Monitoring the Secretion and Activity of Alpha-1 Antitrypsin in Various Mammalian Cell Types.
Monitoring the Secretion and Activity of Alpha-1 Antitrypsin in Various Mammalian Cell Types.
复制标题
监测各种哺乳动物细胞类型中 Alpha-1 抗胰蛋白酶的分泌和活性。
DOI：
10.1007/978-1-0716-3605-3_14
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Hebert,DanielN
中科院分区：
文献类型：
--
作者：
Guay,KevinP;Ke,Haiping;Gierasch,LilaM;Gershenson,Anne;Hebert,DanielN
重组蛋白在哺乳动物细胞中的过表达被广泛用于生产生物制品，因为蛋白质成熟和翻译后修饰与人类细胞相似。一些治疗方法，如mRNA疫苗，针对可能含有低效分泌机制的非天然细胞。例如，α -1抗胰蛋白酶（AAT）是一种通常在肝细胞中产生的糖蛋白，由于易于输送，通常针对肌肉细胞进行基因替代治疗。在本章中，我们定义了在代表性细胞类型（如Huh-7）中表达AAT的方法；肝细胞;中国仓鼠卵巢细胞（CHO）是一种生产生物制剂的常见宿主C2C12，一种肌肉祖细胞系。描述了代谢标记AAT以监测这些细胞系分泌的方法，以及使用蛋白酶抑制激活剂来增加C2C12成肌细胞和分化肌管中AAT的分泌量。还提出了评估过表达AAT的活性和聚糖组成的方法。使用蛋白静止激活剂SAHA可使肌样细胞中活性AAT的表达提高40%，并且可能是mRNA疫苗递送蛋白重组生产的有利佐剂。
Overexpression of recombinant protein in mammalian cells is widely used for producing biologics, as protein maturation and post-translational modifications are similar to human cells. Some therapeutics, such as mRNA vaccines, target nonnative cells that may contain inefficient secretory machinery. For example, gene replacement therapies for alpha-1 antitrypsin (AAT), a glycoprotein normally produced in hepatocytes, are often targeted to muscle cells due to ease of delivery. In this chapter, we define methods for expressing AAT in representative cell types such as Huh-7; hepatocytes; Chinese hamster ovarian cells (CHO), a common host to produce biologics; and C2C12, a muscle progenitor cell line. Methods for metabolically labeling AAT to monitor secretion in these cell lines are described along with the use of proteostasis activators to increase the amount of AAT secreted in both C2C12 myoblasts and differentiated myotubes. Assays to assess the activity and glycan composition of overexpressed AAT are also presented. The usage of the proteostasis activator SAHA provided a 40% improvement in expression of active AAT in muscle-like cells and may be an advantageous adjuvant for recombinant production of proteins delivered by mRNA vaccines.