Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie, Abteilung Biotechnologie, Spielmannstr, 7, 38106 Braunschweig, Germany.
Microb Cell Fact. 2007 Nov 27;6:38. doi: 10.1186/1475-2859-6-38.
The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli.
The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli.
B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.
在研究、诊断和治疗中对抗原结合试剂的需求引发了对新型抗体形式以及合适生产系统的疑问。最近，新型单链 Fab（scFab）抗体形式结合了单链 Fv（scFv）和 Fab 片段的特性，在革兰氏阴性菌大肠杆菌中得到了生产。在这项研究中，我们评估了革兰氏阳性菌巨大芽孢杆菌在重组生产 scFab 和 scFv 方面与大肠杆菌的比较。
特异性针对溶菌酶的 D1.3 scFab 在巨大芽孢杆菌和大肠杆菌中得到了生产。从大肠杆菌的周质部分和培养上清液中纯化获得的 scFab 的总产率略高于从巨大芽孢杆菌的培养上清液中获得的产率。然而，通过分析抗原结合活性来确定功能性 scFab 的产率在这两种生产系统中是相等的。此外，还生产了一种针对人 C 反应蛋白的特异性 scFv 片段。在巨大芽孢杆菌中生产的抗 CRP scFv 的总产量略低于大肠杆菌，而在巨大芽孢杆菌中生产的纯化 scFvs 的比活性高于大肠杆菌。
巨大芽孢杆菌允许包括新型 scFab 抗体形式在内的抗体片段的分泌生产。功能性抗体片段的产量和质量与大肠杆菌中的周质生产相当。
