GLYCOSYLATION PATTERNS OF EXTRACELLULAR PROTEINS OF NEUROSPORA CRASSA

该项目广泛的长期目标是准确地定义 正常和异种胞外糖基化模式的研究 丝状真菌粗枝脉孢菌分泌的糖蛋白。 脉孢霉最近被首席调查者用作宿主 用于生产有用的异源哺乳动物蛋白质的细胞。正在进行中 实验室的研究包括尝试表达人类免疫球蛋白 脉孢子虫中的抗体。众所周知，从生物学上讲， 抗体的“正确”糖基化是对 它们在哺乳动物免疫系统中的全部功能。白花蛇舌草糖型 不同的物种，同一物种的不同细胞类型，甚至 已经显示了在不同培养条件下生长的相同细胞类型 是高度多变的。任何被提议用于 异源表达系统必须产生与之一致的糖形式 由产生蛋白质的细胞类型产生的天然糖类 在自然界中。脉孢菌产生的糖类形式从未被 系统地调查了。同源真菌蛋白和异源真菌蛋白 哺乳动物蛋白在脉孢子虫细胞中表达并分泌到细胞中 表面或进入培养基中将从介质中收集或 在细胞壁膜制剂的颗粒中。粗制制剂和 将对电泳法分离的糖蛋白进行评估 开发的“首选”糖基化分析系统中的糖型 伯林格曼海姆公司对这些糖形式的系统调查将 为评估脉孢子瘤的有效性提供第一步 用于生产哺乳动物糖蛋白的表达系统。

The broad long-term goals of this project are to accurately define the glycosylation patterns of normal and heterologous extra-cellular glycoproteins secreted from the filamentous fungus Neurospora crassa. Neurospora has been recently used by the principal investigator as a host cell for production of useful heterologous mammalian proteins. Ongoing research in the laboratory includes the attempt to express human IgG antibodies in Neurospora. It is well established that the biologically "correct" glycosylation of antibodies is an absolute requirement for their full function in the mammalian immune system. The glycoforms of different species, different cell types from the same species, and even the same cell type grown under various culture conditions have been shown to be highly variable. Any host cell which is proposed for use in a heterologous expression system must produce glycoforms consistent with the natural glycoform produced by the cell type generating the protein in nature. Glycoforms produced by Neurospora have never been systematically investigated. Homologous fungal proteins and heterologous mammalian proteins expressed in Neurospora cells and secreted to the cell surface or into the culture medium will be collected from the media or in pellets of cell wall-membrane preparations. Crude preparations and electrophoretically separated glycoproteins will be evaluated for glycoforms by the "First Choice" glycosylation assay system developed by Boehringer Mannheim Corp. A systematic survey of these glycoforms will provide a first step in evaluating the usefulness of a Neurospora expression system for production of mammalian glycoproteins.