Chemical and Biomolecular Engineering Research and Publications

 

Date of this Version

January 2005

Comments

This paper was originally published in the Journal of “Transgenic Research” Vol. 13Pages 437-450, 2004.And all the copy rights © of this journal belongs to the Kluwer Academic Publisher. The orinal copy of this link can be found at following site springer.metapress Any other further information about this paper can be obtained from the following link of the publisher Kluwer Academic Publishers

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 4.1 kbp 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 μ g/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 chains. Also, the subunit complexes Aαγ2, γ2 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.