Monitoring the Secretion and Activity of Alpha-1 Antitrypsin in Various Mammalian Cell Types.
Methods Mol Biol
; 2750: 143-163, 2024.
Article
in En
| MEDLINE
| ID: mdl-38108975
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biological Products
/
MRNA Vaccines
Limits:
Animals
/
Humans
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2024
Document type:
Article
Affiliation country:
United States