Suderman Richard J, Rice Daren A, Gibson Shane D, Strick Eric J, Chao David M
Nectagen, Inc., 2002 W. 39th Ave, Kansas City, KS 66103, USA.
Nectagen, Inc., 2002 W. 39th Ave, Kansas City, KS 66103, USA.
Protein Expr Purif. 2017 Jun;134:114-124. doi: 10.1016/j.pep.2017.04.008. Epub 2017 Apr 17.
The purification of functional proteins is a critical pre-requisite for many experimental assays. Immunoaffinity chromatography, one of the fastest and most efficient purification procedures available, is often limited by elution conditions that disrupt structure and destroy enzymatic activity. To address this limitation, we developed polyol-responsive antibody mimetics, termed nanoCLAMPs, based on a 16 kDa carbohydrate binding module domain from Clostridium perfringens hyaluronidase. nanoCLAMPs bind targets with nanomolar affinity and high selectivity yet release their targets when exposed to a neutral polyol-containing buffer, a composition others have shown to preserve quaternary structure and enzymatic activity. We screened a phage display library for nanoCLAMPs recognizing several target proteins, produced affinity resins with the resulting nanoCLAMPs, and successfully purified functional target proteins by single-step affinity chromatography and polyol elution. To our knowledge, nanoCLAMPs constitute the first antibody mimetics demonstrated to be polyol-responsive.
功能性蛋白质的纯化是许多实验分析的关键先决条件。免疫亲和色谱法是目前最快且最有效的纯化方法之一，但它常常受到洗脱条件的限制，这些条件会破坏蛋白质结构并使其酶活性丧失。为了解决这一局限性，我们基于产气荚膜梭菌透明质酸酶的一个16 kDa碳水化合物结合模块结构域，开发了一种多元醇响应性抗体模拟物，称为纳米钳。纳米钳以纳摩尔亲和力和高选择性结合靶标，但当暴露于含中性多元醇的缓冲液时会释放其靶标，其他人已证明这种缓冲液成分能够保留蛋白质四级结构和酶活性。我们筛选了一个噬菌体展示文库，以寻找能够识别几种靶蛋白的纳米钳，用所得的纳米钳制备了亲和树脂，并通过单步亲和色谱法和多元醇洗脱成功纯化了功能性靶蛋白。据我们所知，纳米钳是首个被证明具有多元醇响应性的抗体模拟物。
