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

从复杂的溶液中分离重要分子通常是通过蛋白质或肽来完成的，这些蛋白质或肽被设计成具有高亲和力和选择性的结合目标。这种方法的一个挑战是目标的恢复和结合肽的再利用。PI一直在研究一种独特的肽（称为β卷），它在没有钙的情况下是无结构的，在有钙的情况下折叠成扁平的螺旋状。PI之前已经设计了螺旋锥的一面用于自组装，并且有初步数据表明肽可以被设计成与模型目标蛋白（溶菌酶）结合。这个NSF项目的目标是开发一种高通量的方法来设计新的β卷突变体，这些突变体可以结合在生物技术中重要的不同蛋白质靶标上。将这些新的工程肽结合到生物分离平台中是非常有益的，因为它允许目标蛋白在钙存在的情况下结合，然后在钙去除后释放。这一过程可以提高性能并降低与关键蛋白质分子相关的成本，特别是治疗蛋白质。基于亲和分离的关键挑战之一是从亲和结合试剂中洗脱目标分子。PI建议扩展定向进化方法，以大幅增加β - roll肽对所需靶点的亲和力，并扩大分子识别靶点的数量。一种从随机文库中进行选择的方法已经开发出来，但更高亲和力的结合物将需要一种定向进化方法，将遗传多样性纳入文库。将研究与GFP高亲和的新型β - roll肽和两个常见的蛋白表达和纯化标签：麦芽糖结合蛋白（MBP）和谷胱甘肽S转移酶蛋白（GST）。通过将这些进化的高亲和力β - roll肽固定在合适的载体上，PI可能能够证明使用这些肽来亲和纯化MBP和gst标记的蛋白质，并使用钙螯合来洗脱纯化的蛋白质。树脂可以通过加钙再生，并且该系统的性能可以与使用这些融合标签（直链淀粉树脂和谷胱甘肽树脂）的传统纯化方法进行比较。该建议的知识价值源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。钙诱导的β -卷曲肽的构象变化是一个强大的分子开关，可以被利用来可逆地破坏工程生物分子相互作用。使用这种肽作为起始支架，我们将能够生成一系列能够以钙依赖的方式结合靶蛋白的肽，这些新肽将成为亲和生物分离中有价值的生物分子识别元件。这一建议的广泛影响来自于利用蛋白质工程开发一种新的结合基序，用于生物传感器和生物分离等应用。使用内在无序支架进行生物分子识别尚未在文献中报道，该提案将证明这些系统可以被设计成高亲和力的粘合剂，这将有利于那些在生物传感器、智能药物输送、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，并继续在当地社区开展现有的外展活动。

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.