Jordan Eva, Al-Halabi Laila, Schirrmann Thomas, Hust Michael
Institute of Biochemistry and Biotechnology, Technical University Braunschweig, Braunschweig, Germany.
Methods Mol Biol. 2009;525:509-16, xiv. doi: 10.1007/978-1-59745-554-1_27.
The increasing demand for recombinant antibodies as detection reagents in research, diagnostics, and therapy requires appropriate production systems. In contrast to antibody therapies, small recombinant antibody fragments like Fab and scFv are sufficient for most applications in research and diagnostics. These antibody fragments can also be produced in bacterial hosts. Gram-negative bacteria, particularly Escherichia coli, were extensively studied for the recombinant antibody production but they showed only a limited capacity to secrete antibody fragments into the medium--a prerequisite for easy downstream processing. Gram-positive bacteria are known to efficiently secrete recombinant proteins into the medium. Recently, we demonstrated the production of scFv and scFab fragments in Bacillus megaterium. Here, we describe the process in detail from transformation of B. megaterium to production and purification of scFv fragments.
在研究、诊断和治疗中，作为检测试剂的重组抗体需求日益增加，这就需要合适的生产系统。与抗体疗法不同，Fab和scFv等小的重组抗体片段足以满足研究和诊断中的大多数应用。这些抗体片段也可以在细菌宿主中产生。革兰氏阴性菌，尤其是大肠杆菌，已被广泛研究用于重组抗体生产，但它们向培养基中分泌抗体片段的能力有限，而这是易于下游加工的前提条件。已知革兰氏阳性菌能有效地将重组蛋白分泌到培养基中。最近，我们证明了巨大芽孢杆菌能够生产scFv和scFab片段。在此，我们详细描述从巨大芽孢杆菌的转化到scFv片段的生产和纯化的过程。
