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

Abstract

Human glandular kallikrein 2 (hK2) is a serine protease expressed mainly by the prostate gland with 80% identity in primary structure to prostate specific 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 prostate cancer and benign prostate hyperplasia. The studies on hK2 have been hampered by its very low phyciological levels (6 mg´mL 21 ), its close similarity to PSA, and the low expression levels obtained using recombinant procedures to produce hK2 (0.7 mg´L 21 ). We have now generated propeptide mutations of hK2 which can be used to isolate stable, inactive prohK2 mutants. Compared with wild-type hK2, expression of the propeptide hK2 mutants increases the expression levels up to 15±40-fold giving 10±30 mg hK2´L 21 . These results indicate that the low expression levels of wild-type hK2 are related to the activation or autoactivation of the wild-type enzyme and the instability of the active protease in cell culture and possibly also in tissue. The purified mutant hK2 may be activated by either enterokinase or factor Xa to generate an enzyme for use in functional studies with the characteristics of the original wild-type protein. Further, the stable inactive mutant hK2 protein may be used for immunizations to generate novel monoclonal antibodies, used as standard material for clinical assays or in crystallization studies where large quantities of protein are required.Keywords: cancer; expression; kallikrein; prostate; zymogen.The glandular kallikreins are serine proteases encoded by a multigene family with a highly varying number of members in different species [1]. The human glandular kallikrein gene family consists of tissue kallikrein (hK1), human glandular kallikrein 2 (hK2), and prostate-specific antigen (PSA or hK3) [2]. The glandular kallikreins are, in general, involved in processing polypeptide precursors to their bioactive forms and have attracted interest as the releasers of kinin from kininogen and murine epidermal growth factor and nerve growth factor from their respective precursors [3]. As all the human glandular kallikreins have evolved from a single precursor after the separation of the human and murine lineages it is difficult to predict to which extent the function of these proteins has remained similar [4]. hK2 was the last of the three members of the human glandular kallikrein gene family to be identified in 1987 [2]. Recently, hK2 has proven to be a useful marker of prostate cancer which can be used in combination with PSA to better discriminate between prostate cancer and benign prostate hyperplasia ([5,6] C. Becker, T. Piironen, K. Pettersson, T. Bjo Èrk, K. J. Wojno, P. A. Abrahamsson, J. E. Oesterling & H. Lilja, unpublished data). The highest hK2 expression is found in the prostate, but it is also found in the endometrium, breast, and salivary glands. The hK2 levels in seminal plasma are < 6 mg´mL 21 while the levels in amniotic fluid, milk and saliva are < 0.02 ng´mL 21 , 0.05 ng´mL 21 and...

Identification of Breast Cancer - Specific Proteolytic Activities for Targeted Prodrug Activation

“…Compared with wild-type hK2, expression of the propeptide hK2 mutant increases the expression levels up to 15-40-fold (34). The generation and characteristics of this mutant hK2 have been previously described (34). Fmoc amino acids were purchased either from Advanced Chemtech or Novabiochem.…”

“…The objective of Task 3 (months 9-24) is to synthesize thapsigargin prodrugs by coupling thapsigargin analog to lead peptide substrates identified in tasks 1-3. The objective of Task 4 (months [24][25][26][27][28][29][30][31][32][33][34][35][36] is to identify specific protease(s) responsible for proteolytic activity using peptide substrates identified in tasks 1-2.…”

Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2

“…The recombinant hK2 (rhK2) forms fXahK2 and ekhK2 used for immunization have been described elsewhere (39 ). BALB/c mice were immunized by intraperitoneal injections with 30 -40 g of rhK2.…”

Standardization of Two Immunoassays for Human Glandular Kallikrein 2

“…The recombinant hK2 forms were generated using specific cell lines described by Lö vgren et al (30 ). The supernatants of the cell lines were purified and separated into stock solutions.…”