Affinity Purification of Biologically Active and Inactive Forms of Recombinant Human Protein C Produced in Porcine Mammary Gland

Kevin E. Van Cott, Barry Williams, William H. Velander, Frank Gwazdauskas, Tim Lee, Henryk Lubon, William N. Drohan

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Recombinant human protein C (rhPC) secreted in the milk of transgenic pigs was studied. Transgenes having different regulatory elements of the murine milk protein, whey acidic protein, were used with cDNA and genomic human protein C (hPC) DNA sequences to obtain lower and higher expressing animals. The cDNA pigs had a range of expression of about 0.1-0.5 g/l milk. Two different genomic hPC pig lines have expressed 0.3 and 1-2 g/l, respectively. The rhPC was first purified at yields greater than 60 per cent using a monoclonal antibody (mAb) to the activation site on the heavy chain of hPC. Subsequent immunopurification with a calcium-dependent mAb directed to the γ-carboxyglutamic acid domain of the light chain of hPC was used to fractionate a population having a higher specific anticoagulant activity in vitro. The higher percentages of Ca2+-dependent conformers isolated from the total rhPC by immunopurification correlated well with higher specific activity and lower expression. A rate limitation in γ-carboxylation of rhPC was clearly identified for the higher expressing animals. Thus, transgenic animals with high expression levels of complex recombinant proteins produced a lower percentage of biologically active protein.

Original languageEnglish (US)
Pages (from-to)407-414
Number of pages8
JournalJournal of Molecular Recognition
Volume9
Issue number5-6
DOIs
StatePublished - 1996

Keywords

  • Affinity chromatography
  • Mammary gland
  • Porcine
  • Protein C
  • Transgenic

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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