Abstract:
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.
摘要:
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,可能含有低效分泌机制的目标非天然细胞。例如,α-1抗胰蛋白酶(AAT)的基因替代疗法,一种通常在肝细胞中产生的糖蛋白,由于易于递送,通常靶向肌肉细胞。在这一章中,我们定义了在代表性细胞类型中表达AAT的方法,如Huh-7;肝细胞;中国仓鼠卵巢细胞(CHO),产生生物制剂的常见宿主;和C2C12,一种肌肉祖细胞系。描述了代谢标记AAT以监测这些细胞系中的分泌的方法,以及使用蛋白抑制激活剂以增加C2C12成肌细胞和分化肌管中分泌的AAT的量。还呈现了评估过表达的AAT的活性和聚糖组成的测定。蛋白抑制激活剂SAHA的使用提供了肌肉样细胞中活性AAT表达的40%改善,并且可以是用于通过mRNA疫苗递送的蛋白质的重组生产的有利佐剂。