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Biotechnology Journal

Ankita Singh, Helene F Kildegaard, Mikael R Andersen
Chinese hamster ovary (CHO) cell lines can fold, assemble and modify proteins post-translationally to produce human-like proteins; as a consequence, it is the single most common expression systems for industrial production of recombinant therapeutic proteins. A thorough knowledge of cultivation conditions of different CHO cell lines has been developed over the last decade, but comprehending gene or pathway-specific distinctions between CHO cell lines at transcriptome level remains a challenge. To address these challenges, we compiled a compendium of 23 RNA-Seq studies from public and in-house data on CHO cell lines, i...
May 15, 2018: Biotechnology Journal
Jana Adamíková, Monika Antošová, Milan Polakovič
Early-phase development of industrial immobilized biocatalysts has to address the selection of the best candidates from dozens of available carriers and binding methods. This work presents a simple selection method for the immobilization of industrial-grade Aspergillus oryzae β-galactosidase suitable for the production of galactooligosaccharides. Immobilization efficiency and yield of a variety of immobilized biocatalysts were evaluated using simple activity measurements and mathematical modelling of intraparticle kinetics and mass transfer...
May 9, 2018: Biotechnology Journal
Cory Schwartz, Nicholas Curtis, Ann-Kathrin Löbs, Ian Wheeldon
The yeast Yarrowia lipolytica has been widely studied for its ability to synthesize and accumulate intracellular lipids to high levels. Recent studies have identified native genes that enable growth on biomass-derived sugars, but these genes are not sufficiently expressed to facilitate robust metabolism. In this work, we developed a CRISPR-dCas9 activation (CRISPRa) system in Y. lipolytica and used it to activate native β-glucosidase expression to support growth on cellobiose. A series of different transcriptional activators were compared for their effectiveness in Y...
May 5, 2018: Biotechnology Journal
Lindsay Arnold, Kenneth Lee, Joanna Rucker-Pezzini, Jeong H Lee
The changing landscape of the biopharmaceutical market is driving a paradigm shift towards continuous manufacturing. To date, integrated continuous bioprocessing has not been realized as enabling technologies are nascent. In this work, a fully integrated continuous process was successfully demonstrated from pilot scale bioreactor to drug substance. Comparable product quality was observed between the continuous process and a 500 L fed-batch conventional process. The continuous process generated material at a rate of one kilogram of purified mAb every 4 days, achieving a 4...
May 5, 2018: Biotechnology Journal
Joosu Kuivanen, Sami Holmström, Birgitta Lehtinen, Merja Penttilä, Jussi Jäntti
Due to the rapidly increasing sequence information on gene variants generated by evolution and our improved abilities to engineer novel biological activities, microbial cells can be evolved for the production of a growing spectrum of compounds. For high productivity, efficient carbon channeling towards the end product is a key element. In large scale production systems the genetic modifications that ensure optimal performance cannot be dependent on plasmid-based regulators, but need to be engineered stably into the host genome...
May 5, 2018: Biotechnology Journal
Lan-Yu Cui, Shan-Shan Wang, Chang-Ge Guan, Wei-Fan Liang, Zheng-Lian Xue, Chong Zhang, Xin-Hui Xing
Methylobacterium extorquens AM1, which can be used as a methylotrophic cell factory (MeCF) for the production of fine chemicals from methanol, is the most extensively studied model methylotrophic strain. However, its low tolerance for methanol limits the development of bioprocesses and there have been no reports of improved methanol tolerance of M. extorquens AM1. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis, in combination with adaptive laboratory evolution (ALE), was used to generate a mutant with high methanol tolerance (referred to as CLY-2533)...
May 5, 2018: Biotechnology Journal
Sungyeon Jang, Sungho Jang, Gyoo Yeol Jung
CRISPR interference (CRISPRi) is widely utilized for regulation of target gene expression by repressing transcription. Simple design rules for the single guide RNA (sgRNA) and multiplexity won this method immense popularity. However, quantitative control of the expression levels at varying degrees in a dynamic manner using CRISPRi has been regarded difficult. To deal with this limitation, Fontana et al. [1] modulated the expression levels of the components of CRISPRi, the deactivated Cas9 (dCas9) and the sgRNAs, using various constitutive or inducible promoters...
May 1, 2018: Biotechnology Journal
Hoang C Nguyen, Amie L Langland, John P Amara, Michael Dullen, David S Kahn, Joseph A Costanzo
Biologic manufacturing processes typically employ clarification technologies like depth filtration to remove insoluble and soluble impurities. Conventional depth filtration media used in these processes contain naturally-derived components like diatomaceous earth and cellulose. These components may introduce performance variability and contribute extractable/leachable components like beta-glucans that could interfere with limulus amebocyte lysate endotoxin assays. Recently a novel, all-synthetic depth filtration media was developed (Millistak+® HC Pro X0SP) that may improve process consistency, efficiency, and drug substance product quality by reducing soluble process impurities...
April 30, 2018: Biotechnology Journal
Roland Wohlgemuth
The small molecule domain of biological cellular systems is closely related to the synthesis and breakdown of larger molecules such as DNA, RNA, proteins or polysaccharides. Although the analysis, identification, characterization and synthesis of metabolites has a long history of milestone discoveries, it continues to be of great interest in the search for novel biological activities, metabolic pathways, diagnostic and therapeutic applications. Biologically active metabolites benefit from advantages in diffusion and transport for various interactions with proteins and nucleic acids and regulatory events...
April 30, 2018: Biotechnology Journal
Sebastian P Schwaminger, Silvia A Blank-Shim, Isabell Scheifele, Vitaliy Pipich, Paula Fraga-García, Sonja Berensmeier
Superparamagnetic nanoparticles have recently gained much attention due to their broad range of applicability including medical in vivo technologies, sensors and as supports for catalysts. As magnetic affinity materials, they can be utilized for the development of new purification strategies for pharmaceuticals and other target molecules from crude lysates. Here, a short peptide tag based on a glutamate sequence is introduced and the adsorption of pure protein as well as protein from crude cell lysate at different conditions is demonstrated...
April 28, 2018: Biotechnology Journal
J Jansing, J F Buyel
The yield of recombinant proteins in plants determines their economic competitiveness as a production platform compared to microbes and mammalian cells. The promoter, untranslated regions (UTRs) and codon usage can all contribute to the yield, but potential interactions among these components have not been examined in detail. Here we investigated the effect of two promoters (35SS and nos) and four 5'UTRs on the spatiotemporal expression of DsRed mRNA and the accumulation of DsRed protein during transient expression in tobacco (Nicotiana tabacum) mediated by Agrobacterium tumefaciens...
April 27, 2018: Biotechnology Journal
David Reinhart, Lukas Damjanovic, Christian Kaisermayer, Wolfgang Sommeregger, Andreas Gili, Bernhard Gasselhuber, Andreas Castan, Patrick Mayrhofer, Clemens Grünwald-Gruber, Renate Kunert
Chinese hamster ovary (CHO) cells comprise a variety of lineages including CHO-DXB11, CHO-K1, CHO-DG44 and CHO-S. Despite all CHO cell lines sharing a common ancestor, extensive mutagenesis and clonal selection has resulted in substantial genetic heterogeneity among them. Data from sequencing shows that different genes are missing in individual CHO cell lines and each cell line harbors a unique set of mutations with relevance to the bioprocess. However, not much literature is available about the influence of genetic differences of CHO on the performance of bioprocess operations...
April 27, 2018: Biotechnology Journal
Xue Wang, Yoshinori Kawabe, Takeshi Hada, Akira Ito, Masamichi Kamihira
Bacterial backbone sequences of conventional plasmid vectors have been reported to exhibit negative effects on transgene expression in mammalian cells, such as cytotoxicity and gene silencing. Minicircle DNA vectors can be employed to overcome these issues and to improve the transfection efficiency because of their smaller size. In this study, transgenes were integrated into the hypoxanthine phosphoribosyltransferase (hprt) locus of Chinese hamster ovary (CHO) cells by the Cre-loxP system using minicircle DNA vectors as transgene donors...
April 27, 2018: Biotechnology Journal
Bernhard Schoenenberger, Agata Wszolek, Roland Meier, Henrike Brundiek, Markus Obkircher, Roland Wohlgemuth
Shikimic acid 3-phosphate, as a central metabolite of the shikimate pathway, is of high interest as enzyme substrate for 5-enolpyruvoyl-shikimate 3-phosphate synthase, a drug target in infectious diseases and a prime enzyme target for the herbicide glyphosate. As the important substrate shikimic acid 3-phosphate was only accessible via a chemical multi-step route, a new straightforward preparative one-step enzymatic phosphorylation of shikimate using a stable recombinant shikimate kinase has been developed for the selective phosphorylation of shikimate in the 3-position...
April 26, 2018: Biotechnology Journal
Kyeong-Jun Kim, Hyeon-Yeol Cho, Won-Jun Lee, Jeong-Woo Choi
Circulating tumor cell (CTC) which recently arisen as potential sources for monitoring and characterizing non-haematologic cancers and their metastatic derivatives. Immunomagnetic microbeads and magnetic nanoparticles (MNPs) have been extensively explored to isolate CTCs from blood samples. However, MNPs attached on the membrane protein are interrupted further analysis to distinguish the cancer subtype by consumption or blocking the target surface marker. Here, we describe an MNP-mediated analysis method for surface marker expression profile by magnetic force microscopy (MFM)...
April 26, 2018: Biotechnology Journal
Zhiqiang Wu, Jinlin Zhao, Minghan Qiu, Zeyun Mi, Maobin Meng, Yu Guo, Hui Wang, Zhiyong Yuan
Accurately identifying and quantifying cellular autophagy is very important as the significance of autophagy in physiological and pathological processes becomes increasingly evident. Ectopically expressed fluorescent-tagged microtubule-associated protein light chain 3B (MAP1LC3B, LC3) is the most widely used reporter for monitoring autophagy activity thus far. However, this approach ignored the influence of constitutively overexpressed LC3 on autophagy itself and autophagy-related processes and its accuracy in indicating autophagy is questionable...
April 19, 2018: Biotechnology Journal
Monica Bianco, Daniele Vergara, Stefania De Domenico, Michele Maffia, Antonio Gaballo, Valentina Arima
Several techniques can be used to monitor cell dynamism after a perturbation. Among these, Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) offers the great advantage to study the mechanical properties of cells in real-time and with a great sensitivity. Here, we used QCM-D to investigate the effects of two cytoskeleton-targeting agents, cytochalasin D (CytoD) and Y27632, on human MCF-7 cells. Cell adhesion on the sensor surface, crucial for in-flow experiments, was obtained by covalent adsorption of a fibronectin (FN) film, an extracellular matrix (ECM) protein...
April 16, 2018: Biotechnology Journal
Fumiya Iwata, Hidehiko Hirakawa, Teruyuki Nagamune
Bacterial cytochrome P450 monooxygenases (P450s) are promising biocatalysts for chemical syntheses because they catalyze a variety of oxidations on non-activated hydrocarbons using O2 . However, the requirement of two auxiliary proteins, an electron transfer protein and a reductase, for the catalysis is a major bottleneck for in vitro applications of these monooxygenases. Our previous study showed that artificial assembly of a bacterial P450 with its auxiliary proteins using a heterotrimeric proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus yields a self-sufficient P450, but partial dissociation of P450 from the complex at catalytic concentrations reduces the apparent specific activity of this self-sufficient P450...
April 16, 2018: Biotechnology Journal
Matthew Deaner, Allison Holzman, Hal S Alper
Metabolic engineering typically utilizes a suboptimal step-wise gene target optimization approach to parse a highly connected and regulated cellular metabolism. While the endonuclease-null CRISPR/Cas system has enabled gene expression perturbations without genetic modification, it has been mostly limited to small sets of gene targets in eukaryotes due to inefficient methods to assemble and express large sgRNA operons. In this work, we develop a TEF1p-tRNA expression system and demonstrate that the use of tRNAs as splicing elements flanking sgRNAs provides higher efficiency than both Pol III and ribozyme-based expression across a variety of single sgRNA and multiplexed contexts...
April 16, 2018: Biotechnology Journal
Kristina Kemter, Jens Altrichter, Roland Derwand, Thomas Kriehuber, Eva Reinauer, Martin Scholz
To develop highly concentrated therapeutic antibodies enabling convenient subcutaneous application, well stabilizing pharmaceutical formulations with low viscosities are considered to be key. The purpose of this study was to select specific amino acid combinations that reduce and balance aggregation, fragmentation and chemical degradation and also lower viscosity of highly concentrated liquid antibodies. As a model, the therapeutically well-established antibody trastuzumab (25 - >200 mg/mL) in liquid formulation was used...
April 16, 2018: Biotechnology Journal
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