPase pump, inducing apoptosis in mammalian cells. This mechanism has been used to develop a thapsigargin-based cancer drug first by GenSpera and later Inspyr Therapeutics (Westlake Village, California). However, a stable production of thapsigargin is not established., Results: In vitro regeneration from leaf explants, shoot multiplication and rooting of T. garganica was obtained along with the production of thapsigargins in temporary immersion bioreactors (TIBs). Thapsigargin production was enhanced using reduced nutrient supply in combination with methyl jasmonate elicitation treatments. Shoots grown in vitro were able to produce 0.34% and 2.1% dry weight of thapsigargin and nortrilobolide, respectively, while leaves and stems of wild T. garganica plants contain only between 0.1 and 0.5% of thapsigargin and below detectable levels of nortrilobolide. In addition, a real-time reverse transcription PCR (qRT-PCR) study was performed to study the regulatory role of the biosynthetic genes HMG-CoA reductase ( HMGR ), farnesyl diphosphate synthase ( FPPS ), epikunzeaol synthase ( TgTPS2 ) and the cytochrome P450 ( TgCYP76AE2 ) of stem, leaf and callus tissues. Nadi staining showed that the thapsigargins are located in secretory ducts within these tissues., Conclusions: Shoot regeneration, rooting and biomass growth from leaf explants of T. garganica were achieved, together with a high yield in vitro production of thapsigargin in TIBs.

Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant. Co-expression of the CpSEs with four different triterpene cyclases, either transiently in Nicotiana benthamiana or constitutively in yeast, showed that CpSEs boost triterpene production. CpSE2 was the best performing in this regard, which could reflect either increased substrate production or superior channeling of the substrate to the triterpene cyclases. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis with C. pepo cucurbitadienol synthase (CpCPQ) revealed a specific interaction with CpSE2 but not with the other CpSEs. When CpSE2 was transformed into C. pepo hairy root lines, cucurbitacin E production was increased two folds compared to empty vector control lines. This study provides new insight into the importance of SEs in triterpene biosynthesis, suggesting that they may facilitate substrate channeling, and demonstrates that SE overexpression is a new tool for increasing triterpene production in plants and yeast., (Copyright © 2018. Published by Elsevier Inc.)

Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane., Competing Interests: The authors declare no conflict of interest.

TriForC is an innovative EU-funded collaborative project that has established an integrative pipeline for the exploitation of plant triterpenes for commercialization in agriculture and pharmacology. We discuss the main outcomes of TriForC and reflect on its potential long-term impact and on the importance of EU projects for science, industry, and society., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Plastid transformation has emerged as an alternative platform to generate transgenic plants. Attractive features of this technology include specific integration of transgenes-either individually or as operons-into the plastid genome through homologous recombination, the potential for high-level protein expression, and transgene containment because of the maternal inheritance of plastids. Several issues associated with nuclear transformation such as gene silencing, variable gene expression due to the Mendelian laws of inheritance, and epigenetic regulation have not been observed in the plastid genome. Plastid transformation has been successfully used for the production of therapeutics, vaccines, antigens, and commercial enzymes, and for engineering various agronomic traits including resistance to biotic and abiotic stresses. However, these demonstrations have usually focused on model systems such as tobacco, and the technology per se has not yet reached the market. Technical factors limiting this technology include the lack of efficient protocols for the transformation of cereals, poor transgene expression in non-green plastids, a limited number of selection markers, and the lengthy procedures required to recover fully segregated plants. This article discusses the technology of transforming the plastid genome, the positive and negative features compared with nuclear transformation, and the current challenges that need to be addressed for successful commercialization., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A key step in the repair of photoinactivated oxygen-evolving photosystem II (PSII) complexes is the selective recognition and degradation of the damaged PSII subunit, usually the D1 reaction center subunit. FtsH proteases play a major role in D1 degradation in both cyanobacteria and chloroplasts. In the case of the cyanobacterium Synechocystis sp. PCC 6803, analysis of an N-terminal truncation mutant of D1 lacking 20 amino-acid residues has provided evidence that FtsH complexes can remove damaged D1 in a processive reaction initiated at the exposed N-terminal tail. To test the importance of the N-terminal D1 tail in higher plants, we have constructed the equivalent truncation mutant in tobacco using chloroplast transformation techniques. The resulting mutant grew poorly and only accumulated about 25% of wild-type levels of PSII in young leaves which declined as the leaves grew so that there was little PSII activity in mature leaves. Truncating D1 led to the loss of PSII supercomplexes and dimeric complexes in the membrane. Extensive and rapid non-photochemical quenching (NPQ) was still induced in the mutant, supporting the conclusion that PSII complexes are not required for NPQ. Analysis of leaves exposed to high light indicated that PSII repair in the truncation mutant was impaired at the level of synthesis and/or assembly of PSII but that D1 could still be degraded. These data support the idea that tobacco plants possess a number of back-up and compensatory pathways for removal of damaged D1 upon severe light stress.

Despite the largely maternal inheritance of plastid genomes, the risk of transgene dissemination from transplastomic plants can limit the scope for field cultivation. There is a need for a cost-effective, scalable process to grow large quantities of transplastomic plant biomass for biosynthesis of biopharmaceuticals and other high-value heterologous proteins. Temporary immersion culture is a means of achieving this under fully contained conditions. This method describes the organogenesis of transplastomic Nicotiana tabacum callus in RITA(®) temporary immersion bioreactors to produce rootless leafy biomass, and subsequent total soluble protein extraction, SDS-PAGE, and Western immunoblot analysis of heterologous protein expression. This method can be used for propagation of plastid or nuclear transformants, though is especially suitable for transplastomic biomass, as organogenesis leads to greater expression and accumulation of transplastomic proteins due to increases in chloroplast number and size.

Members of the Psb28 family of proteins are accessory factors implicated in the assembly and repair of the photosystem II complex. We present here the crystal structure of the Psb28 protein (Tlr0493) found in the thermophilic cyanobacterium Thermosynechococcus elongatus at a resolution of 2.3 Å. Overall the crystal structure of the Psb28 monomer is similar to the solution structures of C-terminally His-tagged Psb28-1 from Synechocystis sp. PCC 6803 obtained previously by nuclear magnetic resonance spectroscopy. One new aspect is that Escherichia coli-expressed T. elongatus Psb28 is able to form dimers in solution and packs as a dimer of dimers in the crystal. Analysis of wild type and mutant strains of Synechocystis 6803 by blue native-polyacrylamide gel electrophoresis suggests that Psb28-1, the closest homologue to T. elongatus Psb28 in this organism, also exists as an oligomer in vivo, most likely a dimer. In line with the prediction based on the crystal structure of T. elongatus Psb28, the addition of a 3× Flag-tag to the C-terminus of Synechocystis 6803 Psb28-1 interferes with the accumulation of the Psb28-1 oligomer in vivo. In contrast, the more distantly related Psb28-2 protein found in Synechocystis 6803 lacks the residues that stabilize dimer formation in the T. elongatus Psb28 crystal and is detected as a monomer in vivo. Overall our data suggest that the dimer interface in the Psb28 crystal might be physiologically relevant.

Chloroplast transformation technology is a promising approach for the production of foreign proteins in plants with expression levels of up to 70 % of total soluble protein (TSP) achieved in tobacco. However, expression of foreign protein in the chloroplast can lead to drastic or even lethal effects in transplastomic plants grown in soil, thereby potentially limiting the applicability of this technology. For instance, previous attempts to express the outer surface protein A (OspA) from Borrelia burgdorferi in tobacco chloroplasts led to plant death when expressed at 10 % TSP. We show here that this earlier transplastomic line, as well as a new plant line, OspA:YFP, expressing OspA fused to the yellow fluorescent protein, can be propagated in temporary immersion bioreactors (TIBs) using AlkaBurst™ technology to produce leafy biomass that expressed OspA at levels of up to 7.6 % TSP, to give a maximum yield of OspA of about 108 mg/L. Our results show that TIBs provide an alternative method for the production of transplastomic biomass expressing proteins toxic for plants and is a particularly useful approach when 'absolute' containment is required.

Chloroplast transformation offers an exciting platform for the safe, inexpensive and large-scale production of recombinant proteins in plants. An important advantage for the isolation of proteins produced in the chloroplast would be the use of affinity tags for rapid purification by affinity chromatography. To date, only His-tags have been used. In this study, we have tested the feasibility of expressing two additional affinity tags: glutathione-S-transferase (GST) and a His-tagged derivative of the maltose-binding protein (His₆-MBP). By using the chloroplast 16S rRNA promoter and 5' untranslated region of phage T7 gene 10, GST and His₆-MBP were expressed in homoplastomic tobacco plants at approximately 7% and 37% of total soluble protein, respectively. GST could be purified by one-step-affinity purification using a glutathione column. Much better recoveries were obtained for His₆-MBP by using a twin-affinity purification procedure involving first immobilised nickel followed by binding to amylose. Interestingly, expression of GST led to cytoplasmic male sterility. Overall, our work expands the tools available for purifying recombinant proteins from the chloroplast.

The biogenesis and oxygen-evolving activity of cyanobacterial Photosystem II (PSII) is dependent on a number of accessory proteins not found in the crystallised dimeric complex. These include Psb27, a small lipoprotein attached to the lumenal side of PSII, which has been assigned a role in regulating the assembly of the Mn(4)Ca cluster catalysing water oxidation. To gain a better understanding of Psb27, we have determined in this study the crystal structure of the soluble domain of Psb27 from Thermosynechococcus elongatus to a resolution of 1.6 Å. The structure is a four-helix bundle, similar to the recently published solution structures of Psb27 from Synechocystis PCC 6803 obtained by nuclear magnetic resonance (NMR) spectroscopy. Importantly, the crystal structure presented here helps us resolve the differences between the NMR-derived structural models. Potential binding sites for Psb27 within PSII are discussed in light of recent biochemical data in the literature.

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5'UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.

Chloroplast transformation is a promising approach for the commercial production of recombinant proteins in plants. However, gene containment still remains an issue for the large-scale cultivation of transplastomic plants in the field. Here, we have evaluated the potential of using tobacco transplastomic cell suspensions for the fully contained production of a modified form of the green fluorescent protein (GFP+) and, a vaccine antigen, fragment C of tetanus toxin (TetC). Expression of these proteins in cell suspension cultures (and calli) was much less than in leaves, reaching 0.5%-1.5% of total soluble protein (TSP), but still produced 2.4-7.2 mg/L of liquid culture. Much better expression levels were achieved with a novel protein production platform in which transgenic cell suspension cultures were placed in a temporary immersion bioreactor in the presence of Thidiazuron to initiate shoot formation. GFP+ yield reached 660 mg/L of bioreactor (33% TSP), and TetC accumulated to about 95 mg/L (8% TSP). This new production platform, combining the rapid generation of transplastomic cell suspension cultures and the use of temporary immersion bioreactors, is a promising route for the fully contained low-cost production of recombinant proteins in chloroplasts., (© 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

We present here the crystal structure of CyanoP (Tlr2075) from Thermosynechococcus elongatus at 2.8 A. CyanoP is a substoichiometric component of the isolated cyanobacterial Photosystem II (PSII) complex, distantly related to the PsbP extrinsic subunit of the oxygen-evolving PSII complex in higher plants and green algae. Despite the relatively low degree of sequence similarity, we have found that CyanoP adopts the same beta-sandwich fold as higher-plant PsbP and contains a well-conserved metal (zinc)-binding site that is also present in plant PsbP. Our results support the idea that CyanoP represents the basal structural fold of the PsbP superfamily.

Background: Photosystem II (PSII) is the light-driven water:plastoquinone oxidoreductase of oxygenic photosynthesis and is found in the thylakoid membrane of chloroplasts and cyanobacteria. Considerable attention is focused on how PSII is assembled in vivo and how it is repaired following irreversible damage by visible light (so-called photoinhibition). Understanding these processes might lead to the development of plants with improved growth characteristics especially under conditions of abiotic stress., Scope: Here we summarize recent results on the assembly and repair of PSII in cyanobacteria, which are excellent model organisms to study higher plant photosynthesis., Conclusions: Assembly of PSII is highly co-ordinated and proceeds through a number of distinct assembly intermediates. Associated with these assembly complexes are proteins that are not found in the final functional PSII complex. Structural information and possible functions are beginning to emerge for several of these 'assembly' factors, notably Ycf48/Hcf136, Psb27 and Psb28. A number of other auxiliary proteins have been identified that appear to have evolved since the divergence of chloroplasts and cyanobacteria. The repair of PSII involves partial disassembly of the damaged complex, the selective replacement of the damaged sub-unit (predominantly the D1 sub-unit) by a newly synthesized copy, and reassembly. It is likely that chlorophyll released during the repair process is temporarily stored by small CAB-like proteins (SCPs). A model is proposed in which damaged D1 is removed in Synechocystis sp. PCC 6803 by a hetero-oligomeric complex composed of two different types of FtsH sub-unit (FtsH2 and FtsH3), with degradation proceeding from the N-terminus of D1 in a highly processive reaction. It is postulated that a similar mechanism of D1 degradation also operates in chloroplasts. Deg proteases are not required for D1 degradation in Synechocystis 6803 but members of this protease family might play a supplementary role in D1 degradation in chloroplasts under extreme conditions.

Class A penicillin-binding proteins (A-PBPs) are high-molecular weight membrane-bound bifunctional enzymes that catalyze the penicillin-sensitive transpeptidation and transglycosylation reaction steps involved in peptidoglycan assembling. We have over-expressed and characterized a soluble form of the glycosyltransferase domain of PBP1a (GT-PBP1a*) from the hyperthermophilic bacteria Thermotoga maritima. GT-PBP1a* efficiently catalyses peptidoglycan biosynthesis, as shown using an in vitro biosynthetized dansylated-lipid II substrate and a HPLC-coupled assay, and is specifically inhibited by moenomycin. GT-PBP1a* tends to spontaneously aggregate in detergent-free solution, a feature that supports existence of a secondary site for membrane association, distinct from the N-terminal transmembrane anchoring region. Overall, our preliminary data document the biochemical properties of GT-PBP1a* and should guide further studies aimed at deciphering the structural determinants involved into membrane binding by this class of enzymes.

Background and Aims: Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized., Methods: The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT-PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2)., Key Results: The CcDH1 and CcDH2 genes encode Y(3)SK(2) dehydrins and the CcDH3 gene encodes an SK(3) dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized., Conclusions: cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences presented here will facilitate future studies on the induction and control of the osmotic stress response in coffee. The unique expression pattern observed for CcLEA1, and the expression of a related gene in other plants, suggests that this gene may play an important role in the development of grain endosperm tissue. Genomic DNA containing the grain-specific CcDH2 promoter region has been cloned. Sequence analysis indicates that this promoter contains several putative regulatory sites implicated in the control of both seed- and osmotic stress-specific gene expression. Thus, the CcDH2 promoter is likely to be a useful tool for basic studies on the control of gene expression during both grain maturation and osmotic stress in coffee.

Coffee grains have an oil content between 10% and 16%, with these values associated with Coffea canephora (robusta) and C. arabica (arabica), respectively. As the majority of the oil stored in oil seeds is contained in specific structures called oil bodies, we were interested in determining whether there are any differences in the expression of the main oil body proteins, the oleosins, between the robusta and arabica varieties. Here, we present the isolation, characterization and quantitative expression analysis of six cDNAs representing five genes of the coffee oleosin family (CcOLE-1 to CcOLE-5) and one gene of the steroleosin family (CcSTO-1). Each coffee oleosin cDNA encodes for the signature structure for oleosins, a long hydrophobic central sequence containing a proline KNOT motif. Sequence analysis also indicates that the C-terminal domain of CcOLE-1, CcOLE-3 and CcOLE-5 contain an 18-residue sequence typical of H-form oleosins. Quantitative RT-PCR showed that the transcripts of all five oleosins were predominantly expressed during grain maturation in robusta and arabica grain, with CcOLE-1 and CcOLE-2 being more highly expressed. While the relative expression levels of the five oleosins were similar for robusta and arabica, significant differences in the absolute levels of expression were found between the two species. Quantitative analysis of oleosin transcripts in germinating arabica grain generally showed that the levels of these transcripts were lower in the grain after drying, and then further decreased during germination, except for a small spike of expression for CcOLE-2 early in germination. In contrast, the levels of CcSTO-1 transcripts remained relatively constant during germination, in agreement with suggestions that this protein is actively involved in the process of oil body turnover. Finally, we discuss the implications of the coffee oleosin expression data presented relative to the predicted roles for the different coffee oleosins during development and germination.