Development of a Novel Method to Enhance Therapeutic Protein Production

描述（由申请人提供）：任何蛋白质治疗练习的第一步是蛋白质的表达。在某些情况下，这一步并不麻烦。然而，随着设计蛋白数量的增加和翻译后修饰的要求，增强表达和提高蛋白产量已成为非常具有挑战性的任务。商品成本是一个重要因素。在某些情况下，由于商品成本不合理，治疗性蛋白质项目被放弃。公司花12-18个月的时间来优化细胞系，提高重组蛋白的产量，这可能会让人感到惊讶。已经采取了几种方法来克服表达问题；然而，很有可能没有单一的技术或程序能够克服难以表达的蛋白质问题。宿主的选择、培养条件和下游加工都对产品蛋白质的质量和数量起作用。LifeSensors之前描述了一种基于相扑的新型融合系统。SUMO附着在无活性或低表达蛋白的n端上，可以显著改善蛋白质的功能，并使蛋白质产量提高10-500倍。在原核生物中的表达产生完整的融合，通过SUMO的n端多种亲和标签纯化。SUMO融合体在体外被高效的SUMO蛋白酶裂解，产生具有所需n -末端的天然蛋白。然而，类似的系统不能用于高等真核细胞，例如：主要用于生产治疗性抗体和蛋白质。在本提案中，我们描述了相关融合系统的发展，该系统将增强CHO细胞中难以表达的蛋白的表达和分泌。随着大量工程化抗体和治疗性蛋白作为临床候选物的出现，蛋白表达已成为一个真正的瓶颈。开发一种将蛋白质生产质量和数量提高10倍的系统将是蛋白质治疗学的一大进步。

DESCRIPTION (provided by applicant): The first step in any protein therapeutic exercise is expression of the protein. In some cases, this step is not troublesome. For increasing numbers of designer proteins and requirements for post-translation modifications, however, enhanced expression and increasing protein yield have become very challenging tasks. Cost of goods is an important factor. In some cases therapeutic protein projects are abandoned because of an unreasonable cost of goods. It may come as a surprise that companies spend 12-18 months to optimize the cell lines and improve the yield of recombinant proteins. Several approaches have been taken to overcome the expression problems; nevertheless, it is highly likely that no single technology or procedure will be able to overcome the difficult-to-express protein problem. Selection of host, culture conditions and downstream processing can all play a role in the quality and quantity of product protein. LifeSensors has previously described a novel SUMO based fusion system. Attachment of SUMO to N-termini of inactive or poorly expressed proteins has led to a remarkable improvement in the functionality of proteins and 10-500-fold enhancement in protein production. Expression in prokaryotes yields an intact fusion that is purified by virtue of a variety of affinity tags at the N-terminus of SUMO. SUMO-fusions are cleaved in vitro by a highly efficient SUMO protease to generate native protein with desired N-termini. However, a similar system is not available for higher eukaryotic cells ,e.g., CHO that are primarily used for production of therapeutic antibodies and proteins. In this proposal we describe the development of related fusion system that will enhance the expression and secretion of difficult-to-express protein in CHO cells. With the advent of large numbers of engineered antibodies and therapeutic proteins as clinical candidates, protein expression has become a real bottleneck. Development of a system that improves quality and quantity of protein production by 10x fold will be a great advance in protein therapeutics. PUBLIC HEALTH RELEVANCE: Production of recombinant protein is a costly process. Increasing numbers of proteins are engineered to develop therapeutic products. It is becoming difficult to produce some of the proteins at a reasonable cost to the point that clinical projects are abandoned due to high cost of protein production. We have proposed the novel use of our previously demonstrated fusion proteins. Such a strategy would likely increase the quantity and quality of protein expression in therapeutics development.