Sastry L, Mubaraki M, Janda K D, Benkovic S J, Lerner R A
Department of Molecular Biology & Chemistry, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Ciba Found Symp. 1991;159:145-51; discussion 151-5. doi: 10.1002/9780470514108.ch10.
A bacteriophage lambda vector system for the expression of Fab fragments from the mouse antibody repertoire in Escherichia coli has been described. We have used this system to generate a catalytic antibody from a combinatorial antibody library. Monoclonal antibody 43C9 was raised against a transition state analogue of the hydrolysis of carboxyamide. mRNA from hybridoma cells expressing this antibody was cloned into phage lambda and clones that expressed the mRNA for either the heavy or the light chain of the antibody were isolated. These individual libraries were then crossed to generate a combinatorial library in which clones coexpressed the heavy and light chains. This library was screened for antibodies/Fab fragments that bound to the original antigen with high affinity. DNA sequencing showed that these fragments were the same as those in antibody 43C9. Three different clones were found to catalyse the hydrolysis of carboxyamide. More efficient expression vectors and improved screening techniques should lead to the isolation of many more catalytic antibodies from combinatorial antibody libraries.
已描述了一种用于在大肠杆菌中表达来自小鼠抗体库的Fab片段的噬菌体λ载体系统。我们已使用该系统从组合抗体库中产生一种催化抗体。单克隆抗体43C9是针对羧酰胺水解的过渡态类似物产生的。将表达该抗体的杂交瘤细胞的mRNA克隆到噬菌体λ中，并分离出表达该抗体重链或轻链mRNA的克隆。然后将这些单独的文库杂交以产生一个组合文库，其中克隆共表达重链和轻链。筛选该文库以寻找与原始抗原具有高亲和力结合的抗体/Fab片段。DNA测序表明这些片段与抗体43C9中的片段相同。发现三个不同的克隆催化羧酰胺的水解。更有效的表达载体和改进的筛选技术应能从组合抗体库中分离出更多的催化抗体。
