Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv
Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv
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DOI：
10.1007/s00253-014-6268-4
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发表时间：
2015-03-01
影响因子：
5
通讯作者：
O'Kennedy, Richard
中科院分区：
文献类型：
--
作者：
Ayyar, B. Vijayalakshmi;Hearty, Stephen;O'Kennedy, Richard
兔源性重组抗体传统上被认为是难以处理的分子，因为在V-L结构域的第80位存在半胱氨酸，当存在于单链Fv （scFv）和嵌合Fab格式的“非配对”环境中时，该半胱氨酸会变得“异常”。当兔重组抗体在原核生物系统中产生时，这种异常的Cys80可以严重影响可实现的表达水平。未配对的Cys残基也使纯化变得困难。因此，研究人员往往忽视兔抗体库由于可获得的曲目多样性的感知限制。我们已经证明，通过在真实筛选活动中分离的含有异常cys的兔scFv中切换V-H和V-L结构域的方向，可以大大提高该克隆的表达和纯化产量。此外，通过加入一个新的兔C-kappa恒定融合域，我们能够进一步提高表达水平，并纯化该抗体达到高度的同质性，这是迄今为止使用含有异常cys的野生型scFv无法实现的。总的来说，这些发现表明，简单的重新格式化可以帮助兔抗体库，一个非常宝贵的资源，更容易获得该领域的研究人员。
Rabbit-derived recombinant antibodies have traditionally been viewed as intractable molecules due to the presence of a cysteine in position 80 of the V-L domain that becomes rendered 'aberrant' when present in the 'unpaired' context of a single chain Fv (scFv) and chimeric Fab formats. This aberrant Cys80 can severely impinge on the achievable expression levels when rabbit recombinant antibodies are produced in prokaryote systems. The unpaired Cys residue also renders purification problematic. Consequently, researchers often disregard rabbit antibody libraries due to perceived limitations in accessible repertoire diversity. We have shown that by switching the orientation of the V-H and V-L domains in an aberrant-Cys-containing rabbit scFv isolated in a bona fide screening campaign, it was possible to substantially increase the expression and purification yields of this clone. Furthermore, by incorporating a novel rabbit C-kappa constant fusion domain, we were able to potentiate a further increase in expression level and purify this antibody to a high degree of homogeneity, hitherto impossible to achieve using the aberrant-Cys-containing wild-type scFv. Cumulatively, these findings demonstrate that facile re-formatting can help make the rabbit antibody repertoire, a very valuable resource, more accessible to researchers in the field.