Miller G P, Posner B A, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Bioorg Med Chem. 1997 Mar;5(3):581-90. doi: 10.1016/s0968-0896(96)00271-4.
We employed a chain-shuffling technique to determine if the light chain of the catalytic antibody, 43C9, provides the best partner for the 43C9 heavy chain. Previously, we reported construction and screening of a 43C9 HC CROSS library, where the 43C9 heavy-chain gene was crossed with a library of light-chain genes in a lambda bacteriophage system. The library contained a high frequency of reconstituted antibodies recognizing the transition-state analogue. Here, we report the isolation and characterization of four of these clones. Recovered light-chain proteins share 92-96% sequence identity to the 43C9 light-chain protein. Somatic mutations of these light chains occur randomly at positions distant from the active site. Residues required for binding and catalysis were conserved. Mutations affected the topology of the binding site. Nevertheless, catalysis was not affected. Isolation of these light chains suggests the best partner for the 43C9 heavy chain is the original light chain. These clones attempt to broaden a class of 43C9-like antibodies, where the catalytic residues, His91 and Arg96, have been reproducibly selected. Similar catalytic properties between the 43C9-like antibodies suggests binding has been optimized, thus further maturation of the light chain would not lead to a better catalyst. To improve catalysis, other approaches must be considered.
我们采用了链改组技术来确定催化抗体43C9的轻链是否为43C9重链的最佳搭档。此前，我们报道了43C9 HC CROSS文库的构建与筛选，其中43C9重链基因在λ噬菌体系统中与轻链基因文库进行了杂交。该文库中含有高频率的可识别过渡态类似物的重组抗体。在此，我们报道了其中四个克隆的分离与鉴定。回收的轻链蛋白与43C9轻链蛋白的序列同一性为92% - 96%。这些轻链的体细胞突变在远离活性位点的位置随机发生。结合和催化所需的残基是保守的。突变影响了结合位点的拓扑结构。然而，催化作用并未受到影响。这些轻链的分离表明43C9重链的最佳搭档是原始轻链。这些克隆试图拓宽一类43C9样抗体，其中催化残基His91和Arg96已被反复筛选出来。43C9样抗体之间相似的催化特性表明结合已得到优化，因此轻链的进一步成熟不会产生更好的催化剂。为了提高催化作用，必须考虑其他方法。
