Enhancing mammalian glycoprotein production in the baculovirus expression vector

描述（由申请人提供）：杆状病毒表达载体系统（BEVS）是一种经过验证的、强大的、通用的真核蛋白表达方法。它被用于为众多研究项目生产疫苗、诊断和生物活性蛋白。然而，像所有的表达系统一样，BEVS也有它的缺点。一个是由于病毒诱导的细胞死亡和裂解，表达是短暂的。paratech公司已经将一款解决这一问题的产品商业化。表达病毒锚蛋白基因的细胞系显示杆状病毒感染细胞的延迟死亡和裂解，从而显著提高重组蛋白的生产。这种活性被称为vankyrin-enhanced BEVS (VE-BEVSTM)，根据蛋白质和vankyrin递送方法的不同，可将靶蛋白表达提高20倍。杆状病毒表达的第二个限制是昆虫细胞缺乏产生最终唾液化的复杂n -聚糖的能力，这限制了BEVS用于表达人类治疗性蛋白的有效性。该提案解决了这一问题，旨在开发新的细胞系，在vankyrin增强杆状病毒技术的背景下产生真正的“人源化”n-聚糖。为了实现这一目标，将三种不同的昆虫细胞系与编码vankyrin基因和不同测试糖蛋白的杆状病毒共同感染，这些细胞系先前已被设计用于产生哺乳动物糖基化酶。在几个启动子的控制下，用两种不同的vankyrin基因转化具有最高vankyrin应答的转基因细胞系。将筛选多克隆细胞以增强糖蛋白表达，并克隆最有希望的细胞群。与未转化的亲本系相比，将测试不同的单克隆系是否产生复杂的末端唾液化糖蛋白。这些细胞将提供最高产量的精确加工分泌蛋白和跨膜蛋白，并将销售给从事人类分泌组结构-功能研究和蛋白质疗法开发的个人研究人员和制药公司。这些I期研究将显著扩展ParaTechs的vankyrin增强BEVS技术的应用。paratech人员和Jarvis小组对这些研究中使用的所有技术都有经验，并且预计实现目标不会有困难。该I期申请的资金数据将使ParaTechs启动II期研究，以证明这些细胞系在与潜在最终用户合作生产跨膜和分泌蛋白方面的效用。

DESCRIPTION (provided by applicant): The baculovirus expression vector system (BEVS) is a proven, powerful and versatile method of eukaryotic protein expression. It is used to produce vaccines, diagnostics, and biologically active proteins for a multitude of research projects. Like all expression systems, however, BEVS has its disadvantages. One is the fact that expression is short-lived due to virus-induced cell death and lysis. ParaTechs has already commercialized a product that addresses this shortcoming. Cell lines that express a viral ankyrin gene show delayed death and lysis of baculovirus-infected cells, thereby significantly enhancing recombinant protein production. This activity, referred to as vankyrin-enhanced BEVS (VE-BEVSTM), boosts target protein expression up to 20-fold, depending on the protein and method of vankyrin delivery. A second limitation of baculovirus expression is that insect cells lack the ability to produce terminally sialylated, complex N-glycans, which limits the usefulness of BEVS for the expression of human therapeutic proteins. This proposal addresses that issue and aims to develop new cell lines that produce authentic "humanized" N-glycans in the context of vankyrin-enhanced baculovirus technology. To obtain this goal, three different insect cell lines that have been previously engineered to produce mammalian glycosylation enzymes will be co-infected with baculoviruses that encode a vankyrin gene and different test glycoproteins. The transgenic cell line with the highest vankyrin response will be transformed with two different vankyrin genes under the control of several promoters. Polyclonal cells will be screened for enhanced glycoprotein expression, and the most promising cell population will be cloned. Different monoclonal lines will be tested for production of complex terminally sialylated glycoproteins, as compared to the non-transformed parental line. These cells will provide the highest yields of accurately processed secreted and transmembrane proteins, and will be marketed to individual researchers and pharmaceutical companies engaged in structure-function studies of the human secretome and development of protein therapeutics. These Phase I studies will significantly expand the applications of ParaTechs' vankyrin- enhanced BEVS technology. ParaTechs personnel and the Jarvis group have experience with all of the techniques to be used in these studies and do not anticipate difficulty achieving the objectives. The data derived from funding of this Phase I proposal will position ParaTechs to initiate Phase II studies to demonstrate the utility of these cell lines for production of transmembrane and secreted proteins in collaboration with potential end users. PUBLIC HEALTH RELEVANCE: The inability of insect cells to produce terminally sialylated, complex N-glycans limits the usefulness of baculovirus expression system for the production of human therapeutic proteins. This proposal addresses that deficiency and aims to develop new cell lines that produce authentic "humanized" N-glycans in the context of ParaTechs vankyrin-enhanced baculovirus technology. Successful completion of these objectives will produce cells that provide the highest levels of accurately processed secreted and transmembrane proteins and will be marketed to individual researchers and pharmaceutical companies engaged in structure-function studies of the human secretome and development of protein therapeutics.