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

Authors

Kevin E. Van Cott, University of Nebraska-LincolnFollow
Barry Williams Department of Chemical Engineering,Virginia Tech University, Blacksburg, VA 24061, USA
William H. Velander, University of Nebraska-LincolnFollow
Francis C. Gwazdauskas, Deparment of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VAFollow
Tim Lee Jerome Holland Laboratory, American Red Cross, Rockville, MD 20855, USA
Henryk Lubon, Jerome Holland Laboratory, American Red Cross, Rockville, MD 20855, USA
William N. Drohan, Jerome Holland Laboratory, American Red Cross, Rockville, MD 20855, USA

Date of this Version

9-1-1996

Comments

Originally Published in JOURNAL OF MOLECULAR RECOGNITION, VOL. 9.407-414 (1996): This Articale can be viewd at: http://www3.interscience.wiley.com/cgi-bin/fulltext/22664/PDFSTART

Abstract

Recombinant human protein C (rhPC) secreted in the milk of transgenic pigs was studied. 'Ikansgenes 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 y-carboxyglutamic acid domain of the light chain of hPC was used to fractionate a population having a higher specific anticoagulant activity in vW. 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 y-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.