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

1402656哥伦比亚大学班塔，斯科特1402656从复杂的溶液中分离重要分子通常是使用蛋白质或多肽来完成的，这些蛋白质或多肽被设计成以高亲和力和选择性结合目标。这种方法的一个挑战是目标的回收和结合多肽的重复使用。PI一直在研究一种独特的多肽(称为Beta卷)，这种多肽在没有钙的情况下是无结构的，在有钙的情况下会折叠成扁平的开瓶器形状。PI之前已经设计了用于自组装的开瓶器的一个面，并有初步数据表明，可以将多肽设计为与模型靶蛋白(溶菌酶)结合。该NSF项目的目标是开发一种高通量方法来设计新的Beta卷突变体，该突变体可以与生物技术中重要的不同蛋白质靶标结合。将这些新的工程肽结合到生物分离平台中将是非常有益的，因为它将允许目标蛋白在存在钙的情况下结合，然后在去除钙后释放。这一过程可以提高性能并降低与关键蛋白质分子，特别是治疗性蛋白质相关的成本。亲和分离的关键挑战之一是将目标分子从亲和结合试剂中洗脱出来。PI建议扩展定向进化方法，以显著增加贝塔卷肽与所需靶标的亲和力，并扩大分子识别靶标的数量。已经开发了一种从随机文库中进行选择的方法，但更高亲和力的结合将需要定向进化方法，其中将遗传多样性纳入文库。将研究与绿色荧光蛋白具有高亲和力的新型贝塔卷肽和两种常见的蛋白质表达和纯化标签：麦芽糖结合蛋白和谷胱甘肽S转移酶蛋白。通过将这些进化的、高亲和力的β卷曲多肽固定在合适的载体上，PI可能能够证明使用这些多肽来亲和纯化MBP和GST标记的蛋白质，并使用钙螯合来洗脱纯化的蛋白质。该树脂可以通过添加钙离子进行再生，其性能可以与使用这些融合标签的传统方法(直链淀粉树脂和GSH树脂)进行比较。这一提议的智力价值源于使用一种具有内在触发构象变化的独特多肽作为生物分子识别工程的起始支架。钙诱导的β-卷曲多肽的构象变化是一个强大的分子开关，可以被用来可逆地破坏工程生物分子之间的相互作用。利用这种多肽作为起始支架，我们将能够产生一系列能够以钙依赖的方式与靶蛋白结合的多肽，这些新的多肽将成为亲和生物分离的有价值的生物分子识别元件。这一提议的更广泛影响来自于使用蛋白质工程开发新的结合基序，用于生物传感器和生物分离等应用。利用内在无序的支架进行生物分子识别尚未见文献报道，这一提议将证明这些系统可以被设计成高亲和力的结合剂，这将有利于那些在生物传感器、智能药物递送、生物纳米技术和生物分离等领域工作的人。资金还将用于指导学生和继续在当地社区开展现有的外联活动。

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.