Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS), UMR 7104, Institut National de la Santé et de la Recherche Médicale (INSERM), U1258, Université de Strasbourg, Illkirch, France.
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département of Integrated Structural Biology, Equipe Labellisée Ligue, Centre National de la Recherche Scientifique (CNRS), UMR 7104, Institut National de la Santé et de la Recherche Médicale (INSERM), U1258, Université de Strasbourg, Illkirch, France.
Methods Mol Biol. 2021;2247:17-38. doi: 10.1007/978-1-0716-1126-5_2.
Most cellular processes are mediated by multi-subunit protein complexes which have attracted major interest in both academia and industry. Recombinant production of such entities in quantity and quality sufficient for functional and structural investigations may be extremely challenging and necessitate specific technologies. The baculovirus expression vector system is widely used for the production of eukaryotic multiprotein complexes, and a variety of strategies are available to assemble transfer vectors for the generation of recombinant baculoviruses. Here we detail applications of homology-based cloning techniques for one-step construction of dual promoter baculovirus transfer plasmids and of restriction-free (RF) cloning for the modification of existing constructs.
大多数细胞过程都是由多亚基蛋白复合物介导的，这些复合物在学术界和工业界都引起了极大的兴趣。为了进行功能和结构研究，以足够的数量和质量来重组生产这些实体可能极具挑战性，并且需要特定的技术。杆状病毒表达载体系统被广泛用于真核多蛋白复合物的生产，并且有多种策略可用于组装转移载体以生成重组杆状病毒。在这里，我们详细介绍了基于同源性克隆技术的应用，用于一步构建双启动子杆状病毒转移质粒，以及用于修饰现有构建体的无限制（RF）克隆技术。
