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

Abstract：

: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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Horlick RA,Sperle K,Breth LA,Reid CC,Shen ES,Robbins AK,Cooke GM,Largent BL

doi

10.1006/prep.1996.0701

subject

Has Abstract

pub_date

1997-04-01 00:00:00

pages

301-8

issue

3

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(96)90701-9

journal_volume

9

pub_type

杂志文章

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