Production and activation of recombinant hK2 with propeptide mutations resulting in high expression levels

Human glandular kallikrein 2 (hK2) is a serine protease expressed mainly by the prostate gland with 80% identity in primary structure to prostate spec ific antigen (PSA). hK2 has proven to be a useful marker of prostate cancer which can be used in combination with PSA to better discriminate between p rostate cancer and benign prostate hyperplasia. The studies on hK2 have bee n hampered by its very low phyciological levels (6 mu g.mL(-1)), its close similarity to PSA, and the low expression levels obtained using recombinant procedures to produce hK2 (0.7 mg.L-1). We have now generated propeptide m utations of hK2 which can be used to isolate stable, inactive prohK2 mutant s. Compared with wild-type hK2, expression of the propeptide hK2 mutants in creases the expression levels up to 15-40-fold giving 10-30 mg hK2.L-1. The se results indicate that the low expression levels of wild-type hK2 are rel ated to the activation or autoactivation of the wild-type enzyme and the in stability of the active protease in cell culture and possibly also in tissu e. The purified mutant hK2 may be activated by either enterokinase or facto r Xa to generate an enzyme for use in functional studies with the character istics of the original wild-type protein. Further, the stable inactive muta nt hK2 protein may be used for immunizations to generate novel monoclonal a ntibodies, used as standard material for clinical assays or in crystallizat ion studies where large quantities of protein are required.