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Barry S.M.,University of Warwick | Barry S.M.,Kings College London | Kers J.A.,Cornell University | Kers J.A.,Intrexon Corporation | And 12 more authors.
Nature Chemical Biology | Year: 2012

Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N′-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase, and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the nonproteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O 2. © 2012 Nature America, Inc. All rights reserved. Source

Mamedov T.,Fraunhofer United States Center for Molecular Biotechnology
Bioengineered | Year: 2013

At present, several eukaryotic expression systems including yeast, insect and mammalian cells and plants are used for the production of recombinant proteins. Proteins with potential N-glycosylation sites are efficiently glycosylated when expressed in these systems. However, the ability of the eukaryotic expression systems to glycosylate may be not desirable for some proteins. If target proteins that do not carry N-linked glycans in the native host contain potential N-linked glycosylation sites, they can be aberrantly glycosylated in the eukaryotic expression systems, thus, potentially impairing biological activity. Recently, we have developed a strategy of enzymatic deglycosylation of proteins in vivo by co-introducing bacterial PNGase F via agroinfiltration followed by transient expression in plants. (1) Here, we summarize our work on this topic and its potential implications. Source

Kropat J.,University of California at Los Angeles | Gallaher S.D.,University of California at Los Angeles | Urzica E.I.,University of California at Los Angeles | Urzica E.I.,Westfaelische Wilhelms University | And 6 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retentionof genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper. Source

Wu Y.,National Institute of Allergy and Infectious Diseases | Sinden R.E.,The Jenner Institute | Churcher T.S.,Imperial College London | Tsuboi T.,Ehime University | Yusibov V.,Fraunhofer United States Center for Molecular Biotechnology
Advances in Parasitology | Year: 2015

Despite decades of effort battling against malaria, the disease is still a major cause of morbidity and mortality. Transmission-blocking vaccines (TBVs) that target sexual stage parasite development could be an integral part of measures for malaria elimination. In the 1950s, Huff et al. first demonstrated the induction of transmission-blocking immunity in chickens by repeated immunizations with Plasmodium gallinaceum-infected red blood cells. Since then, significant progress has been made in identification of parasite antigens responsible for transmission-blocking activity. Recombinant technologies accelerated evaluation of these antigens as vaccine candidates, and it is possible to induce effective transmission-blocking immunity in humans both by natural infection and now by immunization with recombinant vaccines. This chapter reviews the efforts to produce TBVs, summarizes the current status and advances and discusses the remaining challenges and approaches. © 2015 Elsevier Ltd. Source

Buyel J.F.,Fraunhofer United States Center for Molecular Biotechnology | Buyel J.F.,RWTH Aachen | Bautista J.A.,Fraunhofer United States Center for Molecular Biotechnology | Fischer R.,RWTH Aachen | And 2 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2012

Several studies indicated that biopharmaceuticals based on the recombinant protein E7 of human papillomavirus (HPV) can serve as therapeutic vaccines preventing the development of cancer in women infected with high-risk types of HPV such as HPV16. Here, we report effective extraction and purification of a plant-produced E7GGG-lichenase fusion protein, an HPV16 subunit vaccine candidate, from Nicotiana benthamiana plants, to a high yield. The target contains the modified HPV16 E7 protein internally fused to the surface loop of a truncated, hexa-His- and KDEL-tagged variant of bacterial lichenase, and has been previously shown to possess anti-cancer activity in an animal model [18]. We purified the protein using a combination of immobilized metal-ion affinity chromatography and gel filtration. The achieved purity of the final product was 99% as confirmed by Coomassie or SYPRO Ruby staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical size exclusion chromatography coupled with multi-angle laser light scattering. The overall yield was 50% corresponding to 0.1. g of protein per 1. kg plant biomass. Only slight changes in these parameters were observed during the process scale-up from 50. g to 1. kg of processed leaf biomass. © 2011 Elsevier B.V. Source

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