Production of a Lung Surfactant SP-B Mimic

描述（由申请人提供）：表面活性剂蛋白B （SP-B）对肺表面活性剂的正常功能很重要。利用已知的“皂苷”类蛋白质的三维结构，早期SP-B模型预测，二硫交联、N端和c端结构域折叠成带电荷的两亲螺旋，并且这些相邻的螺旋可能参与关键的表面活性剂活性。在小范围内，我们化学合成了34个残基，二硫连接的n端- c端肽结构（即“Mini-B”），模拟了肺表面活性物质脂质中79个残基亲本蛋白的体外-体内结构和功能。然而，通过化学合成方法或通过细菌表达系统来扩大Mini-B的合成规模已被证明是有问题的。在这个SBIR拨款申请中，我们建议通过使用无细胞表达系统（CFES）来解决这个问题，这将促进高纯度Mini-B肽的大规模生产。开发一种商业上可行的生产方法将允许Mini-B肽在生物医学应用中的使用，包括肺表面活性剂替代疗法，如新生儿呼吸窘迫综合征（NRDS）、成人呼吸窘迫综合征（ARDS），以及促进抗生素、抗炎药和遗传物质的新型肺部输送系统的开发。

DESCRIPTION (provided by applicant): Surfactant Protein B (SP-B) is important for the normal functioning of lung surfactant. Using the known three-dimensional structures of proteins which also belong to the "saposin" class, early SP-B models predicted that the disulfide cross-linked, N- and C-terminal domains fold as charged amphipathic helices, and further that these adjacent helices may participate in critical surfactant activities. On a small-scale, we have chemically synthesized a 34 residue, disulfide-linked N-terminal - C-terminal peptide construct (i.e., "Mini-B") that emulates the in vitro - in vivo structure and function of the 79 residue parent protein in lung surfactant lipids. However, scale-up of the synthesis of Mini-B by either chemical synthetic methods or through a bacterial expression system has proven to be problematic. In this SBIR grant application, we propose to address this issue by utilizing a cell-free expression system (CFES) that would facilitate the large-scale production of high-purity Mini-B peptide. Development of a commercially viable production method will permit the use of the Mini-B peptide in biomedical applications, including lung surfactant replacement therapies such as Neonatal Respiratory Distress Syndrome (NRDS), Adult Respiratory Distress Syndrome (ARDS), as well as facilitate the development of novel delivery systems to the lung for antibiotics, anti-inflammatory agents, and genetic material.