Purification and characterization of placental protein 5.
This report describes the purification of placental protein 5, PP5, from the human placenta by two affinity chromatography steps, the first with Heparin-Sepharose and the second with Sepharose-linked monoclonal anti-PP5 antibody. The final purification is achieved by reversed-phase high performance liquid chromatography. In SDS-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, PP5 purified in this study migrates as one major band at 36 kD. The previously purified PP5 is more heterogeneous: under nonreducing conditions it migrates at 30 kD and, after reduction, it gives three bands at 16.8 kD, 18.3 kD, and 19.0 kD. In Western blot analysis, both purified proteins react with polyclonal and monoclonal anti-PP5 antibodies. Three N-terminal amino acid sequences are obtained for the previously purified PP5, whereas the N-terminal of PP5 purified in this study is blocked. These results suggest that PP5 previously purified in the absence of protease inhibitors, does not represent the native form of PP5. Computer comparison of the obtained amino acid sequences revealed no significant homology to known protein sequences.
Bützow
R
R
First Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Finland.
Huhtala
M L
ML
Bohn
H
H
Virtanen
I
I
Seppälä
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006023:Glycoproteins; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D011257:Pregnancy Proteins; C021240:tissue-factor-pathway inhibitor 2; D003208:Concanavalin A