Affinity purification of biologically active and inactive forms of recombinant human protein C produced in porcine mammary gland
✍ Scribed by Kevin E. van Cott; Barry Williams; William H. Velander; Frank Gwazdauskas; Tim Lee; Henryk Lubon; William N. Drohan
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 872 KB
- Volume
- 9
- Category
- Article
- ISSN
- 0952-3499
No coin nor oath required. For personal study only.
✦ Synopsis
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.