Rapid generation of stable cell lines expressing corticotropin-releasing hormone receptor for drug discovery

Protein Expr Purif. 1997 Apr;9(3):301-8. doi: 10.1006/prep.1996.0701.


Human HEK293 cells that stably express the Epstein Barr nuclear antigen 1 (EBNA1) support the episomal replication of plasmids containing the Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E) cell line expressing the human corticotropin-releasing hormone receptor subtype I (CRHR1) from an episomal plasmid was generated (293CR1s), analyzed, adapted to spinner culture, and scaled-up for production in less than 6 weeks. Forty-seven stable CHO cell lines transfected with CRHR1 were also isolated. Expression of the receptor in the best of these lines (as judged by CRH-induced cAMP production), CHO-R22, was compared to that in 293CR1s cells. Results indicate that the CRHR1 episomal expression vector in 293E cells (1) rapidly generates stable cell lines suitable for scale-up; (2) is stably maintained during 3 months in culture; (3) expresses high levels of CRHR1 mRNA; and (4) expresses significantly more CRHR1 than the CHO-R22 line. Coexpression of additional G protein alpha subunit (G alpha s) with CRHR1 in 293E cells converts a higher percentage of receptor to the agonist high-affinity G-protein-coupled state. Our data support the idea that using the EBV oriP-driven episomal system for gene expression results in greater production of protein in a relatively short period of time.

MeSH terms

  • Animals
  • CHO Cells
  • Cell Line*
  • Cricetinae
  • Drug Design*
  • Epstein-Barr Virus Nuclear Antigens / genetics
  • Gene Expression
  • Genetic Vectors
  • Herpesvirus 4, Human / genetics
  • Humans
  • Plasmids / genetics
  • RNA / genetics
  • RNA / metabolism
  • Receptors, Corticotropin-Releasing Hormone / biosynthesis
  • Receptors, Corticotropin-Releasing Hormone / classification
  • Receptors, Corticotropin-Releasing Hormone / genetics*
  • Replication Origin


  • Epstein-Barr Virus Nuclear Antigens
  • Receptors, Corticotropin-Releasing Hormone
  • RNA