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Genetic Engineering & Biotechnology News | GENengnews.com | OCTOBER 1, 2016 | 33 nology Core Laboratory, Intramural Research at the National Institute of Diabetes and Di- gestive and Kidney Diseases, National Insti- tutes of Health. "We decided to use it for iden- tifying genes that are currently not known to be involved in protein expression, yet whose knockdown could improve recombinant pro- tein production in mammalian cells." Dr. Shiloach and colleagues performed a genome-scale, high-throughput RNAi screen using HEK293 cells expressing firefly lucif- erase. "The basic idea was to individually knock down approximately 22,000 genes one at a time and look for those that affected ex- pression, but did not affect cell growth," ex- plained Dr. Shiloach. "We utilized three differ- ent siRNAs for each gene and performed the work collaborating with the National Center for Advanced Translational Science." Among the "hits" identified was the gene for ornithine decarboxylase antizyme 1. The protein product helps regulate intracellular polyamines that are needed for cell growth and proliferation. "We also performed a de- tailed investigation to confirm the findings," continued Dr. Shiloach. "We are now assess- ing the long-term effects of knockdown, the mechanisms involved in metabolism, and feasibility of extending this approach to other mammalian cells." According to Dr. Shiloach, the take-home message from these studies is that genome- scale RNAi screens can identify key path- ways and genes whose modulation may help improve the expression and production of recombinant proteins. "These studies are more a beginning than an ending," he stated. "But we have established a different founda- tion for the targeted design of more efficient mammalian cell expression platforms." 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Search ' Cobra Microbiota' for more information Visit our website: www.cobrabio.com Visit us at ICSE CPhI 4-6 Oct 2016 stand 3J67 BIOPROCESSING > Celonic, IBI-Lorenzini Collaborate on Cell-Line Development IBI-Lorenzini has entered into a partnership with the Swiss contract development and manufacturing organization, Celonic, to establish high-performance cell lines for R&D.; To support commercial introduction of biological drugs to the market, Celonic has developed CHOvolution, a cell-line kit that can be used for a range of applications, from non-GMP R&D; testing to GMP development and commercial market supply. "This agreement is a major step for IBI-Lorenzini," commented Camilla Khevenhueller Borghese, president at IBI-Lorenzini. > AGC Acquires Biomeva, a German CMO Asahi Glass (AGC), a manufacturer of glass, chemicals, and high-tech materials, announced that it has ac- quired all of the shares of Biomeva from Biomeva Holding. Now that Biomeva, a biopharmaceutical con- tract manufacturing organization (CMO) in Europe, is a part of AGC, AGC's life science business will be offering wider geographic coverage and a wider range of capacities. Based in Heidelberg, Germany, Biom- eva has expertise in using microbial ex- pression technology. Biomeva's services include process development for highly efficient therapeutic protein expression, cGMP compliant fermentation, purifica- tion, and bulk filling, as well as analytical testing and cell banking. > Recipharm Inks Long-Term Supply Deal with Tillotts Recipharm entered into a long-term supply agreement with Tillotts Pharma that comprises technology transfer and commercial manufacturing of the prod- ucts Entocort and Asacol. The partnership will also involve the commercial scale development of Tillotts' ongoing develop- ment program for TP05. All products are part of Tillotts' focus on gastroenterology. Recipharm will provide advanced pellet coating technology for controlled release from its facility in Pessac, France, tablet and capsule manufacturing, in- cluding final packaging from its facility in Fontaine, France, and bottle filling from its site in Lisbon, Portugal. The total shared investment across the Recipharm network to enable this business amounts to €3.5 million. This includes investments to create new capacity as well as product specific investments. n News Bioprocessing Expression levels of recombinant proteins (simple proteins as well as complex biolog- ics) can be unpredictable in mammalian cells, and this causes major headaches for target discovery and scale-up manufacture, according to Tom Payne, Ph.D., head of cell engineering at Oxford Genetics. Scientists are familiar with finding that sometimes proteins express at unexpectedly low levels, even when under the control of strong pro- moters (e.g., CMV). "To minimize protein-to-protein vari- ability and maximize yield, we adopted a high-throughput approach to screen and optimize >5,000 recombinant promoters by random shuffling, >30 5' UTRs in different configurations and >15 poly-A signals," said Dr. Payne. "We also developed Kozak and stop codon libraries and screened >1,000 genes from diverse organisms encoding putative ancillary protein to generate expres- sion plasmids that consistently produce high yields of diverse proteins in HEK-293 and CHO systems, termed SnapFast Pro." Validating the System To validate this vector system, Dr. Payne and his team compared protein yields to an industry-standard protein-expression vector. To maximize the relevance of the dataset they algorithmically determined the frequency with which each gene in the human genome had been cited in publications (PubMed hits, 2000–2016), identifying those genes consis- tently increasing in hits, as an indication of importance in biomedical research. "The top 150 genes were batch codon- optimized, FLAG-tagged, and sub-cloned into the expression vector or SnapFast Pro," continued Dr. Payne. "Following transient transfection into either CHO-K1 or HEK-293 cells, lysates and/or supernatants (depend- ing on whether the protein is secreted) were subjected to high-throughput auto- mated western blot. For both secreted and nonsecreted proteins, SnapFast Pro gave consistently high-level protein expression, in many cases showing high expression where no protein could be detected with the reference expression vector." n Boosting Expression Oxford Genetics is a specialist synthetic biol- ogy company focused on providing DNA, pro- tein, virus, and cell-line design and develop- ment solutions. Two of the firm's scientists can be seen working on a custom cell-line development project looking to optimize expression of a client's gene of interest.