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Production of the catalytic core of human peptidylglycine ?-hydroxylating monooxygenase (hPHMcc) in Escherichia coli.


ABSTRACT: Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of ?-amidated peptides. ?-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine ?-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the ?-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine ?-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His(6) fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate ?-amidated peptides by the co-expression of hPHMcc and the ?-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed ?-amidated peptide precursors.

SUBMITTER: Handa S 

PROVIDER: S-EPMC3614408 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli.

Handa Sumit S   Spradling Tyler J TJ   Dempsey Daniel R DR   Merkler David J DJ  

Protein expression and purification 20120425 1


Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of  ...[more]

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