Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

  • Shaozheng Song
  • Zhengyi He
  • Junyan Mei
  • Zhengqiang Qi
  • Si Chen
  • Sheng Xu
  • Yuguo Yuan
  • Yong Cheng
Keywords: tPA, Immunoaffinity chromatography, PR-mAb, ELISA-elution, Antibody, Thrombolytic activity

Abstract

Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk.

Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen because of its similarity to rhPA in terms of structure. The PR-mAb was prepared by hybridoma technology and screened by ELISA-elution assay. Screening antibody was performed using rhPA milk in an ELISA-elution assay. The antibody clone C4-PR-mAb was selected for immunoaffinity chromatography. The rhPA was effectively bound to immobilized C4-PR-mAb on the column and was eluted with Tris buffer comprising 0.75 mol/L ammonium sulfate and 40n% propanediol (pH7.9). The rhPA was further purified by passing through Chromdex75 gel filtration column.

Results: There were 12 hybridoma strains selected into the polyol responsive mAbs screen step and three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). The rhPA can be purified from transgenic rabbit milk and maintained a higher thrombolytic activity in vitro by FAPA.

Conclusion: The results demonstrate the suitability of the alternative approach used in this study. Using immunoaffinity chromatography and  gel filtration column is feasible and convenient for extracting rhPA from milk, and should be useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

Keywords: tPA, Immunoaffinity chromatography, PR-mAb, ELISA-elution, Antibody, Thrombolytic activity

Published
2016-05-30
Section
Articles

Journal Identifiers


eISSN: 1596-9827
print ISSN: 1596-5996