Tom Roseanne, Bisson Louis, Durocher Yves
CSH Protoc. 2008 Mar 1;2008:pdb.prot4979. doi: 10.1101/pdb.prot4979.
INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin, due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, other methods suitable for efficient transfection of mammalian cells are easier to use. This protocol describes the steps needed for successful transfection of 293-6E cells in suspension culture in serum-free medium using 293fectin.
引言
重组蛋白（r蛋白）的快速高效生产仍然是学术界和生物制药界面临的一项重大挑战。当作为生物治疗药物进行开发时，通常需要大量（数百毫克至克级）的纯r蛋白，或者在高通量筛选活动以及结构或功能研究中需要少量（毫克级）的纯r蛋白。在表达哺乳动物来源的cDNA时，由于哺乳动物细胞具有进行复杂的翻译后修饰的卓越能力，因此通常比原核系统更受青睐。哺乳动物细胞的大规模转染如今已成为科学界的一项“必备”技术，因为它能够在将cDNA克隆到合适的表达载体后的几天内生产出毫克至克级的r蛋白。尽管钙介导的大规模转染非常有效，但其他适用于高效转染哺乳动物细胞的方法使用起来更加简便。本方案描述了使用293fectin在无血清培养基中对悬浮培养的293-6E细胞进行成功转染所需的步骤。
