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Human tissue kallikrein 9: production of recombinant proteins and specific antibodies

  • Nader Memari , Linda Grass , Terukazu Nakamura , Inci Karakucuk and Eleftherios P. Diamandis
Published/Copyright: June 26, 2006
Biological Chemistry
From the journal Volume 387 Issue 6

Abstract

Human tissue kallikreins (genes, KLKs; proteins, hKs) are a subgroup of hormonally regulated serine proteases. Two tissue kallikreins, namely hK2 and hK3 (prostate-specific antigen, PSA), are currently used as serological biomarkers of prostate cancer. Human tissue kallikrein 9 (KLK9) is a newly identified member of the tissue kallikrein gene family. Recent reports have indicated that KLK9 mRNA is differentially expressed in ovarian and breast cancer and has prognostic value. Here, we report the production of recombinant hK9 (classic form) using prokaryotic and mammalian cells and the generation of polyclonal antibodies. Total testis tissue mRNA was reverse-transcribed to cDNA, amplified, cloned into a pET/200 TOPO plasmid vector, and transformed into E. coli cells. hK9 was purified and used as an immunogen to generate polyclonal antibodies. Full-length KLK9 cDNA was also cloned in the vector pcDNA3.1 and was expressed in CHO cells. The identity of hK9 was confirmed by mass spectrometry. hK9 rabbit antiserum displayed no cross-reactivity with other tissue kallikreins and could specifically recognize E. coli- and CHO-derived hK9 on Western blots. hK9 was mainly detected in testis and seminal vesicles by Western blotting. The reagents generated here will help to define the physiological role of this tissue kallikrein and its involvement in human disease.

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References

Bernett, M.J., Blaber, S.I., Scarisbrick, I.A., Dhanarajan, P., Thompson, S.M., and Blaber, M. (2002). Crystal structure and biochemical characterization of human tissue kallikrein 6 reveals that a trypsin-like tissue kallikrein is expressed in the central nervous system. J. Biol. Chem.277, 24562–24570.10.1074/jbc.M202392200Search in Google Scholar

Borgono, C.A. and Diamandis, E.P. (2004). The emerging roles of human tissue kallikreins in cancer. Nat. Rev. Cancer4, 876–890.10.1038/nrc1474Search in Google Scholar

Borgono, C.A., Michael, I.P., and Diamandis, E.P. (2004). Human tissue kallikreins: physiologic roles and application in cancer. Mol. Cancer Res.2, 257–280.10.1158/1541-7786.257.2.5Search in Google Scholar

Brattsand, M., Stefansson, K., Lundh, C., Haasum, Y., and Egelrud, T. (2005). A proteolytic cascade of tissue kallikreins in the stratum corneum. J. Invest. Dermatol.124, 198–203.10.1111/j.0022-202X.2004.23547.xSearch in Google Scholar

Chang, A., Yousef, G.M., Jung, K., Meyts, E.R., and Diamandis, E.P. (2001). Identification and molecular characterization of five novel tissue kallikrein gene 13 (KLK13; KLK-L4) splice variants: differential expression in human testis and testicular cancer. Anticancer Res.21, 3147–3152.Search in Google Scholar

Chang, A., Yousef, G.M., Scorilas, A., Grass, L., Sismondi, P., Ponzone, R., and Diamandis, E.P. (2002). Human tissue kallikrein gene 13 (KLK13) expression by quantitative RT-PCR: an independent indicator of favourable prognosis in breast cancer. Br. J. Cancer86, 1457–1464.10.1038/sj.bjc.6600283Search in Google Scholar

Christopoulos, T.K. and Diamandis, E.P. (1992). Enzymatically amplified time-resolved fluorescence immunoassay with terbium chelates. Anal. Chem.64, 342–346.10.1021/ac00028a004Search in Google Scholar

Clements, J.A., Willemsen, N.M., Myers, S.A., and Dong Y. (2004). The tissue kallikrein family of serine proteases: functional roles in human disease and potential as clinical biomarkers. Crit. Rev. Clin. Lab. Sci.41, 265–312.10.1080/10408360490471931Search in Google Scholar

Collins, F.S. (1995). Positional cloning moves from perditional to traditional. Nat. Genet.9, 347–350.10.1038/ng0495-347Search in Google Scholar

Diamandis, E.P. (1998). Prostate-specific antigen – its usefulness in clinical medicine. Trends Endocrinol. Metab.9, 310–316.10.1016/S1043-2760(98)00082-4Search in Google Scholar

Diamandis, E.P., Yousef, G.M., Luo, L.Y., Magklara, A., and Obiezu, C.V. (2000a). The new human tissue kallikrein gene family: implications in carcinogenesis. Trends Endocrinol. Metab.11, 54–60.10.1016/S1043-2760(99)00225-8Search in Google Scholar

Diamandis, E.P., Yousef, G.M., Clements, J., Ashworth, L.K., Yoshida, S., Egelrud, T., Nelson, P.S., Shiosaka, S., Little, S., Lilja, H., et al. (2000b). New nomenclature for the human tissue kallikrein gene family. Clin. Chem.46, 1855–1858.10.1093/clinchem/46.11.1855Search in Google Scholar

Diamandis, E.P., Scorilas, A., Fracchioli, S., Van Gramberen, M., de Bruijn, H., Henrik, A., Soosaipillai, A., Grass, L., Yousef, G.M., Stenman, U.H., et al. (2003). Human tissue kallikrein 6 (hK6): a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma. J. Clin. Oncol.21, 1035–1043.10.1200/JCO.2003.02.022Search in Google Scholar

Dong, Y., Kaushal, A., Bui, L., Chu, S., Fuller, P.J., Nicklin, J., Samaratunga, H., and Clements, J.A. (2001). Human tissue kallikrein 4 (KLK4) is highly expressed in serous ovarian carcinomas. Clin. Cancer Res.7, 2363–2371.Search in Google Scholar

Gomis-Ruth, F.X., Bayes, A., Sotiropoulou, G., Pampalakis, G., Tsetsenis, T., Villegas, V., Aviles, F.X., and Coll, M. (2002). The structure of human protissue kallikrein 6 reveals a novel activation mechanism for the tissue kallikrein family. J. Biol. Chem.277, 27273–27281.10.1074/jbc.M201534200Search in Google Scholar

Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual (Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press).Search in Google Scholar

Kumar, A., Mikolajczyk, S.D., Goel, A.S., Millar, L.S., and Saedi, M.S. (1997). Expression of the pro-form of prostate-specific antigen by mammalian cells and its conversion to mature, active form by human tissue kallikrein 2. Cancer Res.57, 3111–3114.Search in Google Scholar

Luo, L.Y., Rajpert-De Meyts, E.R., Jung, K., and Diamandis, E.P. (2001a). Expression of the normal epithelial cell-specific 1 (NES1; KLK10) candidate tumour suppressor gene in normal and malignant testicular tissue. Br. J. Cancer85, 220–224.10.1054/bjoc.2001.1870Search in Google Scholar

Luo, L.Y., Grass, L., Howart, J.C., Thibault, P., Ong, H., and Diamandis, E.P. (2001b). Immunofluorometric assay of human tissue kallikrein 10 and its identification in biological fluids and tissues. Clin. Biochem.47, 237–246.10.1093/clinchem/47.2.237Search in Google Scholar

Luo, L.Y., Diamandis, E.P., Look, M.P., Soosaipillai, A.P., and Foekens, J.A. (2002). Higher expression of human tissue kallikrein 10 in breast cancer tissue predicts tamoxifen resistance. Br. J. Cancer86, 1790–1796.10.1038/sj.bjc.6600323Search in Google Scholar

Luo, L.Y., Katsaros, D., Scorilas, A., Fracchioli, S., Bellino, R., Van Gramberen, M., de Bruijn, H., Henrik, A., Stenman, U.H., Massobrio, M., Van der Zee, A.G., Vergote, I., and Diamandis, E.P. (2003a). The serum concentration of human tissue kallikrein 10 represents a novel biomarker for ovarian cancer diagnosis and prognosis. Cancer Res.63, 807–811.Search in Google Scholar

Luo, L.Y., Yousef, G.M., and Diamandis, E.P. (2003b). Human tissue kallikreins and testicular cancer. APMIS111, 225–232.10.1034/j.1600-0463.2003.11101261.xSearch in Google Scholar

Luo, L.Y., Grass, L., and Diamandis, E.P. (2003c). Steroid hormone regulation of the human tissue kallikrein 10 (KLK10) gene in cancer cell lines and functional characterization of the KLK10 gene promoter. Clin. Chim. Acta337, 115–126.10.1016/j.cccn.2003.07.008Search in Google Scholar

Nakamura, T., Scorilas, A., Stephan, C., Jung, K., Soosaipillai, A.R., and Diamandis, E.P. (2003). The usefulness of serum human tissue kallikrein 11 for discriminating between prostate cancer and benign prostatic hyperplasia. Cancer Res.63, 6543–6546.Search in Google Scholar

Rittenhouse, H.G., Finlay, J.A., Mikolajczyk, S.D., and Partin, A.W. (1998). Human tissue kallikrein 2 (hK2) and prostate-specific antigen (PSA): two closely related, but distinct, tissue kallikreins in the prostate. Crit. Rev. Clin. Lab. Sci.35, 275–368.10.1080/10408369891234219Search in Google Scholar

Stephan, C., Jung, K., Diamandis, E.P., Rittenhouse, H.G., Lein, M., and Loening, S.A. (2002). Prostate-specific antigen, its molecular forms, and other tissue kallikrein markers for detection of prostate cancer. Urology59, 2–8.10.1016/S0090-4295(01)01449-2Search in Google Scholar

Takayama, T.K., Carter, C.A., and Deng, T. (2001a). Activation of prostate-specific antigen precursor (pro-PSA) by prostin, a novel human prostatic serine protease identified by degenerate PCR. Biochemistry40, 1679–1687.10.1021/bi002129rSearch in Google Scholar

Takayama, K.T., McMullen, B.A., Nelson, P.S., Matsumura, M., and Fujikawa, K. (2001b). Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single chain urokinase-type plasminogen activator and degradation of prostates acid phosphatase. Biochemistry40, 15341–15348.10.1021/bi015775eSearch in Google Scholar

Talieri, M., Diamandis, E.P., Gourgiotis, D., Mathioudaki, K., and Scorilas, A. (2004). Expression analysis of the human tissue kallikrein 7 (KLK7) in breast tumors: a new potential biomarker for prognosis of breast carcinoma. Thromb. Haemost.91, 180–186.10.1160/TH03-05-0261Search in Google Scholar

Tanimoto, H., Underwood, L.J., Shigemasa, K., Yan, Y., Clarke, J., and O'Brien, T.J. (1999). The stratum corneum chymotryptic enzyme that mediates shedding and desquamation of skin cells is highly overexpressed in ovarian tumor cells. Cancer86, 2074–2082.10.1002/(SICI)1097-0142(19991115)86:10<2074::AID-CNCR27>3.0.CO;2-3Search in Google Scholar

Yousef, G.M. and Diamandis, E.P. (2000). The expanded human tissue kallikrein gene family: locus characterization and molecular cloning of a new member, KLK-L3 (KLK9). Genomics65, 184–194.10.1006/geno.2000.6159Search in Google Scholar

Yousef, G.M. and Diamandis, E.P. (2001). The new human tissue kallikrein gene family: structure, function, and association to disease. Endocr. Rev.22, 184–204.Search in Google Scholar

Yousef, G.M., Kyriakopoulou, L.G., Scorilas, A., Fracchioli, S., Ghiringhello, B., Zarghooni, M., Chang, A., Diamandis, M., Giardina, G., Hartwick, W.J., et al. (2001a). Quantitative expression of the human tissue kallikrein gene 9 (KLK9) in ovarian cancer: a new independent and favorable prognostic marker. Cancer Res.61, 7811–7818.Search in Google Scholar

Yousef, G.M., Magklara, A., Chang, A., Jung, K., Katsaros, D., and Diamandis, E.P. (2001b). Cloning of a new member of the human tissue kallikrein gene family, KLK14, which is down-regulated in different malignancies. Cancer Res.61, 3425–3431.Search in Google Scholar

Yousef, G.M., Borgono, C.A., Scorilas, A., Ponzone, R., Biglia, N., Iskander, L., Polymeris, M.E., Roagna, R., Sismondi, P., and Diamandis, E.P. (2002a). Quantitative analysis of human tissue kallikrein gene 14 expression in breast tumours indicates association with poor prognosis. Br. J. Cancer87, 1287–1293.10.1038/sj.bjc.6600623Search in Google Scholar

Yousef, G.M., Scorilas, A., Kyriakopoulou, L.G., Rendl, L., Diamandis, M., Ponzone, R., Biglia, N., Giai, M., Roagna, R., Sismondi, P., and Diamandis, E.P. (2002b). Human tissue kallikrein gene 5 (KLK5) expression by quantitative PCR: an independent indicator of poor prognosis in breast cancer. Clin. Chem.48, 1241–1250.10.1093/clinchem/48.8.1241Search in Google Scholar

Yousef, G.M., Obiezu, C.V., Jung, K., Stephan, C., Scorilas, A., and Diamandis, E.P. (2002c). Differential expression of tissue kallikrein gene 5 in cancerous and normal testicular tissues. Urology60, 714–718.10.1016/S0090-4295(02)01811-3Search in Google Scholar

Yousef, G., Scorilas, A., Nakamura, T., Ellatif, M.A., Ponzone, R., Biglia, N., Maggiorotto, F., Roagna, R., Sismondi, P., and Diamandis, E.P. (2003). The prognostic value of the human tissue kallikrein gene 9 (KLK9) in breast cancer. Breast Cancer Res. Treat.78, 149–158.10.1023/A:1022931403825Search in Google Scholar

Published Online: 2006-06-26
Published in Print: 2006-06-01

©2006 by Walter de Gruyter Berlin New York

Articles in the same Issue

  1. The First International Symposium on Kallikreins
  2. A comprehensive nomenclature for serine proteases with homology to tissue kallikreins
  3. The kallikrein world: an update on the human tissue kallikreins
  4. Cellular distribution of human tissue kallikreins: immunohistochemical localization
  5. The tissue kallikrein-kinin system protects against cardiovascular and renal diseases and ischemic stroke independently of blood pressure reduction
  6. Proteinase-mediated cell signalling: targeting proteinase-activated receptors (PARs) by kallikreins and more
  7. Recombinant kallikrein expression: site-specific integration for hK6 production in human cells
  8. Kallikrein-related peptidase (KLK) family mRNA variants and protein isoforms in hormone-related cancers: do they have a function?
  9. The role of kallikrein-related peptidases in prostate cancer: potential involvement in an epithelial to mesenchymal transition
  10. Human kallikrein 10, a predictive marker for breast cancer
  11. Activation and enzymatic characterization of recombinant human kallikrein 8
  12. Human tissue kallikrein 9: production of recombinant proteins and specific antibodies
  13. The human kallikrein 10 promoter contains a functional retinoid response element
  14. Human kallikrein 4: enzymatic activity, inhibition, and degradation of extracellular matrix proteins
  15. Kallikrein-related peptidase 14 may be a major contributor to trypsin-like proteolytic activity in human stratum corneum
  16. A sensitive proximity ligation assay for active PSA
  17. Multiple mechanisms underlie the aberrant expression of the human kallikrein 6 gene in breast cancer
  18. Expression of the human kallikrein genes 10 (KLK10) and 11 (KLK11) in cancerous and non-cancerous lung tissues
  19. mRNA expression analysis of human kallikrein 11 (KLK11) may be useful in the discrimination of benign prostatic hyperplasia from prostate cancer after needle prostate biopsy
  20. The epigenetic basis for the aberrant expression of kallikreins in human cancers
  21. Improved prostate cancer detection with a human kallikrein 11 and percentage free PSA-based artificial neural network
  22. Overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 increases the malignant phenotype of ovarian cancer cells
  23. Inhibition profiles of human tissue kallikreins by serine protease inhibitors
  24. Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs)
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