RII Track-4:NSF: Bioactive Surfaces Through Affinity Tag Protein-Polymer Conjugation

该项目中所涉及的技术有可能缓解与表面交互相关的日常常见挑战。在不方便的时候把咖啡洒在衬衫上，或者在全球大流行期间对触摸门把手感到恐惧，这些都是本研究旨在缓解的挑战的例子。人们日常接触的表面是被动的，需要不断清洁才能方便和安全。然而，如果表面是自清洁的、抗菌的、保护的或治疗的，则与表面的相互作用将是不同的。蛋白质提供许多对健康、农业、国防和食品加工重要的功能。聚合物与蛋白质的永久缀合，产生蛋白质-聚合物缀合物，是广泛使用的策略，以增强这些许多应用的蛋白质稳定性。然而，这种化学方法仍然存在挑战，包括通过不加选择的缀合或通过聚合物破坏蛋白质折叠成其活性结构来降低蛋白质活性的可能性。此外，这些共价策略导致材料浪费。一旦蛋白质失活，蛋白质、聚合物和它们所缀合的任何表面也将由于这种永久的共价化学作用而损失。该项目旨在通过一个简单而强大的非永久性相互作用来实现蛋白质-聚合物结合，以产生再生的，蛋白质修饰的“生物活性”表面。该研究基础设施改善轨道-4 EPSCoR研究员（RII轨道-4）项目将提供奖学金给助理教授，并为南密西西比大学（USM）的研究生提供培训。蛋白质对健康、农业、运输、国防、食品加工和许多其他方面都具有重要的功能。对蛋白质的这种需求导致了寻找将这些生物催化剂掺入合成系统中的策略的愿望的增加，同时提供蛋白质活性的稳定性和寿命。尽管在开发共价蛋白质-聚合物缀合物以实现这一点方面取得了一些成功，但共价修饰的挑战仍然存在，导致由蛋白质变性引起的蛋白质活性降低或丧失。该项目将采用可扩展的组氨酸标签（His-标签）亲和配体化学，开发并常规用于重组蛋白纯化，结合可逆加成片段化链转移（RAFT）聚合作为一种策略，以创建非共价蛋白质-聚合物缀合物，以涂覆表面并赋予生物活性。利用His标签进行缀合将确保聚合物附着在蛋白质的N-或C-末端的位点，避免聚合物附着在蛋白质活性位点，防止变性，同时适用于用该化学纯化的几乎所有重组蛋白。非共价偶联化学将允许构建蛋白质特异性生物活性表面，易于清洗去除和活性重组蛋白的重新偶联，从而产生适合减少废物的再生表面。成功完成这一原理方法的验证将为高性能生物活性涂层提供一个平台技术。PI Clemons将接受石楠梅纳德的指导，她是加州洛杉矶大学（UCLA）的化学教授，也是NSF资助的BioPACIFIC MIP的联合主任，是蛋白质-聚合物结合领域的世界领导者。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

The technology addressed in this project has potential to mitigate everyday common challenges associated with interacting with surfaces. Spilling coffee on a shirt at an inconvenient time or having trepidation about touching a door handle during a global pandemic are examples of challenges that this research aims to mitigate. The surfaces that one interacts with on a daily basis are passive, requiring constant cleaning to be convenient and safe. However, interaction with surfaces would be different if surfaces were self-cleaning, antibacterial, protective, or therapeutic. Proteins provide many functions important to health, agriculture, defense, and food processing. Permanent conjugation of polymers to proteins, creating protein-polymer conjugates, is a widely used strategy to enhance protein stability for these many applications. However, challenges remain with this chemistry, including the potential of reducing protein activity through indiscriminate conjugation, or via the polymer disrupting the protein from folding into its active structure. Further, these covalent strategies contribute to material waste. Once the protein is rendered inactive, the protein, polymer, and any surface these are conjugated to will also be lost due to this permanent covalent chemistry. This project aims to develop strategies for achieving protein-polymer conjugation through a simple yet strong non-permanent interaction to produce regenerative, protein decorated ‘bioactive’ surfaces.This Research Infrastructure Improvement Track-4 EPSCoR Research Fellows (RII Track-4) project would provide a fellowship to an Assistant professor and training for a graduate student at the University of Southern Mississippi (USM). Proteins provide a myriad of functions important to health, agriculture, transportation, defense, food processing and many others. This demand for proteins has led to an increase in the desire to find strategies to incorporate these biological catalysts into synthetic systems, while providing stability and longevity of protein activity. Despite some successes in developing covalent protein-polymer conjugates to achieve this, challenges with covalent modification remain, leading to reduced or loss of protein activity resulting from protein denaturation. This project will employ scalable histidine tag (His-tag) affinity ligand chemistries, developed and routinely used for recombinant protein purification, coupled with reversible-addition fragmentation chain transfer (RAFT) polymerization as a strategy to create non-covalent protein-polymer conjugates to coat surfaces and impart bioactivity. Utilizing the His-tag for conjugation will ensure polymer attachment at the site of the N- or C-terminus of the protein, avoiding polymer attachment at the protein active site, preventing denaturation, while being amenable to almost all recombinant proteins purified with this chemistry. The non-covalent coupling chemistry will allow for the construction of protein specific bioactive surfaces, with the ease of wash removal and recoupling of active recombinant protein, creating a regenerative surface, suitable for waste reduction. Successful completion of this proof of principle approach will provide a platform technology suitable for high performing bioactive coatings. PI Clemons will be mentored by Heather Maynard, Professor of Chemistry at the University of California Los Angeles (UCLA), Co-Director of the NSF-funded BioPACIFIC MIP, and a world-leader in protein-polymer conjugation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.