Laflamme, CarlMcPherson, Peter S.Edwards, Aled M.Bandrowski, Anita E....
8页
查看更多>>摘要：A vast array of commercial antibodies covers a large percentage of human gene products, but determining which among them is most appropriate for any given application is challenging. This leads to use of non-specific antibodies that contributes to issues with reproducibility. It is our opinion that the community of scientists who use commercial antibodies in their biomedical research would benefit from third-party antibody characterization entities that use standardized operating procedures to assess and compare antibody performance. Ideally, such entities would follow the principles of open science, such that all antibodies against any given protein target would be tested in parallel, and all data generated released to the public domain without bias. Furthermore, there should be no financial incentive for the entity beyond cost-recovery. Such non-profit organizations, combined with other scientific efforts, could catalyse new discoveries by providing scientists with better validated antibody tools.
查看更多>>摘要：The transition from preclinical biological drug development into clinical trials requires an efficient upscaling process. In this context, bispecific antibody drugs are particularly challenging due to their propensity to form aggregates and generally produce low titers. Here, the upscaling process for a tetravalent bispecific antibody expressed by a piggyBac transposon-mediated stable HEK293 cell pool has been evaluated. The project was performed as a case study at Testa Center, a non-GMP facility for scale-up testing of biologics in Sweden, and encompassed media adaptation strategies, fed-batch optimization and a novel antibody purification technology. The cell pool was adapted to different culture media for evaluation in terms of cell viability and titers compared to its original Expi293 Expression Medium. These parameters were assessed in both sequential stepwise adaption and direct media exchanges. By this, a more affordable medium was identified that did not require stepwise adaptation and with similar titers and viability as in the Expi293 Expression Medium. Fed-batch optimizations resulted in culture densities reaching up to 20 x 106 viable cells/mL with over 90 % viability 12 days postinoculum, and antibody titers three times higher than corresponding batch cultures. By implementing a novel high-speed protein A fiber technology (Fibro PrismA) with a capture residence time of only 7.5 s, 8 L of supernatant could be purified in 4.5 h without compromising the purity, structural integrity and function of the bispecific antibody. Results from this study related to medium adaptation and design of fed-batch protocols will be highly beneficial during the forthcoming scale-up of this therapeutic antibody.
Howard, Thomas P.Azubuike, Christopher C.Gatehouse, Angharad M. R.
11页
查看更多>>摘要：Cupriavidus necator H16 is a chemolithoautotroph with a range of industrial biotechnological applications. Advanced metabolic engineering in the bacterium, however, is impeded by low transformation efficiency, making it difficult to introduce and screen new genetic functions rapidly. This study systematically characterized the broad host range plasmids pBHR1, pBBR1MCS-2 and pKT230 used frequently for C. necator engineering. Kanamycin resistance cassette (KanR) and a truncated sequence of the replication origin (Rep) are contributing factors to C. necator low electroporation transformation efficiency. Consequently, a series of modular minimal plasmids, named pCAT, were constructed. pCAT vectors transform C. necator H16 with a > 3000-fold higher efficiency (up to 107 CFU/mu g DNA) compared to control plasmids. Further, pCAT vectors are highly stable, expressing reporter proteins over several days of serial cultivation in the absence of selection pressure. Finally, they can be assembled rapidly from PCR or synthesized DNA fragments, and restriction-ligation reactions can be efficiently electroporated directly into C. necator, circumventing the requirement to use Escherichia coli for plasmid maintenance or propagation. This study demonstrates that an understanding of the behaviour of the constituent parts of plasmids in a host is key to efficient propagation of genetic information, while offering new methods for engineering a bacterium with desirable industrial biotechnological features.
查看更多>>摘要：Irrespective of their biological origin, most proteins are composed of several elementary domains connected by linkers. These domains are either functionally independent units, or part of larger multidomain structures whose functions are defined by their spatial proximity. Carbohydrate-degrading enzymes provide examples of a range of multidomain structures, in which catalytic protein domains are frequently appended to one or more non-catalytic carbohydrate-binding modules which specifically bind to carbohydrate motifs. While the carbohydrate-binding specificity of these modules is clear, their function is not fully elucidated. Herein, an original approach to tackle the study of carbohydrate-binding modules using the Jo-In biomolecular welding protein pair is presented. To provide a proof of concept, recombinant xylanases appended to two different carbohydrate-binding modules have been created and produced. The data reveal the biochemical properties of four xylanase variants and provide the basis for correlating enzyme activity to structural properties and to the nature of the substrate and the ligand specificity of the appended carbohydrate-binding module. It reveals that specific spatial arrangements favour activity on soluble polymeric substrates and that activity on such substrates does not predict the behaviour of multimodular enzymes on insoluble plant cell wall samples. The results highlight that the Jo-In protein welding system is extremely useful to design multimodular enzyme systems, especially to create rigid conformations that decrease the risk of intermodular interference. Further work on Jo-In will target the introduction of varying degrees of flexibility, providing the means to study this property and the way it may influence multimodular enzyme functions.
Lai, Liang-ChuanTsai, Mong-HsunLu, Tzu-PinChuang, Eric Y....
11页
查看更多>>摘要：The substantial reduction in experimental cost of next-generation sequencing techniques makes it feasible to assemble a bacterial genome of unknown species de novo and acquire substantial genetic information from environmental samples. Many bioinformatics tools and algorithms have also been developed for prokaryotes, but complex parameter settings and command line-based user interfaces cause a significant entry barrier for novices. Efficient construction of pipelines that integrate all the available genomic data poses a major challenge to the understanding of unknown pathogens. MiDSystem is a comprehensive online system for analyzing genomic data from microbiomes. With a user-friendly interface, MiDSystem supports both de novo assembly and metagenomic analysis pipelines. It is designed to automatically analyze whole genome shotgun sequencing data of bacteria submitted by users. Multiple analytical steps can be performed directly on the system, and the results generated from the embedded tools are visualized in an online summary report to make it more interpretable. Constructing a genome de novo has gradually become the foundation of bacterial studies. Taking both single species and metagenomic samples into consideration, MiDSystem can greatly reduce the time and effort for analysis of bacterial genomic data. Use of MiDSystem will enable more focus to be placed on understanding the etiology of bacterial infections and microorganism ecologies.
Dempfle, DavidKrocher, OliverStuder, Michael Hans-Peter
8页
查看更多>>摘要：Lignocellulose-based biofuels are of major importance to mitigate the impact of international traffic and transport on climate change while sustaining agricultural land for food supply. Highly integrated systems like consolidated bioprocessing (CBP), where enzyme production, enzymatic hydrolysis and fermentation of the released sugars are carried out in one reactor, offer the highest potential to save costs and to make lignocellulosebased biofuels economically competitive. The work described here showed that CBP based on a microbial consortium operated at full-scale (2000 t/d) saves up to 27.5 % of the total ethanol production costs compared to conventional ethanol production from lignocellulose in individual process steps. The cost savings are mainly achieved through lower CAPEX due to less apparatus requirements because of the integrated process, as well as through lower OPEX since no glucose is needed for enzyme production. A comparison with literature estimations of cost savings of CBP based on genetically modified microorganisms results in approximately the same range. As a result of a detailed sensitivity analysis, scale and yield were identified as the main cost-pushers from a process point of view, whereas the price level of the plant location has the highest impact on the investment conditions. In the EU, CBP yields enough margin for profitable production and the possibility to decentralize biomass valorization, whereas in the world's largest ethanol market, the U.S, profitable production of lignocellulosic ethanol can only be achieved by CBP combined with other cost saving techniques, such as utilization of cost-free waste feedstocks, since ethanol has undergone a considerable price slump.
查看更多>>摘要：Microalgae produce a broad range of organic compounds that are increasingly being recognised for their value in novel product production and biotechnological applications. Most microalgae are photoautotrophic, but some are capable of either mixotrophy or heterotrophy. Reported enhanced biomass yields or contrasting metabolite profiles compared to autotrophic growth improve the economics of large-scale production of microalgae, which currently limits industrial applications. Here, the potential of a high-throughput method for the rapid screening of microalgal metabolism was assessed against 95 different carbon sources, using the cost-effective Biolog plate. Of the 5 microalgae tested, Desmodesmus communis (30 carbon sources) and Chlorella vulgaris (19 carbon sources) had the highest number of positive responses to carbon sources, whereas Chlorella sorokiniana had the most negative (toxic) response to the various carbon sources (77 carbon sources). Comparison of Biolog plate results with traditional culture techniques showed good agreement. Species with a high number of positive responses on the Biolog plate exhibited the highest biomass yield under heterotrophic conditions, whilst those with low number of positive responses exhibited the highest biomass yield under autotrophic conditions, using traditional culturing techniques. While the use of these plates is limited to obtaining axenic lines of microalgal species, the method provided a high-throughput assessment of carbon source metabolism, without the expense of undertaking large, laborious traditional culturing assessments. Such high-throughput assessments can be regarded as useful tools for progressing species selection, metabolic capacity and optimal culture conditions for microalgal biotechnology applications.
NSTL
Elsevier