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.
Nishimura
O
O
Biotechnology Laboratories, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan.
Moriya
T
T
Suenaga
M
M
Tanaka
Y
Y
Itoh
T
T
Koyama
N
N
Fujii
R
R
Hinuma
S
S
Kitada
C
C
Fujino
M
M
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