[Procaryotic expression, purification and identification of recombinant human prostate-specific antigen].
OBJECTIVE
To produce recombinant human prostate-specific antigen (PSA) by molecular cloning technology and to identify its activity.


METHODS
The human PSA cDNA and PET-12a vector were digested by NdeI and BamH1 before ligated by T4 ligase. The correct sequence was verified and transformed into high competent E. coli BL21 (DE3). Recombinant PSA was expressed and purified by hydrophobic interaction phenyl Sepharose column and activated by trypsin digestion. Enzymatic activation assay was done by hydrolysis of the substrate S-2586 and semenogelin.


RESULTS
Non-active recombinant PSA was digested by trypsin and demonstrated enzyme activity. The activated PSA hydrolyzed S-2586 and its physiological substrate semenogelin (Sg).


CONCLUSIONS
Recombinant pro-PSA can be an active serine protease by trypsin digestion and demonstrate native PSA enzymatic activity.
Wang
Zeng-Jun
ZJ
Department of Urology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China. zengjunwang2002@hotmail.com
Zhang
Wei
W
Wu
Hong-Fei
HF
Sui
Yuan-Geng
YG
D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis; D008297:Male; D009842:Oligopeptides / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D029607:Seminal Vesicle Secretory Proteins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D018076:DNA, Complementary; D009842:Oligopeptides; D011994:Recombinant Proteins; C414985:S 2586; D029607:Seminal Vesicle Secretory Proteins; C424482:seminal vesicle-specific antigen; D014357:Trypsin; D017430:Prostate-Specific Antigen