Microsystem-based Formation of Recombinant Protein Fibers

描述（由申请人提供）：拟议项目的目标是使用集成微流体装置研究蛋白质纤维的形成，并开发“绿色”工艺来生成用于生物医学应用的高性能蛋白质纤维。尽管蛋白质合成和制造技术取得了重大进展，但蜘蛛仍然是最好的工程师，可以在低压、室温和水作为溶剂下生产出非常坚固的丝纤维。据推测，蜘蛛能很好地控制丝蛋白的种类、蛋白质溶液的生理条件以及丝线的机械变形，从而纺出强度非凡的丝纤维。在这里，我们假设微系统技术的使用为研究丝纤维形成的机制以及随后开发生产高性能蛋白质纤维的“绿色”工艺提供了一个重要的范例。具体来说，重组丝绸弹性蛋白样蛋白（selp）由来自丝绸和弹性蛋白的多肽序列组成，将被用作拟议项目的模型材料。具体目标确定影响纤维形成和纤维二级结构的蛋白质溶液特性。为了研究SELP纤维的形成，将制作一个由pdm微通道和铝微加热器组成的集成微流控系统，用于局部温度控制。SELP水溶液通过主通道泵入微流控系统，盐和酸性或碱性溶液通过侧通道驱动，可控地调节SELP溶液的离子强度和pH值。pH、离子和温度对纤维形成和纤维二级结构的影响将使用光学显微镜进行监测，并使用各种光谱方法进行表征。具体目标2。研究纺丝管道的流动特性，这些特性决定了纤维的形成、二级结构和selp的机械性能。SELP螺纹的机械变形可以通过缩小通道宽度和改变流量来控制。所得到的SELP纤维的力学性能和二级结构将被表征。在这项工作中，计算流体动力学（CFD）模拟和本构分析模型将被用来加强我们对剪切/伸长流动对纤维形成、结构和性能的影响的理解。具体目标3。探索蛋白质初级结构在调节SELP纤维的形成、结构和特性中的作用。selp由不同长度的丝/弹性蛋白样块和交联位点组成，并将研究其形成纤维的能力。将研究加入赖氨酸残基、类丝块的大小以及类丝与弹性蛋白块的比例对SELP纤维形成、结构和性能的影响。

DESCRIPTION (provided by applicant): The goal of the proposed project is to study protein fiber formation using an integrated microfluidic device, and to develop "green" processes for generating performance protein fibers for biomedical applications. Despite significant advances in protein synthesis and fabrication technology, spiders are still the best engineers producing extraordinarily strong silk fibers at low pressures, ambient temperatures, and with water as solvent. It has been postulated that spiders finely control the types of silk proteins, the physiological conditions of protein solutions, as well as the mechanical deformation of the silk thread in order to spin silk fibers of exceptional strength. Here, we hypothesize that the use of microsystem technology provides an important paradigm for studying the mechanisms involved in silk fiber formation and, subsequently, developing "green" processes for producing performance protein fibers. Specifically, recombinant silk-elastin-like proteins (SELPs) comprised of polypeptide sequences derived from silk and elastin will be used as a model material for the proposed project. Specific Aim 1. Determine the protein solution characteristics that influence the fiber formation and the fiber secondary structures. An integrated microfluidic system consisting of PDMS-based microchannels and an aluminum micro heater, for local temperature control, will be fabricated for studying SELP fiber formation. SELP aqueous solution will be pumped into the microfluidic system through the main channel, while salt and acidic or basic solutions will be driven through side channels to adjust the ionic strength and pH value of the SELP solution in a controlled manner. The effects of pH, ions and temperature on the fiber formation and the fiber secondary structures will be monitored using an optical microscope and characterized using a variety of spectroscopic methodologies. Specific Aim 2. Investigate the flow properties of the spinning duct that determine the fiber formation, secondary structures, and mechanical properties of SELPs. Mechanical deformation of the SELP thread will be controlled by narrowing the channel width and varying the flow rates. The resulting mechanical properties and secondary structures of SELP fibers will be characterized. In this effort, computational fluid dynamics (CFD) simulations and a constitutive analytical model will be utilized to enhance our understanding of the effects of shear/elongational flows on fiber formation, structure, and properties. Specific Aim 3. Explore the effects of the protein primary structures in modulating the formation, structure, and properties of SELP fibers. SELPs composed of varying lengths of silk-/elastin-like blocks and crosslinking sites will be synthesized, and their capability to form fibers will be examined. The influence of incorporating lysine residues, the size of silk-like blocks, as well as the ratio of silk- to elastin- like blocks on the formation, structure, and properties of SELP fibers will be investigated. PUBLIC HEALTH RELEVANCE: Microsystem-Based Formation of Recombinant Protein Fibers Spiders have amazed scientists by producing extraordinarily strong silk fibers using "green" processes at ambient temperatures, low pressures and with water as solvent. We propose to use an integrated microfluidic device as an important tool for studying the formation mechanisms of silk fibers and for developing "green" fiber fabrication techniques. Significantly, the demystification of spiders' remarkable silk spinning process and the development of "green" fabrication techniques may lead to the engineering of performance protein fibers for many biomedical applications.