Expression of active, secreted human prostate-specific antigen by recombinant baculovirus-infected insect cells on a pilot-scale.
We have expressed human prostate-specific antigen (PSA) on a pilot-scale in Spodoptera frugiperda Sf9 insect cells using recombinant baculovirus system. Infected cells secreted PSA into culture medium at a concentration of 2-4 mg per liter. PSA was expressed both in active and inactive forms which were separated in a final purification step using cation-exchange chromatography eluted with a low salt gradient. The N-terminus of active PSA was correctly cleaved; two amino acids of the propeptide remained, however, at the N-terminus of the inactive PSA. Purified recombinant PSA showed a chymotrypsin-like activity with the synthetic substrate MeO-Suc-Arg-Pro-Tyr-pNA, but did not have a trypsin-like activity when Pro-Phe-Arg-pNA was used. The molecular mass of active PSA was 31.0 kDa in reduced SDS-PAGE, 26.0 kDa in nonreduced SDS-PAGE and 26.5 kDa in ion spray mass spectrometry. The active protein formed complexes with alpha 1-antichymotrypsin (ACT) and alpha 2-macroglobulin (alpha 2M) in vitro similar to the commercial PSA purified from human seminal fluid.
Kurkela
R
R
Biocenter Oulu, University of Oulu, Finland.
Herrala
A
A
Henttu
P
P
Nai
H
H
Vihko
P
P
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D010455:Peptides / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016622:Silver Staining; D018411:Spodoptera / Q000378:metabolism*; D013379:Substrate Specificity
D003470:Culture Media; D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D002918:Chymotrypsin; D017430:Prostate-Specific Antigen