Use of recombinant DNA derived human relaxin to probe the structure of the native protein.
This report describes the physical, chemical, and biological characterization of recombinant human relaxin (rhRlx) used as a probe to establish the disulfide pairing in native human relaxin. This strategy is necessary since native human relaxin is only available in the nanogram range. The relaxin molecule is composed of two nonidentical peptide chains, an A-chain 24 amino acids in length and a B-chain of 29 amino acids, linked by two disulfide bridges with an additional disulfide linkage in the A-chain. Native relaxin isolated from human corpora lutea was compared to rhRlx by reversed-phase chromatography, partial sequence analysis, mass spectroscopy, and bioassay. The potency of rhRlx was established by its ability to stimulate cAMP from primary human uterine endometrial cells. Native relaxin isolated from human corpora lutea was equipotent to chemically synthesized relaxin, which in turn was equipotent to rhRlx. A tryptic map was developed for rhRlx to confirm the complete amino acid sequence and assignment of the disulfide bonds. The three disulfide bonds (CysA10-CysA15, CysA11-CysB11, and CysA24-CysB23) were assigned by mass spectrometric analysis of the tryptic peptides and by comparison to chemically synthesized peptides disulfide linked in the two most probable configurations. In addition, the observed amino acid composition and sequence of rhRlx was in agreement with that predicted from the cDNA sequence with the exception that the A-chain amino terminal was pyroglutamic acid. The migration of rhRlx upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis was consistent with a monomeric structure, and the identity of the band was demonstrated by immunoblotting.
Canova-Davis
E
E
Medicinal and Analytical Chemistry Department, Genentech, Inc., South San Francisco, California 94080.
Kessler
T J
TJ
Lee
P J
PJ
Fei
D T
DT
Griffin
P
P
Stults
J T
JT
Wade
J D
JD
Rinderknecht
E
E
D000595:Amino Acid Sequence; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012065:Relaxin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014357:Trypsin
D004220:Disulfides; D011994:Recombinant Proteins; D012065:Relaxin; D014357:Trypsin