Hust Michael, Steinwand Miriam, Al-Halabi Laila, Helmsing Saskia, Schirrmann Thomas, Dübel Stefan
Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie, Abteilung Biotechnologie, Spielmannstr. 7, 38106 Braunschweig, Germany.
N Biotechnol. 2009 Sep;25(6):424-8. doi: 10.1016/j.nbt.2009.03.004.
The new era of functional genomics demands several antibodies as specific detection reagents for proteins, their complexes and post-translational modifications. Only in vitro antibody selection technologies are able to provide the required throughput to generate these large numbers. Phage display is the most widely used technology for in vitro selection of antibodies. The major bottleneck of a phage display selection pipeline is the production of monoclonal antibody fragments for screening and further analysis. In this study, we describe the development of improved protocols for the production of single chain Fv (scFv) antibody fragments in 96-well microtitre plates (MTPs) in Escherichia coli. Four scFvs were expressed using the antibody expression vector pOPE101-XP to analyse the influence of a set of different parameters on their production. Further, six scFvs were expressed using the phage display vector pHAL14 to investigate the effect on the production of functional scFvs using those parameters that improved production from pOPE101-XP. Yield in MTPs was influenced by a variety of conditions and was also strongly dependent on the individual scFv clone. Although it was not possible to deduce a single set of optimal parameters applicable to all the tested scFvs, a combined protocol was developed which improved the expression of scFv fragments over standard methods.
功能基因组学的新时代需要多种抗体作为蛋白质、其复合物及翻译后修饰的特异性检测试剂。只有体外抗体筛选技术能够提供所需的通量来产生大量此类抗体。噬菌体展示是体外筛选抗体最广泛使用的技术。噬菌体展示筛选流程的主要瓶颈是用于筛选和进一步分析的单克隆抗体片段的生产。在本研究中，我们描述了在大肠杆菌中于96孔微量滴定板（MTP）中生产单链Fv（scFv）抗体片段的改进方案的开发。使用抗体表达载体pOPE101-XP表达了四种scFv，以分析一组不同参数对其生产的影响。此外，使用噬菌体展示载体pHAL14表达了六种scFv，以研究使用那些改善了pOPE101-XP生产的参数对功能性scFv生产的影响。MTP中的产量受多种条件影响，并且也强烈依赖于各个scFv克隆。尽管不可能推导出适用于所有测试scFv的单一最佳参数集，但开发了一种组合方案，该方案相对于标准方法提高了scFv片段的表达。
