The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Peter Neubauer - One of the best experts on this subject based on the ideXlab platform.
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reconstituted biosynthesis of the nonribosomal macrolactone antibiotic valinomycin in escherichia coli
ACS Synthetic Biology, 2014Co-Authors: Jennifer Jaitzig, Roderich D. Süssmuth, Peter NeubauerAbstract:The structural complexity of nonribosomal peptides (NRPs) impeding economic chemical synthesis and poor cultivability of source organisms limits the development of bioprocesses for novel bioactive compounds. Since nonribosomal peptide synthetases (NRPSs) assemble NRPs from simple amino acid building blocks, heterologous Expression of NRPSs in a robust and easy to manipulate Expression Host is an attractive strategy to make pharmaceutically relevant NRPs more accessible and is also a basis for engineering of these enzymes to generate novel synthetic bioactive compounds. Here we show a systematic approach for the heterologous Expression of the 654 kDa heterodimeric valinomycin synthetase (VlmSyn) from Streptomyces tsusimaensis in a soluble and active form in Escherichia coli. VlmSyn activity and precursor requirements were determined in vitro and provided evidence for a previously proposed model of valinomycin biosynthesis. In vivo production of recombinant valinomycin, a macrolactone antibiotic with reported antifungal, antibacterial, and antiviral activities, was achieved using an engineered E. coli strain growing in inexpensive media and independent of the supplementation with precursors and further optimization of the cultivation conditions. Tailoring of VlmSyn in E. coli paves the way to the production of novel valinomycin analogues in the future.
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fed batch process for the psychrotolerant marine bacterium pseudoalteromonas haloplanktis
Microbial Cell Factories, 2010Co-Authors: Boris Wilmes, Angelika Hartung, Michael Lalk, Manuel Liebeke, Thomas Schweder, Peter NeubauerAbstract:Background: Pseudoalteromonas haloplanktis is a cold-adapted g-proteobacterium isolated from Antarctic sea ice. It is characterized by remarkably high growth rates at low temperatures. P. haloplanktis is one of the model organisms of cold-adapted bacteria and has been suggested as an alternative Host for the soluble overproduction of heterologous proteins which tend to form inclusion bodies in established Expression Hosts. Despite the progress in establishing P. haloplanktis as an alternative Expression Host the cell densities obtained with this organism, which is unable to use glucose as a carbon source, are still low. Here we present the first fed-batch cultivation strategy for this auspicious alternative Expression Host. Results: The key for the fed-batch cultivation of P. haloplanktis was the replacement of peptone by casamino acids, which have a much higher solubility and allow a better growth control. In contrast to the peptone medium, on which P. haloplanktis showed different growth phases, on a casamino acids-containing, phosphate-buffered medium P. haloplanktis grew exponentially with a constant growth rate until the stationary phase. A fed-batch process was established by feeding of casamino acids with a constant rate resulting in a cell dry weight of about 11 g l -1 (OD540 = 28) which is a twofold increase of the highest densities which have been obtained with P. haloplanktis so far and an eightfold increase of the density obtained in standard shake flask cultures. The cell density was limited in the fed-batch cultivation by the relatively low solubility of casamino acids (about 100 g l -1 ), which was proven by pulse addition of casamino acid powder which increased the cell density to about 20 g l -1 (OD540 = 55). Conclusion: The growth of P. haloplanktis to higher cell densities on complex medium is possible. A first fed-batch fermentation strategy could be established which is feasible to be used in lab-scale or for industrial purposes. The substrate concentration of the feeding solution was found to influence the maximal biomass yield considerably. The bottleneck for growing P. haloplanktis to high cell densities still remains the availability of a highly concentrated substrate and the reduction of the substrate complexity. However, our results indicate glutamic acid as a major carbon source, which provides a good basis for further improvement of the fed-batch process.
Renate Kunert - One of the best experts on this subject based on the ideXlab platform.
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transient pentameric igm fulfill biological function effect of Expression Host and transfection on igm properties
PLOS ONE, 2020Co-Authors: Julia Hennicke, Linda Schwaigerlehner, Clemens Grunwaldgruber, Isabelle Bally, Wai Li Ling, Nicole M Thielens, J B Reiser, Renate KunertAbstract:Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient Expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable Expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient Expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient Expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function.
Brian R. Mcnaughton - One of the best experts on this subject based on the ideXlab platform.
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Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1.
ACS infectious diseases, 2019Co-Authors: Terumasa Ikeda, Rachel L. Tennyson, Susanne N. Walker, Reuben S. Harris, Brian R. McnaughtonAbstract:Drugs that block HIV-1 entry are relatively limited. Enfuvirtide is a 36-residue synthetic peptide that targets gp41 and blocks viral fusion. However, Enfuvirtide-resistant HIV has been reported, and this peptide drug requires daily injection. Previously, we have reported helix-grafted display proteins, consisting of HIV-1 gp41 C-peptide helix grafted onto Pleckstrin Homology domains. Some of these biologics inhibit HIV-1 entry with relatively modest and varied potency (IC50 = 190 nM to >1 μM). Here, we report that gp41 C-peptide helix-grafted Sac7d (Sac7d-Cpep) potently suppresses HIV-1 entry in a live virus assay (IC50 = 1.9–12.4 nM). Yeast display sequence optimization of solvent exposed helix residues led to new biologics with improved Expression in E. coli (a common biosimilar Expression Host), with no appreciable change in entry inhibition. Evolved proteins inhibit the entry of a clinically relevant mutant of HIV-1 that is gp41 C-peptide sensitive and Enfuvirtide resistant. Fusion proteins designed ...
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Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV‑1
2019Co-Authors: Terumasa Ikeda, Rachel L. Tennyson, Susanne N. Walker, Reuben S. Harris, Brian R. McnaughtonAbstract:Drugs that block HIV-1 entry are relatively limited. Enfuvirtide is a 36-residue synthetic peptide that targets gp41 and blocks viral fusion. However, Enfuvirtide-resistant HIV has been reported, and this peptide drug requires daily injection. Previously, we have reported helix-grafted display proteins, consisting of HIV-1 gp41 C-peptide helix grafted onto Pleckstrin Homology domains. Some of these biologics inhibit HIV-1 entry with relatively modest and varied potency (IC50 = 190 nM to >1 μM). Here, we report that gp41 C-peptide helix-grafted Sac7d (Sac7d-Cpep) potently suppresses HIV-1 entry in a live virus assay (IC50 = 1.9–12.4 nM). Yeast display sequence optimization of solvent exposed helix residues led to new biologics with improved Expression in E. coli (a common biosimilar Expression Host), with no appreciable change in entry inhibition. Evolved proteins inhibit the entry of a clinically relevant mutant of HIV-1 that is gp41 C-peptide sensitive and Enfuvirtide resistant. Fusion proteins designed for serum stability also potently suppress HIV-1 entry. Collectively, we report several evolved biologics that are functional against an Enfuvirtide-resistant strain and are designed for serum stability
Renato Fani - One of the best experts on this subject based on the ideXlab platform.
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Genome-scale metabolic reconstruction and constraint-based modelling of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.
Environmental microbiology, 2014Co-Authors: Marco Fondi, Ermenegilda Parrilli, Maria Luisa Tutino, Isabel Maida, Elena Perrin, Alessandra Mellera, Stefano Mocali, Pietro Liò, Renato FaniAbstract:Summary The Antarctic strain Pseudoalteromonas haloplanktis TAC125 is one of the model organisms of cold-adapted bacteria and is currently exploited as a new alternative Expression Host for numerous biotechnological applications. Here, we investigated several metabolic features of this strain through in silico modelling and functional integration of –omics data. A genome-scale metabolic model of P. haloplanktis TAC125 was reconstructed, encompassing information on 721 genes, 1133 metabolites and 1322 reactions. The predictive potential of this model was validated against a set of experimentally determined growth rates and a large dataset of growth phenotypic data. Furthermore, evidence synthesis from proteomics, phenomics, physiology and metabolic modelling data revealed possible drawbacks of cold-dependent changes in gene Expression on the overall metabolic network of P. haloplanktis TAC125. These included, for example, variations in its central metabolism, amino acid degradation and fatty acid biosynthesis. The genome-scale metabolic model described here is the first one reconstructed so far for an Antarctic microbial strain. It allowed a system-level investigation of variations in cellular metabolic fluxes following a temperature downshift. It represents a valuable platform for further investigations on P. haloplanktis TAC125 cellular functional states and for the design of more focused strategies for its possible biotechnological exploitation.
Clemens Grunwaldgruber - One of the best experts on this subject based on the ideXlab platform.
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transient pentameric igm fulfill biological function effect of Expression Host and transfection on igm properties
PLOS ONE, 2020Co-Authors: Julia Hennicke, Linda Schwaigerlehner, Clemens Grunwaldgruber, Isabelle Bally, Wai Li Ling, Nicole M Thielens, J B Reiser, Renate KunertAbstract:Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient Expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable Expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient Expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient Expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function.
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reduced paucimannosidic n glycan formation by suppression of a specific β hexosaminidase from nicotiana benthamiana
Plant Biotechnology Journal, 2017Co-Authors: Yunji Shin, Clemens Grunwaldgruber, Friedrich Altmann, Alexandra Castilho, Martina Dicker, Flavio Sadio, Ulrike Vavra, Taeho Kwon, Herta Steinkellner, Richard StrasserAbstract:Plants are attractive Hosts for the production of recombinant glycoproteins for therapeutic use. Recent advances in glyco-engineering facilitate the elimination of nonmammalian-type glycosylation and introduction of missing pathways for customized N-glycan formation. However, some therapeutically relevant recombinant glycoproteins exhibit unwanted truncated (paucimannosidic) N-glycans that lack GlcNAc residues at the nonreducing terminal end. These paucimannosidic N-glycans increase product heterogeneity and may affect the biological function of the recombinant drugs. Here, we identified two enzymes, β-hexosaminidases (HEXOs) that account for the formation of paucimannosidic N-glycans in Nicotiana benthamiana, a widely used Expression Host for recombinant proteins. Subcellular localization studies showed that HEXO1 is a vacuolar protein and HEXO3 is mainly located at the plasma membrane in N. benthamiana leaf epidermal cells. Both enzymes are functional and can complement the corresponding HEXO-deficient Arabidopsis thaliana mutants. In planta Expression of HEXO3 demonstrated that core α1,3-fucose enhances the trimming of GlcNAc residues from the Fc domain of human IgG. Finally, using RNA interference, we show that suppression of HEXO3 Expression can be applied to increase the amounts of complex N-glycans on plant-produced human α1-antitrypsin.
