Synthesis of new peptides with prolactin-releasing activity by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction.
A newly isolated peptide from bovine hypothalamus with prolactin-releasing activity (prolactin-releasing peptide; PrRP) was synthesized by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction, together with rat and human homologs. The peptides were expressed in the form of fusion proteins with basic fibroblast growth factor mutein, which were purified by heparin-affinity chromatography. The fusion proteins were cleaved at the cysteine residues of the junction site by cyanylation, followed by treatment with ammonia for C-terminal amidation. Purification of the resulting crude peptides was performed using chromatography on a gel-filtration column, a cation-exchange column, and a reversed-phase column. As an example, about 90 mg of bovine PrRP (bPrRP) was obtained from 201 of culture bloth. The purified b PrRP showed full biological activities in binding to its receptor expressed on CHO cells and releasing arachidonic acid metabolite from the same cells, while the C-terminal acid form of bPrRP had little of these activities. These results indicate that the C-terminal amide structure is very important for expressing biological activity. The peptides obtained here might be very useful for studies on their biological significance and roles in vivo.
Biotechnology Laboratories, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan.
O
O
Nishimura
T
T
Moriya
M
M
Suenaga
Y
Y
Tanaka
T
T
Itoh
N
N
Koyama
R
R
Fujii
S
S
Hinuma
C
C
Kitada
M
M
Fujino
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D006224:Cricetinae; D003486:Cyanides; D003545:Cysteine / Q000737:chemistry*; D004274:DNA, Recombinant / Q000737:chemistry*; D004926:Escherichia coli; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007028:Hypothalamic Hormones / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D011388:Prolactin / Q000032:analysis; D056690:Prolactin-Releasing Hormone; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000302:isolation & purification
D003486:Cyanides; D004274:DNA, Recombinant; D007028:Hypothalamic Hormones; D009479:Neuropeptides; C534377:PRLH protein, human; C534379:Prlh protein, rat; D056690:Prolactin-Releasing Hormone; D014409:Tumor Necrosis Factor-alpha; C109425:mutein 3; D016222:Fibroblast Growth Factor 2; D011388:Prolactin; D003545:Cysteine