Production of recombinant human relaxin 3 in AtT20 cells.
Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.
Kizawa
Hideki
H
Discovery Research Laboratories I, Pharmaceutical Research Division, Takeda Chemical Industries Ltd, Wadai 10, Ibaraki Tsukuba 300-4293, Japan.
Nishi
Kazunori
K
Ishibashi
Yoshihiro
Y
Harada
Masataka
M
Asano
Tsuneo
T
Ito
Yasuaki
Y
Suzuki
Nobuhiro
N
Hinuma
Shuji
S
Fujisawa
Yukio
Y
Onda
Haruo
H
Nishimura
Osamu
O
Fujino
Masahiko
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D012065:Relaxin / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013552:Swine; D013997:Time Factors
C480238:RLN3 protein, human; D011994:Recombinant Proteins; D012065:Relaxin; D000242:Cyclic AMP