Epitope mapping of human prostate specific antigen and glandular kallikrein expressed in insect cells

Rajakoski, Kristiina; Piironen, Timo; Pettersson, Kim; Lövgren, Janita; Karp, Matti

doi:10.1038/sj.pcan.4500206

Cited by 18 publications

(19 citation statements)

References 14 publications

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“…Most of the protein expressed in E. coli is in inclusion bodies and needs to be refolded, it lacks glycosylation but is enzymatically active [11]. We have earlier found it difficult to express high levels of recombinant hK2 in both mammalian cells and insect cells [17,18]. The expression levels obtained have been 0.2 mg·L −1 and 0.7 mg·L −1 , respectively.…”

mentioning

confidence: 99%

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

Lövgren

Tian

Lundwall

et al. 1999

European Journal of Biochemistry

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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...

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confidence: 99%

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

Lövgren

Tian

Lundwall

et al. 1999

European Journal of Biochemistry

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“…One nucleotide overhang was added to the reporter B sequence containing the Eu 3+ ion carrier chelate because it was previously found to enhance the signal-to-background ratios of the mixed chelate complex [15]. Recombinant PSA and Mab H50 were produced as described previously [33,34]. Polyclonal rabbit anti-mouse antibody (RaM) used in the characterization of the fusion protein was obtained from DakoCytomation (Glostrup, Denmark) and labeled with Eu 3+ chelate as described previously [35].…”

Section: Methodsmentioning

confidence: 99%

Precise construction of oligonucleotide–Fab fragment conjugate for homogeneous immunoassay using HaloTag technology

Päkkilä

Peltomaa

Lamminmäki

et al. 2015

Analytical Biochemistry

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“…For calibration, we used affinity-purified recombinant precursor form of PSA, proPSA, which was produced in a baculovirus expression system as described previously (Rajakoski et al, 1997) and purified with affinity chromatography (Nurmikko et al, 2000; Vaisanen et al, 1999). The concentration of the recombinant proPSA was calibrated against the World Health Organization (WHO) standard (Rafferty et al, 2000).…”

Section: Methodsmentioning

confidence: 99%

Immunoassay for the discrimination of free prostate-specific antigen (fPSA) forms with internal cleavages at Lys145 or Lys146 from fPSA without internal cleavages at Lys145 or Lys146

Peltola

Niemelä

Alanen

et al. 2011

Journal of Immunological Methods

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Total levels of circulating prostate-specific antigen (tPSA) are strongly associated with prostate cancer (PCa) risk and outcome but benign prostate disease is the most frequent cause of a moderately elevated PSA level. Free PSA (fPSA) forms are independently associated with PCa risk and contribute modest diagnostic enhancements above and beyond tPSA alone. We developed an immunoassay for fPSA subfractions containing internal cleavages at Lys145 or Lys146 (fPSA-N). The assay was based on blocking intact single-chain fPSA (fPSA-I) with antibody 4D4 which does not detect PSA containing internal cleavages at Lys145 or Lys146. We also measured fPSA-N in blood from healthy volunteers and in anti-coagulated plasma from 76 men with or without evidence of PCa at biopsy. The analytical and functional detection limits of this assay were 0.016 ng/mL and 0.10 ng/mL, respectively. The median recovery of male fPSA-N from female plasma was 95.0 %. All 12 female samples (average age 28 years) had fPSA-N concentrations at or below the analytical detection limit. The median fPSA-N concentration (0.050 ng/mL) in 9 healthy male volunteers (age < 40 years) was below the functional detection limit, 0.420 ng/mL in 27 patients with benign prostate conditions and 0.239 ng/mL in 49 patients with PCa. Deming regression analysis of the patient samples showed that the measured fPSA-N concentrations were generally 23% lower than the previously calculated (fPSA minus fPSA-I) concentrations, likely due to differences in the antibody combinations used. In conclusion, we have developed a sensitive, specific and direct immunoassay for fPSA-N which can be used to study the clinical relevance of this PSA isoform.

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Epitope mapping of human prostate specific antigen and glandular kallikrein expressed in insect cells

Cited by 18 publications

References 14 publications

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

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

Precise construction of oligonucleotide–Fab fragment conjugate for homogeneous immunoassay using HaloTag technology

Immunoassay for the discrimination of free prostate-specific antigen (fPSA) forms with internal cleavages at Lys145 or Lys146 from fPSA without internal cleavages at Lys145 or Lys146

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