Secretion of recombinant human fibrinogen by the murine mammary gland

Stephen Butler, Tanya O'Sickey, Susan Lord, Henryk Lubon, Francis Gwazdauskas, William Velander

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aα, Bβ and γ fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 μg/ml, with total secretion of subunits approaching 700 μg/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bβ and γ chains were rate limiting. Both the Bβ and γ chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for γ chains over Bβ chains. Also, the subunit complexes γ 2, A αγ2 and the individual subunits Aα, Bβ and γ were found as secretion products. When the Bβ was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of Bβ chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.

Original languageEnglish (US)
Pages (from-to)437-450
Number of pages14
JournalTransgenic Research
Volume13
Issue number5
DOIs
StatePublished - Oct 2004

Keywords

  • Mammary gland expression
  • Recombinant human fibrinogen
  • Transgenic mouse
  • WAP promoter

ASJC Scopus subject areas

  • Biotechnology
  • Animal Science and Zoology
  • Agronomy and Crop Science
  • Genetics

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