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  • 1
    Online Resource
    Online Resource
    A soybean plastid‐targeted NADH‐malate dehydrogenase: cloning and expression analyses
    Wiley ; 2001
    In:  American Journal of Botany Vol. 88, No. 12 ( 2001-12), p. 2136-2142
    Details
    In: American Journal of Botany, Wiley, Vol. 88, No. 12 ( 2001-12), p. 2136-2142
    Abstract: A typical soybean ( Glycine max ) plant assimilates nitrogen rapidly both in active root nodules and in developing seeds and pods. Oxaloacetate and 2‐ketoglutarate are major acceptors of ammonia during rapid nitrogen assimilation. Oxaloacetate can be derived from the tricarboxylic acid (TCA) cycle, and it also can be synthesized from phosphoenolpyruvate and carbon dioxide by phosphoenolpyruvate carboxylase. An active malate dehydrogenase is required to facilitate carbon flow from phosphoenolpyruvate to oxaloacetate. We report the cloning and sequence analyses of a complete and novel malate dehydrogenase gene in soybean. The derived amino acid sequence was highly similar to the nodule‐enhanced malate dehydrogenases from Medicago sativa and Pisum sativum in terms of the transit peptide and the mature subunit (i.e., the functional enzyme). Furthermore, the mature subunit exhibited a very high homology to the plastid‐localized NAD‐dependent malate dehydrogenase from Arabidopsis thaliana , which has a completely different transit peptide. In addition, the soybean nodule‐enhanced malate dehydrogenase was abundant in both immature soybean seeds and pods. Only trace amounts of the enzyme were found in leaves and nonnodulated roots. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to the mature subunit, which has a molecular mass of ∼34 kDa. We propose that this new malate dehydrogenase facilitates rapid nitrogen assimilation both in soybean root nodules and in developing soybean seeds, which are rich in protein. In addition, the complete coding region of a geranylgeranyl hydrogenase gene, which is essential for chlorophyll synthesis, was found immediately upstream from the new malate dehydrogenase gene.
    Type of Medium: Online Resource
    ISSN: 0002-9122 , 1537-2197
    URL: Article
    DOI: 10.2307/3558374
    RVK:
    WA 15000
    Language: English
    Publisher: Wiley
    Publication Date: 2001
    detail.hit.zdb_id: 2053581-8
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    Integrated process development ‐ quality by design compliant evaluation of upstream variations at the microscale level
    Wiley ; 2018
    In:  Journal of Chemical Technology & Biotechnology Vol. 93, No. 7 ( 2018-07), p. 2021-2032
    Details
    In: Journal of Chemical Technology & Biotechnology, Wiley, Vol. 93, No. 7 ( 2018-07), p. 2021-2032
    Abstract: Evaluation of optimized upstream conditions is often solely based on titers that do not reflect the downstream process and, consequently, the final purity and yield. This is especially critical for proteins expressed as inclusion bodies (IBs) because the subsequent downstream process is more complex than for soluble proteins. A miniaturized process development platform representing the entire downstream process at a microscale level combined with a multi‐fermenter system for bioprocess screening enables fast investigation, using quality by design (QbD) measures, of the quality of feedstock compositions that are altered by upstream conditions. RESULTS High‐throughput methods were integrated for an entire process chain, including upstream and downstream processing, for production of a recombinant protein as IBs. The miniaturized process chain consisted of cell disruption, IB harvesting, solubilization, refolding, and chromatographic purification. This enables processing and evaluation of 16 different fermentation conditions within 7 days. Only 2 g of initial biomass was required for evaluation of an individual fermentation. This resulted in a 15‐fold reduction of time and a 100‐fold reduction of material compared with common bench scale experiments. CONCLUSION This microscale process chain mimics the bench process and is able to differentiate purity variations as low as 2%. © 2017 Society of Chemical Industry
    Type of Medium: Online Resource
    ISSN: 0268-2575 , 1097-4660
    URL: Issue
    URL: Article
    DOI: 10.1002/jctb.2018.93.issue-7
    DOI: 10.1002/jctb.5407
    Language: English
    Publisher: Wiley
    Publication Date: 2018
    detail.hit.zdb_id: 1479465-2
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  • 3
    Online Resource
    Online Resource
    Advanced purification platform using circularly permuted caspase‐2 for affinity fusion‐tag removal to produce native fibroblast growth factor 2
    Wiley ; 2021
    In:  Journal of Chemical Technology & Biotechnology Vol. 96, No. 6 ( 2021-06), p. 1515-1522
    Details
    In: Journal of Chemical Technology & Biotechnology, Wiley, Vol. 96, No. 6 ( 2021-06), p. 1515-1522
    Abstract: Recombinant proteins produced for use as biopharmaceuticals need to harbor their native N‐terminus. A drawback in expression of recombinant proteins as fusion proteins with an affinity fusion‐tag is that enzymatic or chemical processing is required to trim the artificial tag and release the true protein of interest. In many cases, however, this processing step generates an incorrect N‐terminus. RESULTS Human fibroblast growth factor 2 (FGF2) was expressed as a fusion protein in Escherichia coli fed‐batch cultivations. The protein of interest (POI) carried an N‐terminal affinity fusion‐tag which enabled purification via affinity chromatography. After enzymatic removal of the affinity fusion‐tag with a circularly permuted human caspase‐2 (cpCasp2), the POI was further purified using subtractive affinity chromatography. Mass spectrometric analysis confirmed the authentic N‐terminus of the POI. The generated POI was highly pure with 42 ppm host cell protein, 3.7 μg mL −1 dsDNA and ~ 1000 EU mL −1 endotoxin. Only a small number of E. coli host cell proteins were co‐purified with the POI. Because of the high specificity of the novel protease cpCasp2, no off‐target cleavage could be observed. CONCLUSION Our findings demonstrate that cpCasp2 can be used for the production of native proteins using a fusion‐protein process. This represents a first case study at large laboratory scale for the production of an industrially relevant protein. This technology constitutes the basis of a highly scalable cpCasp2‐based platform fusion protein process (CASPON technology) purification platform. © 2021 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
    Type of Medium: Online Resource
    ISSN: 0268-2575 , 1097-4660
    URL: Issue
    URL: Article
    DOI: 10.1002/jctb.v96.6
    DOI: 10.1002/jctb.6666
    Language: English
    Publisher: Wiley
    Publication Date: 2021
    detail.hit.zdb_id: 1479465-2
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  • 4
    Online Resource
    Online Resource
    Real‐time monitoring and model‐based prediction of purity and quantity during a chromatographic capture of fibroblast growth factor 2
    Wiley ; 2019
    In:  Biotechnology and Bioengineering Vol. 116, No. 8 ( 2019-08), p. 1999-2009
    Details
    In: Biotechnology and Bioengineering, Wiley, Vol. 116, No. 8 ( 2019-08), p. 1999-2009
    Abstract: Process analytical technology combines understanding and control of the process with real‐time monitoring of critical quality and performance attributes. The goal is to ensure the quality of the final product. Currently, chromatographic processes in biopharmaceutical production are predominantly monitored with UV/Vis absorbance and a direct correlation with purity and quantity is limited. In this study, a chromatographic workstation was equipped with additional online sensors, such as multi‐angle light scattering, refractive index, attenuated total reflection Fourier‐transform infrared, and fluorescence spectroscopy. Models to predict quantity, host cell proteins (HCP), and double‐stranded DNA (dsDNA) content simultaneously were developed and exemplified by a cation exchange capture step for fibroblast growth factor 2 expressed in Escherichia coli Online data and corresponding offline data for product quantity and co‐eluting impurities, such as dsDNA and HCP, were analyzed using boosted structured additive regression. Different sensor combinations were used to achieve the best prediction performance for each quality attribute. Quantity can be adequately predicted by applying a small predictor set of the typical chromatographic workstation sensor signals with a test error of 0.85 mg/ml (range in training data: 0.1–28 mg/ml). For HCP and dsDNA additional fluorescence and/or attenuated total reflection Fourier‐transform infrared spectral information was important to achieve prediction errors of 200 (2–6579 ppm) and 340 ppm (8–3773 ppm), respectively.
    Type of Medium: Online Resource
    ISSN: 0006-3592 , 1097-0290
    URL: Issue
    URL: Article
    DOI: 10.1002/bit.v116.8
    DOI: 10.1002/bit.26984
    Language: English
    Publisher: Wiley
    Publication Date: 2019
    detail.hit.zdb_id: 1480809-2
    detail.hit.zdb_id: 280318-5
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    Preparative crystallization of a single chain antibody using an aqueous two‐phase system
    Wiley ; 2014
    In:  Biotechnology and Bioengineering Vol. 111, No. 11 ( 2014-11), p. 2192-2199
    Details
    In: Biotechnology and Bioengineering, Wiley, Vol. 111, No. 11 ( 2014-11), p. 2192-2199
    Abstract: A simultaneous crystallization and aqueous two‐phase extraction of a single chain antibody was developed, demonstrating process integration. The process conditions were designed to form an aqueous two‐phase system, and to favor crystallization, using sodium sulfate and PEG‐2000. At sufficiently high concentrations of PEG, a second phase was generated in which the protein crystallization occurred simultaneously. The single chain antibody crystals were partitioned to the top, polyethylene glycol‐rich phase. The crystal nucleation took place in the sodium sulfate‐rich phase and at the phase boundary, whereas crystal growth was progressing mainly in the polyethylene glycol‐rich phase. The crystals in the polyethylene glycol‐rich phase grew to a size of 〉 50 µm. Additionally, polyethylene glycol acted as an anti‐solvent, thus, it influenced the crystallization yield. A phase diagram with an undersaturation zone, crystallization area, and amorphous precipitation zone was established. Only small differences in polyethylene glycol concentration caused significant shifts of the crystallization yield. An increase of the polyethylene glycol content from 2% (w/v) to 4% (w/v) increased the yield from approximately 63–87%, respectively. Our results show that crystallization in aqueous two‐phase systems is an opportunity to foster process integration. Biotechnol. Bioeng. 2014;111: 2192–2199. © 2014 Wiley Periodicals, Inc.
    Type of Medium: Online Resource
    ISSN: 0006-3592 , 1097-0290
    URL: Issue
    URL: Article
    DOI: 10.1002/bit.v111.11
    DOI: 10.1002/bit.25287
    Language: English
    Publisher: Wiley
    Publication Date: 2014
    detail.hit.zdb_id: 1480809-2
    detail.hit.zdb_id: 280318-5
    SSG: 12
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