Expression, purification, and characterization of deglycosylated human pro-prostate-specific antigen.
Wild-type and deglycosylated forms of human prostate-specific antigen were expressed in Chinese hamster ovary (CHO) cells as zymogens. ProPSA was collected from conditioned medium and purified using a single cation-exchange chromatographic step for the deglycosylated form and cation-exchange followed by gel filtration chromatography for the wild-type form. Recombinant wild-type proPSA produced in CHO cells has an average MW of 34.5 kDa, whereas the deglycosylated proPSA has a MW of 32.4 kDa. Both forms of proPSA were activated in vitro and the kinetic properties measured for the deglycosylated PSA are very similar to those of the wild-type recombinant PSA and the native PSA isolated from seminal fluid. These results suggest that deglycosylated PSA is likely to be very similar to native PSA with respect to its three-dimensional structure and will provide a homogeneous protein preparation necessary for X-ray crystallographic analysis.
Bowman
K K
KK
Department of Protein Expression, Axys Pharmaceuticals, Inc., 180 Kimball Way, South San Francisco, California 94080, USA.
Clark
J
J
Yu
L
L
Mortara
K
K
Radika
K
K
Wang
J
J
Zhan
H
H
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D003001:Cloning, Molecular; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D016296:Mutagenesis; D017430:Prostate-Specific Antigen / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011498:Protein Precursors; D011994:Recombinant Proteins; D017430:Prostate-Specific Antigen