Directed Evolution of peptides that bind protein targets only in the presence of calcium: A new tool for bioseparations

仅在钙存在的情况下结合蛋白质靶标的肽的定向进化：生物分离的新工具

• 批准号: 1402656
• 负责人: Scott Banta
• 金额: $ 35.98万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1402656&HistoricalAwards=false
• 关键词: Directed; Evolution; peptides; bind; protein

Banta, Scott 1402656 Columbia University The separation of important molecules from complex solutions is often accomplished using proteins or peptides that have been engineered to bind the target with high affinity and selectivity. One challenge in this approach is the recovery of the target, and reuse of the binding peptide. The PI has been working with a unique peptide (called the beta roll) that is unstructured in the absence of calcium, and folds into a flattened corkscrew shape in the presence of calcium. The PI has previously engineered one face of the corkscrew for self-assembly, and has preliminary data showing the peptide can be engineered to bind to a model target protein (lysozyme). The goal of this NSF project is to develop a high throughput method to engineer new beta roll mutants that can bind to different protein targets that are important in biotechnology. The incorporation of these new engineered peptides into a bioseparations platform would be very beneficial, as it would allow for target proteins to be bound in the presence of calcium and then released upon calcium removal. This process could improve performance and reduce the costs associated with critical protein molecules, especially therapeutic proteins. One of the key challenges in affinity-based separations is the elution of the target molecule from the affinity binding reagent. The PI proposes to expand the directed evolution approach to substantially increase the affinity of the beta roll peptides to desired targets and expand the number of targets for molecular recognition. A method for selection from a randomized library has been developed, but higher affinity binders will require a directed evolution approach where genetic diversity is incorporated into the library. New beta roll peptides with high affinity for GFP and two common protein expression and purification tags will be researched: the maltose binding proteins (MBP) and the glutathione S transferase protein (GST). By immobilizing these evolved, high-affinity beta roll peptides on a suitable support, the PI may be able to demonstrate the use of these peptides to affinity purify MBP- and GST-tagged proteins, and use calcium chelation to elute the purified proteins. The resin can be regenerated via calcium addition and the performance of this system can be compared to traditional methods for purification with these fusion tags (amylose resin and GSH resin). The Intellectual Merit of this proposal results from the use of a unique peptide with an intrinsic triggered conformational change as a starting scaffold for the engineering of biomolecular recognition. The calcium-induced conformational change of the beta roll peptides is a powerful molecular switch that can be exploited to reversibly disrupt engineered biomolecular interactions. Using this peptide as a starting scaffold, we will be able to generate a collection of peptides that can bind target proteins in a calcium-dependent fashion, and these new peptides will be valuable biomolecular recognition elements for affinity bioseparations. The Broader Impact of this proposal arises from the use of protein engineering to develop a new binding motif for use in applications such as biosensors and bioseparations. The use of intrinsically disordered scaffolds for biomolecular recognition has not yet been reported in the literature, and this proposal will demonstrate that these systems can be engineered to be high affinity binders, which will be boon to those working in areas such as biosensors, smart drug delivery, bionanotechnology, and bioseparations. Funding will also be used for the mentoring of students and to continue existing outreach activities in the local community.

Banta，Scott 1402656哥伦比亚大学，通常使用经过设计以高亲和力和选择性结合目标的蛋白质或肽来完成重要分子与复杂溶液的分离。在这种方法中，一个挑战是恢复靶标，并重复使用结合肽。 PI一直在使用独特的肽（称为β卷），该肽在没有钙的情况下是非结构的，并在钙的存在下折叠成扁平的开瓶器形状。 PI先前已经设计了开瓶器的一张面孔进行自组装，并具有初步数据，表明该肽可以被设计成与模型靶蛋白（溶菌酶）结合。该NSF项目的目的是开发一种高通量方法，以设计新的β卷突变体，该方法可以与生物技术重要的不同蛋白质靶标结合。将这些新工程肽掺入生物序列平台中非常有益，因为它可以在钙存在下结合靶蛋白，然后在去除钙后释放。该过程可以提高性能并降低与关键蛋白质分子相关的成本，尤其是治疗蛋白。基于亲和力的分离的主要挑战之一是从亲和力结合试剂中洗脱靶分子。 PI提议扩大有向进化的方法，以大大增加β掷肽对所需靶标的亲和力，并扩大分子识别的靶标数。已经开发了一种从随机库中选择的方法，但是较高的亲和力粘合剂将需要一种定向进化方法，其中遗传多样性纳入了库中。将研究对GFP高亲和力的新β卷肽，将研究两个常见的蛋白质表达和纯化标签：麦芽糖结合蛋白（MBP）和谷胱甘肽的转移酶蛋白（GST）。通过将这些进化的高亲和力β胶卷固定在合适的支撑上，PI可能能够证明使用这些肽来纯化MBP和GST标记的蛋白质，并使用钙螯合以洗脱纯化的蛋白质。可以通过钙添加钙再生树脂，并可以将该系统的性能与这些融合标签（淀粉糖树脂和GSH树脂）进行纯化的传统方法进行比较。该提案的智力优点是由于使用具有内在触发构象变化的独特肽作为生物分子识别工程的起始支架。 β胶卷肽的钙诱导的构象变化是一种强大的分子开关，可以利用可逆地破坏工程的生物分子相互作用。我们将使用该肽作为起始支架，我们将能够生成一系列可以以钙依赖性的方式结合靶蛋白的肽，并且这些新肽将是有价值的生物分子识别元素的亲和力生物脱位。该提案的更广泛影响是由使用蛋白质工程来开发用于在生物传感器和生物释放等应用中使用的新结合基序。文献中尚未报告固有无序的脚手架识别的固有脚手架识别，该提案将证明这些系统可以设计为高亲和力粘合剂，这将为生物传感器，智能药物递送，BionAnotechnology和Bioseparparations等领域的工作恩赐。资金还将用于指导学生，并继续在当地社区中进行现有的外展活动。