Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling.
Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling.
复制标题
DOI：
10.1186/1756-6606-4-2
复制
发表时间：
2011-01-07
期刊：
影响因子：
3.6
通讯作者：
Kubo T
中科院分区：
文献类型：
--
作者：
Naimuddin M;Kobayashi S;Tsutsui C;Machida M;Nemoto N;Sakai T;Kubo T
生物分子如DNA、RNA和蛋白质的定向进化已经成为获得用于各种目的的分子的有效方法。近年来，来自非免疫球蛋白的蛋白引起了人们的注意，因为它们在结合潜力方面模拟抗体并提供进一步的潜在优势。在这方面，我们试图探索一种三指神经毒素蛋白（3F）。3F蛋白质小（~7 kDa），结构明确，热稳定并且对蛋白水解具有抗性，这使它们成为定向进化的有希望的候选者。我们通过随机化与乙酰胆碱受体结合的环中的残基，并利用cDNA展示来获得白细胞介素-6受体（IL-6 R）调节剂，从而设计了一种属于3F家族的蛇α-神经毒素。所选候选物对IL-6 R具有高度特异性，解离常数和IC 50在纳摩尔范围内。在IL-6依赖性细胞增殖测定中鉴定拮抗剂以及激动剂。大小最小化产生的肽的分子量约为原始蛋白质的三分之一，而没有显着的活动损失，此外，导致识别负责功能的环。这项研究表明，3F蛋白适合在环中引入氨基酸变化，从而能够制备可用于获得针对大分子的配体的高多样性文库。我们相信这是第一个报告的蛋白质工程，以转换神经毒素受体配体以外的父母受体，从非免疫球蛋白的激动剂的鉴定，肽模拟IL-6的建设，并成功地减少单链蛋白的大小。
Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F). 3F proteins are small (~7 kDa), structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution. We have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R). Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function. This study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.
登录
查看更多内容
影响因子：
3.8
作者：
BOCK, GH;LONG, CA;NELSON, DL
通讯作者：
NELSON, DL
影响因子：
5.6
作者：
Binz, HK;Stumpp, MT;Plückthun, A
通讯作者：
Plückthun, A
影响因子：
3.8
作者：
Fee, D;Grzybicki, D;Fabry, Z
通讯作者：
Fabry, Z
DOI：
10.1073/pnas.93.12.5688
发表时间：
1996-06-11
影响因子：
11.1
作者：
Braisted, AC;Wells, JA
通讯作者：
Wells, JA
DOI：
10.1073/pnas.0602658103
发表时间：
2006-09-26
影响因子：
11.1
作者：
Frederickson, Shana;Renshaw, Mark W.;Bowdish, Katherine S.
通讯作者：
Bowdish, Katherine S.