title: Affinity purification of biologically active and inactive forms of recombinant human protein C produced in porcine mammary gland.
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 gamma-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 gamma-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.
authors.affiliation: Department of Chemical Engineering, Virginia Tech University, Blacksburg 24061, USA.
authors.forename: K E
authors.initials: KE
authors.lastname: Van Cott
authors.forename: B
authors.initials: B
authors.lastname: Williams
authors.forename: W H
authors.initials: WH
authors.lastname: Velander
authors.forename: F
authors.initials: F
authors.lastname: Gwazdauskas
authors.forename: T
authors.initials: T
authors.lastname: Lee
authors.forename: H
authors.initials: H
authors.lastname: Lubon
authors.forename: W N
authors.initials: WN
authors.lastname: Drohan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D002122:Calcium Chloride; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D008321:Mammary Glands, Animal / Q000378:metabolism; D008969:Molecular Sequence Data; D011486:Protein C / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
chemical_list: D011486:Protein C; D011994:Recombinant Proteins; D002122:Calcium Chloride