Procaryotic expression, purification and identification of recombinant human prostate-specific antigen / 中华男科学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To produce recombinant human prostate-specific antigen (PSA) by molecular cloning technology and to identify its activity.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>Non-active recombinant PSA was digested by trypsin and demonstrated enzyme activity. The activated PSA hydrolyzed S-2586 and its physiological substrate semenogelin (Sg).</p><p><b>CONCLUSION</b>Recombinant pro-PSA can be an active serine protease by trypsin digestion and demonstrate native PSA enzymatic activity.</p>