Secretion of Fc-amidated peptide fusion proteins by Chinese hamster ovary cells.
BMC Biotechnol
; 15: 61, 2015 Jun 27.
Article
in En
| MEDLINE
| ID: mdl-26116580
ABSTRACT
BACKGROUND:
The therapeutic use of α-amidated peptides (e.g. calcitonin, glucagon-like peptide) has increased dramatically, but there are major impediments to wider use of such peptides. Larger peptides are expensive to synthesize, and short plasma half-lives frequently limit the clinical circumstances in which the peptides would be useful. Both problems are potentially solved by producing peptides as fusions with the Fc region of human immunoglobulin.METHODS:
Glucagon-like peptide 1 (GLP1), peptide YY (PYY) and neuromedin U (NMU) were expressed and purified from stable CHO lines; since the α-amide group is essential for full biological potency of many peptides, Fc-fusion peptides were expressed in CHO lines stably expressing the α-amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM EC 1.14.17.3). Purified fusion proteins were analyzed intact and after HRV3C rhinovirus protease cleavage, at a site in the linker separating the Fc region from the peptide, by mass spectrometry and amide-specific immunoassays.RESULTS:
The Fc fusions were expressed at 1-2.5 µg/mg cell protein and secreted at 5-20% of cell content per hour, in a peptide-specific manner. CHO cells express measurable endogenous PAM activity, amidating 25% of Fc-PYY and almost 90% of Fc-GLP1. Expression of exogenous PAM increased the level of peptide amidation to 50% of Fc-PYY and 95 % of Fc-NMU. The Fc-GLP1 fusions were 10,000-fold less active than synthetic GLP1 in a cell-receptor cyclic AMP-based assay, as expected since the amino terminal of GLP1 is essential for full biological activity. The Fc-PYY fusions were 100-fold less active than PYY-NH2 but 10-fold more active than non-amidated PYY-Gly.CONCLUSIONS:
This type of approach can be used for the production of stabilized α-amidated peptides aimed at clinical trials.
Full text:
1
Database:
MEDLINE
Main subject:
Recombinant Fusion Proteins
/
Immunoglobulin Fc Fragments
/
Amides
Limits:
Animals
/
Humans
Language:
En
Year:
2015
Type:
Article