Purification of pregnancy zone protein and its receptor binding domain from human plasma.
A new significantly improved method for purification of pregnancy zone protein (PZP), alpha 2-macroglobulin (alpha 2M), and the C-terminal PZP receptor binding domain is presented. Several steps in an earlier procedure have been deleted, and modifications in the gradients in the DEAE step leave most of the contaminants bound to a DEAE-Sephacel gel. This procedure makes possible the rapid, simultaneous purification of both of these closely related unstable proteins in native form from human plasma, with no thiolester cleavage or formation of tetrameric PZP. The final preparations of both alpha 2M and PZP are pure as determined by nonreducing and reducing polyacrylamide gel electrophoresis following silver staining and no cross-contamination can be observed. The yield has been significantly improved and typically more than 500 mg PZP can be obtained from 1 liter pregnancy plasma. Furthermore, the stability of PZP at different temperatures on storage was studied. In liquid nitrogen PZP can be maintained in native dimeric form with intact thiolester for many years. The storage of native PZP with intact functional properties during and after purification is an obligatory prerequisite to elucidate the biological role of PZP. The receptor binding domain of PZP can be cleaved from the PZP-methylamine complex by papain and isolated from the other peptides by S-200 gel filtration. The cleavage site was determined and the C-terminal fragment was identified with several site-specific monoclonal antibodies against PZP.
Arbelaéz
L F
LF
Department of Immunology, Umeå University, Sweden.
Stigbrand
T
T
D001665:Binding Sites; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000378:metabolism; D015925:Cryopreservation; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008744:Methylamines / Q000378:metabolism; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000302:isolation & purification
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008744:Methylamines; D010446:Peptide Fragments; D011257:Pregnancy Proteins; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins; C027451:methylamine; D002918:Chymotrypsin; D010206:Papain