Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells

BMC Biotechnology

Research Article

Omid Vafa¹  Steven C. Pomerantz¹  Michael Naso¹  William Strohl¹  Richard E. Mains²  Kristina Carlson²  Betty A. Eipper³ 

[1] Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, 19477, Spring House, PA, USA;Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, 06030-3401, Farmington, CT, USA;Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, 06030-3401, Farmington, CT, USA;Department of Molecular Biology and Biophysics, University of Connecticut Health Center, 06030, Farmington, CT, USA;

关键词: CHO cell;    Glucagon-like peptide 1;    Peptide YY;    Neuromedin U;    Mass spectrometry;   

DOI  :  10.1186/s12896-015-0210-4

 received in 2015-05-10, accepted in 2015-10-01,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundAmidation 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.MethodsTo 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.ResultsPAM 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.ConclusionsPeptide 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.

【 授权许可】

CC BY   
© Carlson et al. 2015