Rate limitations in posttranslational processing by the mammary gland of transgenic animals

Anuradha Subramanian, Rekha K. Paleyanda, Henryk Lubon, Barry L. Williams, Francis C. Gwazdauskas, James W. Knight, William N. Drohan, William H. Velander

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Abstract

Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine γ-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of γ-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 ± 4 μg rhPC/ml of milk and 895 ± 21 μg rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 μg rhPC/ml of milk and 800 to 2000 μg rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in γ-carboxylation by the mammary gland occurs at expression levels about > 20 μg/ml in mice and > 500 μg/ml in pigs.

Original languageEnglish (US)
Pages (from-to)87-96
Number of pages10
JournalAnnals of the New York Academy of Sciences
Volume782
DOIs
StatePublished - Jan 1 1996

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ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • History and Philosophy of Science

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