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 alpha-amidated peptides alpha-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine alpha-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the alpha-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine alpha-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 alpha-amidated peptides by the co-expression of hPHMcc and the alpha-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed alpha-amidated peptide precursors. (c) 2012 Elsevier Inc. All rights reserved.