Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation

Javier Garcia-Pardo, Sergi Montané, Francesc Xavier Avilés, Sebastian Tanco, Julia Lorenzo

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a representative example, we performed a preparative purification of this enzyme. The purified protein is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput functional and structural studies.

Original languageEnglish
Article number2138
Number of pages14
JournalPharmaceutics
Volume14
Issue number10
DOIs
Publication statusPublished - 8 Oct 2022

Keywords

  • ECM proteins
  • HEK 293F cells
  • carboxypeptidase A6
  • carboxypeptidase Z
  • carboxypeptidases
  • enhanced protein expression
  • heparin binding
  • mammalian cells
  • sodium heparin
  • thrombin-activable fibrinolysis inhibitor

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