Efficient production of peptidylglycine α-hydroxylating monooxygenase in yeast for protein C-terminal functionalization.
Int J Biol Macromol
; 263(Pt 2): 130443, 2024 Apr.
Article
em En
| MEDLINE
| ID: mdl-38417749
ABSTRACT
Peptidylglycine α-hydroxylating monooxygenase (PHM) is pivotal for C-terminal amidation of bioactive peptides in animals, offering substantial potential for customized protein synthesis. However, efficient PHM production has been hindered by the complexity of animal cell culture and the absence of glycosylation in bacterial hosts. Here, we demonstrate the recombinant expression of Caenorhabditis elegans PHM in the yeast Pichia pastoris, achieving a remarkable space-time yield of 28.8 U/L/day. This breakthrough surpasses prior PHM production rates and eliminates the need for specialized cultivation equipment or complex transfection steps. Mass spectrometry revealed N-glycosylation at residue N182 of recombinant CePHM, which impacts the enzyme's activity as indicated by biochemical experiments. To showcase the utility of CePHM, we performed C-terminal amidation on ubiquitin at a substrate loading of 30 g/L, a concentration meeting the requirements for pharmaceutical peptide production. Overall, this work establishes an efficient PHM production method, promising advancements in scalable manufacturing of C-terminally modified bioactive peptides and probe proteins.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Proteína C
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Complexos Multienzimáticos
Limite:
Animals
Idioma:
En
Revista:
Int J Biol Macromol
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China