KLK15 Human, sf9

What is KLK15 and where is it primarily expressed in human tissues?

KLK15 (Kallikrein-related peptidase 15) is the latest identified member of the kallikrein-related peptidase family. Tissue distribution studies using both immunoassay (ELISA) and immunohistochemistry techniques have revealed that KLK15 is predominantly expressed in the testis and seminal fluid, with lower expression levels detected in the prostate and other tissues . Specifically, immunohistochemical analysis demonstrates strong expression in mature spermatids but not in immature germ cells, suggesting a potential role in spermatogenesis . In prostatic tissue, KLK15 displays a distinctive nuclear localization pattern primarily in the basal cell layer of the epithelium . This specific expression profile provides initial insights into its potential physiological functions.

What is the molecular structure of recombinant KLK15 Human produced in Sf9 cells?

Recombinant KLK15 Human produced in Sf9 baculovirus expression systems is a single, glycosylated polypeptide chain containing 249 amino acids (positions 17-256 of the native sequence) . The protein has a molecular mass of 27.4 kDa, though it typically appears at approximately 28-40 kDa when analyzed by SDS-PAGE due to post-translational modifications, particularly glycosylation . Commercial preparations typically include a 6-amino acid histidine tag at the C-terminus to facilitate purification through proprietary chromatographic techniques . The complete amino acid sequence begins with ADPQDGDKLL and terminates with WIRETMKRNHHHHHH (including the His tag) . Understanding these structural properties is essential for designing experiments involving recombinant KLK15 protein.

How do expression patterns differ between KLK15 mRNA and protein in normal versus cancerous prostate tissue?

An intriguing dichotomy exists between KLK15 mRNA and protein expression patterns in prostate cancer. While mRNA expression is elevated in tumor tissue compared to adjacent normal tissue (as quantified by RT-PCR in laser-capture microdissected samples), protein levels demonstrate an inverse relationship . Specifically, immunohistochemical analysis of 193 prostatic adenocarcinoma samples revealed reduced KLK15 protein levels in cancerous tissue compared to both adjacent normal tissue and prostatic intraepithelial neoplasia . This discordance between transcriptional and translational expression suggests complex post-transcriptional regulatory mechanisms affecting KLK15 in prostate cancer progression. Both mRNA and protein expression levels correlate with pathological tumor stage and Gleason score, reinforcing KLK15's potential clinical significance .

What are the optimal storage and handling conditions for recombinant KLK15 Human produced in Sf9 cells?

For optimal stability of recombinant KLK15 Human produced in Sf9 cells, researchers should adhere to the following storage protocol:

• Short-term storage (2-4 weeks): Store at 4°C in the supplied buffer.

• Long-term storage: Store at -20°C in aliquots to minimize freeze-thaw cycles .

• Buffer composition: Typically, KLK15 protein solution (0.25 mg/ml) is supplied in Phosphate Buffered Saline (pH 7.4) with 10% glycerol .

• Stability enhancement: For long-term storage, addition of carrier protein (0.1% HSA or BSA) is recommended to prevent protein adsorption to surfaces and maintain activity .

• Avoid repeated freeze-thaw cycles as these can lead to protein denaturation and activity loss .

Following these guidelines ensures maintenance of protein integrity and enzymatic activity for experimental applications.

What detection methods have been validated for KLK15 expression analysis in tissue samples?

Research has validated multiple complementary approaches for detecting KLK15 expression in clinical and research specimens:

• Immunoassay (ELISA): A newly developed ELISA has been validated for quantitative measurement of KLK15 in tissue extracts, cell culture supernatants, and biological fluids. This method demonstrates high sensitivity for detecting physiological concentrations of KLK15 in testis, seminal fluid, and prostate tissues .

• Immunohistochemistry (IHC): This technique has been successfully employed to localize KLK15 within cellular compartments, revealing nuclear localization in prostatic epithelium and expression in mature spermatids . For IHC studies, appropriate negative controls and validation of antibody specificity are essential for result interpretation.

• RT-PCR for mRNA expression: Laser capture microdissection followed by reverse-transcriptase polymerase chain reaction has been effectively used to quantify KLK15 mRNA expression in cancerous versus adjacent normal prostatic tissue .

Selection of the appropriate detection method should be guided by the specific research question, sample type availability, and whether protein localization or quantification is the primary objective.

How can researchers evaluate the functional activity of recombinant KLK15 Human protein?

As a serine protease belonging to the kallikrein family, KLK15's functional activity can be assessed through multiple approaches:

• Enzymatic activity assays: Using synthetic peptide substrates containing specific cleavage sites for KLK15. The catalytic activity is classified under EC 3.4.21 (serine endopeptidases) .

• Substrate specificity determination: Testing various physiological substrates to determine KLK15's preferred cleavage sites and kinetic parameters (Km, Vmax, kcat).

• Inhibition studies: Evaluating the effects of general serine protease inhibitors (like PMSF or aprotinin) and specific kallikrein inhibitors on KLK15 activity.

• Cell-based assays: Assessing the biological effects of adding purified KLK15 to prostate cancer cell lines, including potential impacts on proliferation, migration, or invasion.

• Post-translational modification analysis: Determining how glycosylation patterns affect enzymatic activity by comparing differently processed forms of the protein.

When designing these assays, researchers should account for the presence of the C-terminal His-tag, which may potentially influence activity in some experimental contexts.

How does KLK15 expression correlate with prostate cancer progression and clinical outcomes?

KLK15 has emerged as an independent prognostic biomarker for prostate cancer progression. In a comprehensive study of 193 prostatic adenocarcinoma samples, several significant correlations were established:

• Expression correlation with pathological parameters: KLK15 expression levels correlate with pathological tumor stage and Gleason score, both at mRNA and protein levels .

• Prognostic value: Univariate Kaplan-Meier analysis demonstrated a significant association between dichotomized KLK15 levels and disease progression defined by prostate-specific antigen (PSA) relapse (p = 0.001) .

• Independent prognostic factor: Multivariate analysis using the Cox proportional hazards regression model identified dichotomized KLK15 expression as an independent prognostic factor for poor outcome, even after correcting for conventional parameters including age, preoperative PSA level, pathological tumor stage, Gleason score, and surgical margin status (inclusion model, hazard ratio 1.802, 95% confidence interval 1.037-3.132, p = 0.037) .

These findings establish KLK15 as a promising independent marker for identifying patients at elevated risk for disease progression following radical prostatectomy.

How does KLK15 expression in prostate cancer differ from other kallikreins, particularly KLK3 (PSA)?

KLK15 demonstrates distinct expression characteristics from the well-established prostate cancer marker KLK3 (PSA):

• Hormone responsiveness: Unlike KLK3, which exhibits clear androgen-dependent expression, KLK15 expression appears to be hormone-independent in prostate cancer cell lines . This fundamental difference suggests different regulatory mechanisms and potential utility in monitoring hormone-refractory prostate cancers.

• Expression pattern in cancer: While KLK3 protein levels typically increase in prostate cancer, KLK15 shows a more complex pattern with elevated mRNA but reduced protein levels in cancerous tissue compared to adjacent normal tissue .

• Cellular localization: KLK15 displays predominantly nuclear localization in the basal cell layer of prostatic epithelium , whereas KLK3 is primarily secreted.

• Prognostic value: KLK15 has demonstrated independent prognostic value beyond established markers like KLK3 (PSA), suggesting it captures different aspects of tumor biology and progression .

These distinguishing characteristics position KLK15 as a complementary biomarker to KLK3, potentially improving diagnostic and prognostic accuracy when used in combination.

What methodological approaches are recommended for investigating KLK15's role in tumor progression mechanisms?

To effectively investigate KLK15's mechanistic contributions to tumor progression, researchers should consider these methodological approaches:

• Gene expression modulation studies:

  • Overexpression of KLK15 in prostate cell lines using lentiviral or plasmid vectors

  • siRNA or CRISPR-based knockdown/knockout of KLK15 to assess loss-of-function effects

  • Analysis of resultant phenotypes regarding proliferation, migration, invasion, and resistance to apoptosis

• Identification of proteolytic substrates:

  • Proteomic analysis of KLK15-treated versus untreated cell culture supernatants

  • Terminal amine isotopic labeling of substrates (TAILS) to identify direct KLK15 cleavage targets

  • Validation of putative substrate processing using in vitro cleavage assays with recombinant proteins

• Signaling pathway analysis:

  • Western blotting for phosphorylation changes in key signaling nodes after KLK15 treatment

  • Pathway inhibitor studies to determine which signaling pathways mediate KLK15's effects

  • Transcriptomic analysis to identify gene expression changes triggered by KLK15 activity

• In vivo studies:

  • Xenograft models with KLK15-manipulated cancer cell lines

  • Analysis of tumor growth, angiogenesis, and metastatic potential

  • Correlation with KLK15 levels in patient-derived xenografts

These approaches provide a comprehensive framework for dissecting KLK15's functional role in cancer biology.

What are the current challenges in translating KLK15 research findings to clinical applications?

Despite promising results linking KLK15 to prostate cancer progression, several challenges impede clinical translation:

• Biological complexity: The discordance between KLK15 mRNA (elevated) and protein levels (reduced) in prostate cancer tissues complicates interpretation of expression data and necessitates careful consideration of which analyte (mRNA or protein) provides more reliable prognostic information.

• Assay standardization: Current research employs various detection methods for KLK15, including newly developed ELISAs and immunohistochemistry protocols . Standardization of these assays is essential for comparable results across laboratories and clinical settings.

• Specificity considerations: As a member of the kallikrein family with potential sequence homology to other kallikreins, ensuring antibody and detection method specificity for KLK15 remains challenging.

• Clinical validation requirements: Larger, prospective multi-center studies with diverse patient populations are needed to validate KLK15's prognostic value before clinical implementation.

• Functional understanding gaps: Better characterization of KLK15's physiological and pathological roles is needed to determine whether it represents a causal factor in cancer progression or merely a correlative biomarker.

Addressing these challenges requires coordinated efforts across basic science, assay development, and clinical research domains.

How might post-translational modifications affect KLK15 function in physiological versus pathological contexts?

Post-translational modifications (PTMs) likely play crucial roles in regulating KLK15 function:

• Glycosylation: Recombinant KLK15 produced in Sf9 cells is glycosylated , and the pattern of glycosylation may differ between expression systems and between normal and cancer tissues. These differences could affect protein stability, enzymatic activity, or recognition by binding partners.

• Proteolytic activation: Like other kallikreins, KLK15 is likely synthesized as an inactive zymogen requiring proteolytic cleavage for activation. The enzymes responsible for this activation may be differentially expressed in cancer, potentially altering KLK15 activity levels independent of expression.

• Phosphorylation: Nuclear localization of KLK15 in prostatic epithelium suggests potential involvement in nuclear processes, possibly regulated by phosphorylation events that could be dysregulated in cancer.

• Protein-protein interactions: Binding to inhibitors or other regulatory proteins may be affected by cancer-associated PTMs, altering KLK15's functional availability.

Research investigating these modifications in normal versus cancerous tissues would provide valuable insights into KLK15's regulatory mechanisms and potentially explain the observed discrepancy between mRNA and protein levels in cancer.

What complementary biomarkers might enhance the prognostic utility of KLK15 in prostate cancer?

Enhancing KLK15's prognostic value could be achieved through combination with complementary biomarkers:

• Other kallikrein family members: A "kallikrein panel" including KLK2, KLK3 (PSA), KLK15, and others might provide superior prognostic information than individual markers. Research should focus on which combinations offer optimal performance.

• Androgen-regulated genes: Since KLK15 appears hormone-independent unlike KLK3, combining KLK15 with androgen-regulated markers could capture both hormone-dependent and independent aspects of tumor biology.

• Epithelial-mesenchymal transition (EMT) markers: As KLK15 correlates with tumor aggressiveness , combining it with EMT markers might enhance prediction of metastatic potential.

• Genetic/genomic markers: Integrating KLK15 expression with genomic alterations (TMPRSS2-ERG fusion, PTEN loss, etc.) could create more robust prognostic signatures.

• Metabolic biomarkers: Combining KLK15 with metabolic markers might provide complementary information about tumor phenotype and aggressiveness.

Multivariate statistical approaches, machine learning algorithms, and decision tree analyses would be valuable in determining optimal marker combinations and their respective weightings for maximizing prognostic accuracy.