title: Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells.
abstract: BACKGROUND
  Amidation of the carboxyl terminal of many peptides is essential for full biological potency, often increasing receptor binding and stability. The single enzyme responsible for this reaction is peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3), a copper- and ascorbate-dependent Type I membrane protein.
  
  
  METHODS
  To make large amounts of high molecular weight amidated product, Chinese hamster ovary (CHO) cells were engineered to express exogenous PAM. To vary access of the enzyme to its substrate, exogenous PAM was targeted to the endoplasmic reticulum, trans-Golgi network, endosomes and lysosomes or to the lumen of the secretory pathway.
  
  
  RESULTS
  PAM was equally active when targeted to each intracellular location and assayed in homogenates. Immunocytochemical analyses of CHO cells and a pituitary cell line demonstrated that targeting of exogenous PAM was partially successful. PAM substrates generated by expressing peptidylglycine substrates (glucagon-like peptide 1-Gly, peptide YY-Gly and neuromedin U-Gly) fused to the C-terminus of immunoglobulin Fc in CHO cell lines producing targeted PAM. The extent of amidation of the Fc-peptides was determined by mass spectrometry and amidation-specific enzyme immunoassays. Amidation was inhibited by copper chelation, but was not enhanced by the addition of additional copper or ascorbate.
  
  
  CONCLUSIONS
  Peptide amidation was increased over endogenous levels by exogenous PAM, and targeting PAM to the endoplasmic reticulum or trans-Golgi network increased peptide amidation compared to endogenous CHO PAM.
authors.affiliation: Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030-3401, USA. kristinarcarlson@gmail.com.
authors.forename: Kristina
authors.initials: K
authors.lastname: Carlson
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, Spring House, PA, 19477, USA. spomeran@its.jnj.com.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Pomerantz
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, Spring House, PA, 19477, USA. ovafa@its.jnj.com.
authors.forename: Omid
authors.initials: O
authors.lastname: Vafa
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, Spring House, PA, 19477, USA. mnaso@its.jnj.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Naso
authors.affiliation: Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, Spring House, PA, 19477, USA. wstrohl@its.jnj.com.
authors.forename: William
authors.initials: W
authors.lastname: Strohl
authors.affiliation: Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030-3401, USA. mains@uchc.edu.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Mains
authors.affiliation: Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030-3401, USA. eipper@uchc.edu.|Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT, 06030, USA. eipper@uchc.edu.
authors.forename: Betty A
authors.initials: BA
authors.lastname: Eipper
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D060847:Metabolic Engineering / Q000379:methods*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000577:Amides; D007141:Immunoglobulin Fc Fragments; D009097:Multienzyme Complexes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase