Shibui T, Munakata K, Matsumoto R, Ohta K, Matsushima R, Morimoto Y, Nagahari K
Biosciences Laboratory, Mitsubishi Kasei Corporation, Kanagawa, Japan.
Appl Microbiol Biotechnol. 1993 Mar;38(6):770-5. doi: 10.1007/BF00167143.
A high-level secretion system for the production of mouse-human chimeric antibody 21B2 (MHC 21B2) Fab fragment specific for human carcino embryonic antigen (hCEA) in Escherichia coli has been constructed. The genes encoding a light chain and an Fd fragment (a variable region and the CH1 domain of a heavy chain) of a mouse-human chimeric antibody were directly fused to the signal peptide of the E. coli ompF gene sequence. E. coli cells containing expression vectors in which each of the two genes are located downstream of a separate tac promoter were able to secrete the light chain and Fd fragment as two of their major cellular proteins. The signal peptides were efficiently removed from the primary products by post-translational processing, although they formed insoluble aggregates, possibly in the periplasm. In high-cell-density culture experiments using a jar fermentor, the amount of light chain and Fd fragment produced was at levels of up to 2.88 g/l and 1.28 g/l culture, respectively. By optimizing the conditions that encourage correct folding, formation of disulphide bonds, and association of the light chain with the Fd fragment, we have established a procedure that can purify, re-fold, and combine aggregated products to electrophoretically homogeneous Fab fragment with a yield of approximately 47%. Fab fragment produced in this manner shows essentially the same antigen-binding activity and specificity to hCEA as the parental mouse antibody 21B2 (MoAb 21B2).
构建了一种用于在大肠杆菌中生产对人癌胚抗原（hCEA）具有特异性的小鼠 - 人嵌合抗体21B2（MHC 21B2）Fab片段的高效分泌系统。编码小鼠 - 人嵌合抗体轻链和Fd片段（重链可变区和CH1结构域）的基因直接与大肠杆菌ompF基因序列的信号肽融合。含有表达载体的大肠杆菌细胞，其中两个基因各自位于单独的tac启动子下游，能够将轻链和Fd片段作为其主要细胞蛋白之一分泌出来。信号肽在翻译后加工过程中从初级产物中被有效去除，尽管它们形成了不溶性聚集体，可能存在于周质中。在使用罐式发酵罐的高细胞密度培养实验中，产生的轻链和Fd片段的量分别达到高达2.88 g/l和1.28 g/l培养物。通过优化促进正确折叠、二硫键形成以及轻链与Fd片段缔合的条件，我们建立了一种可以纯化、重折叠并将聚集产物组合成电泳纯的Fab片段的方法，产率约为47％。以这种方式产生的Fab片段显示出与亲本小鼠抗体21B2（MoAb 21B2）基本相同的抗原结合活性和对hCEA的特异性。
