GLYCOSYLATION PATTERNS OF EXTRACELLULAR PROTEINS OF NEUROSPORA CRASSA

该项目的广泛长期目标是准确定义 正常和异源细胞外的糖基化模式 由丝状真菌粗糙脉孢菌分泌的糖蛋白。 脉孢菌最近被主要研究者用作宿主 用于生产有用的异源哺乳动物蛋白的细胞。 正在进行 实验室的研究包括尝试表达人IgG 链孢菌中的抗体。 众所周知，在生物学上， 抗体的“正确的”糖基化是抗体的“正确的”糖基化的绝对要求。 它们在哺乳动物免疫系统中的全部功能。 的糖型 不同的物种，来自同一物种的不同细胞类型，甚至 已经显示在不同培养条件下生长相同细胞类型 是高度可变的 任何拟用于以下用途的宿主细胞： 异源表达系统必须产生符合 由产生蛋白质的细胞类型产生的天然糖型 在自然界中。 脉孢菌产生的糖型从未被 系统调查。 同源真菌蛋白和异源真菌蛋白 在脉孢菌细胞中表达并分泌到细胞中的哺乳动物蛋白质 将从培养基中收集表面或进入培养基中， 在细胞壁膜制备的小球中。 粗品制剂和 将评价经电泳分离的糖蛋白， 通过“First Choice”糖基化测定系统（由 Boehringer曼海姆公司。对这些糖型的系统调查将 提供了评估脉孢菌有效性的第一步， 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.