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Affinity purification of biologically active and inactive forms of recombinant human protein C produced in porcine mammary gland
Author(s) -
van Cott Kevin E.,
Williams Barry,
Velander William H.,
Gwazdauskas Frank,
Lee Tim,
Lubon Henryk,
Drohan William N.
Publication year - 1996
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/(sici)1099-1352(199634/12)9:5/6<407::aid-jmr277>3.0.co;2-x
Subject(s) - recombinant dna , monoclonal antibody , complementary dna , microbiology and biotechnology , transgene , biological activity , biology , population , biochemistry , genomic dna , in vitro , chemistry , antibody , gene , immunology , demography , sociology
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 Ca 2+ ‐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.