A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin-Calmodulin Binding Peptide Interaction.
A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin-Calmodulin Binding Peptide Interaction.
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DOI：
10.1016/j.jmb.2015.06.018
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发表时间：
2015-08-14
影响因子：
5.6
通讯作者：
Kossiakoff AA
中科院分区：
文献类型：
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作者：
Mukherjee S;Ura M;Hoey RJ;Kossiakoff AA
可逆的，高亲和力的固定标签是无数生物应用的关键工具。然而，固有的问题与一些目前的固定方法有关。特别是，噬菌体展示分选的一个关键因素是靶蛋白的功能固定化。为了避免这些问题，我们使用了钙调素结合肽（CBP）的突变体（N5A）作为噬菌体展示分选的固定标签。这种固定依赖于钙（KD ~ 2pM）存在时，钙调素对N5A突变体CBP （RWKKNFIAVSAANRFKKIS）的超高亲和力，这种亲和力可以通过EDTA逆转，从而控制特定结合物的“捕获和释放”。为了评估该系统的能力，我们选择了8个靶标，其中一些难以用其他标签过表达和纯化，一些在分选实验中失败。在所有情况下，使用具有CBP融合结构的Fab噬菌体展示库生成特异性结合物。这些fab的KD在亚至低纳摩尔（nM）范围内，在细胞实验中成功地用于选择性识别抗原。即使没有任何标签，其中一些目标也是有问题的，所以所有这些都导致了成功的选择终点，这意味着可以以高概率的积极结果处理边缘情况。结合构象、表位和结构域特异性fab的生成等成功案例的高水平应用，我们认为CBP标签体现了共价固定标签的所有属性，但没有它们的一些众所周知的缺点。
Reversible, high affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD ~ 2pM), which can be reversed by EDTA allowing controlled “capture and release” of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP fused constructs. KD of the Fabs were in sub to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag, so the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of positive outcome. Taken together with examples of successful case specific high level applications like generation of conformation, epitope and domain specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags, but does not suffer from some of their well documented drawbacks.