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Posttranslational processing of recombinant human interferon-gamma in animal expression systems

机译：动物表达系统中重组人干扰素-γ的翻译后加工

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We have characterized the heterogeneity of recombinant human interferon-gamma (IFN-gamma) produced by three expression systems: Chinese hamster ovary cells, the mammary gland of transgenic mice, and baculovirus-infected Spodoptera frugiperda (Sf9) insect cells. Analyses of whole IFN-gamma proteins by electrospray ionization-mass spectrometry (ESI-MS) from each recombinant source revealed heterogeneous populations of IFN-gamma molecules resulting from variations in N-glycosylation and C-terminal polypeptide cleavages. A series of more specific analyses assisted interpretation of maximum entropy deconvoluted ESI-mass spectra of whole IFN-gamma proteins; MALDI-MS analyses of released, desialylated N-glycans and of deglycosylated IFN-gamma polypeptides were combined with analyses of 2-aminobenzamide labeled sialylated N-glycans by cation-exchange high-performance liquid chromatography. These analyses enabled identification of specific polypeptide cleavage sites and characterization of associated N-glycans. Production of recombinant IFN-gamma in the mammalian expression systems yielded polypeptides C-terminally truncated at dibasic amino acid sites. Mammalian cell derived IFN-gamma molecules displayed oligosaccharides with monosaccharide compositions equivalent to complex, sialylated, or high-mannose type N-glycans. In contrast, IFN-gamma derived from baculovirus-infected Sf9 insect cells was truncated further toward the C-terminus and was associated with neutral (nonsialylated) N-glycans. These data demonstrate the profound influence of host cell type on posttranslational processing of recombinant proteins produced in eukaryotic systems.
机译：我们已经表征了由三种表达系统产生的重组人干扰素-γ（IFN-γ）的异质性：中国仓鼠卵巢细胞，转基因小鼠的乳腺和杆状病毒感染的草地贪夜蛾（Sod9 frugiperda）（Sf9）昆虫细胞。通过每个重组来源的电喷雾电离质谱（ESI-MS）对整个IFN-γ蛋白进行分析，结果显示，N-糖基化和C端多肽裂解的差异导致了IFN-γ分子的异质性。一系列更具体的分析有助于解释整个IFN-γ蛋白的最大熵去卷积ESI-质谱。通过阳离子交换高效液相色谱对释放的，脱唾液酸化的N-聚糖和脱糖基化的IFN-γ多肽的MALDI-MS分析与2-氨基苯甲酰胺标记的唾液酸化的N-聚糖的分析相结合。这些分析使得能够鉴定特定的多肽切割位点并鉴定相关的N-聚糖。在哺乳动物表达系统中重组IFN-γ的产生产生了在二元氨基酸位点C-末端被截短的多肽。哺乳动物细胞来源的IFN-γ分子展示的寡糖具有与复杂，唾液酸化或高甘露糖型N-聚糖等效的单糖组成。相反，源自杆状病毒感染的Sf9昆虫细胞的IFN-γ被进一步截断至C末端，并与中性（非唾液酸化的）N-聚糖相关。这些数据证明了宿主细胞类型对真核系统中产生的重组蛋白的翻译后加工的深远影响。

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