title: Production of the catalytic core of human peptidylglycine α-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 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 the catalytic core of human peptidylglycine α-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 α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors.
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL 33620, USA.
authors.forename: Sumit
authors.initials: S
authors.lastname: Handa
authors.forename: Tyler J
authors.initials: TJ
authors.lastname: Spradling
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Dempsey
authors.forename: David J
authors.initials: DJ
authors.lastname: Merkler
mesh_terms: D020134:Catalytic Domain; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003062:Codon; C471213:His-His-His-His-His-His; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D013879:Thioredoxins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase