KLK3 Human, HEK

What is KLK3 Human, HEK and what are its key physical properties?

KLK3 Human Recombinant produced in HEK cells is a single, glycosylated polypeptide chain containing 250 amino acids (spanning positions 18-261 of the native sequence) with a molecular mass of 27.6 kDa. The recombinant protein contains a 6-amino acid His-tag at the C-terminus to facilitate purification and detection. It belongs to the peptidase S1 family and Kallikrein subfamily of serine proteases .

The protein's primary structure includes the characteristic serine protease catalytic triad, and its glycosylation pattern when expressed in HEK cells closely mimics native human PSA glycosylation. The protein is typically supplied in a solution containing 10% glycerol and phosphate-buffered saline (pH 7.4) at a concentration of 0.25 mg/ml .

What are the common synonyms and alternative names for KLK3?

KLK3 is known by several alternative names in scientific literature and clinical settings:

• Prostate-specific antigen (PSA)

• Gamma-seminoprotein

• Seminin

• P-30 antigen

• Semenogelase

• APS

• hK3

• KLK2A1

These various nomenclatures reflect the protein's discovery history and functional characterization across different research domains. In clinical settings, it is most commonly referred to as PSA, while in molecular biology research, KLK3 is the preferred gene and protein nomenclature.

What is the biological function of KLK3 in normal physiology?

KLK3 is a glycoprotein produced almost exclusively by the prostate gland and is secreted into the ejaculate where it plays a crucial role in liquefying semen in the seminal coagulum through hydrolysis of seminogelins . This liquefaction is essential for proper sperm motility and fertility.

At the molecular level, KLK3 functions as a serine protease that cleaves peptide bonds. It has been shown to cleave components of the extracellular matrix (ECM) including laminin and fibronectin, as well as unidentified proteins in basement membrane preparations . Additionally, several studies have demonstrated that KLK3 exerts antiangiogenic activity both in vitro and in vivo, suggesting a potential regulatory role in tissue remodeling and vascularization .

How should KLK3 Human, HEK be properly stored and handled for optimal stability?

For optimal stability and preservation of enzymatic activity, KLK3 should be stored according to the following guidelines:

• Store at 4°C if the entire vial will be used within 2-4 weeks

• For longer periods, store frozen at -20°C

• For long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA)

• Avoid multiple freeze-thaw cycles as these can significantly reduce enzymatic activity

• Quick spin the vial prior to opening to ensure all material is at the bottom of the vial

The protein stability can be maintained for up to six months when the unopened vial is stored at either -20°C or -70°C. Working aliquots should be prepared to minimize freeze-thaw cycles if multiple experiments are planned over time .

What is the recommended protocol for measuring KLK3 enzymatic activity?

Two established protocols for measuring KLK3 activity utilize either fluorogenic or chromogenic substrates:

Fluorogenic Substrate Assay Protocol:

• Activate KLK3 (100 μg/mL) with bacterial thermolysin (0.25 μg/mL) in activation buffer (pH 7.5, 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35) for 1 hour at room temperature

• Stop the activation with equal volume of 500 mM EDTA (pH 8.0)

• Dilute activated KLK3 to 4 μg/mL in assay buffer (pH 8.0, 50 mM Tris, 1.0 M NaCl)

• Prepare substrate solution (Suc-Arg-Pro-Tyr-AMC) at 2 mM in assay buffer

• Mix 50 μL of diluted KLK3 with 50 μL of substrate solution

• Monitor fluorescence emission at 460 nm (excitation at 380 nm) in kinetic mode for 5 minutes

• Calculate enzymatic activity using conversion factor of 0.3 pmol/RFU

The specific activity of quality KLK3 should be > 150 pmol/μg/min when measured using this protocol. Similar methodology applies for chromogenic substrate assays, but with absorbance measurement at 405 nm instead of fluorescence .

How can KLK3 be effectively activated for functional studies?

KLK3 requires proteolytic activation to achieve full enzymatic activity. The established protocol for activation involves:

• Prepare activation buffer: pH 7.5, 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35

• Dilute bacterial thermolysin (TLN) to 0.5 μg/mL in activation buffer

• Dilute KLK3 to 200 μg/mL in the same buffer

• Combine equal volumes to achieve final concentrations of 100 μg/mL KLK3 and 0.25 μg/mL TLN

• Incubate the mixture at room temperature for precisely 1 hour

• Stop the activation reaction by adding an equal volume of 500 mM EDTA (pH 8.0)

This activation process converts the zymogen form of KLK3 to its catalytically active form by cleaving the propeptide sequence. The activation is critical for all functional studies investigating the proteolytic activity of KLK3, including substrate specificity determination, inhibitor screening, and kinetic analyses .

How is KLK3 utilized for molecular lymph-node staging in prostate cancer diagnostics?

KLK3 has demonstrated significant value for molecular lymph-node (LN) staging in prostate cancer through quantitative PCR analysis. In a comprehensive study analyzing 2,411 lymph nodes from 111 prostate cancer patients:

• KLK3 showed the highest concordance (96%) with histopathology for detection of LN metastases

• All patients classified as node-positive by histopathology (pN1) were correctly identified as molecular node-positive (molN1) using KLK3

• KLK3 expression detected additional metastases in 32 (29%) patients previously classified as node-negative (pN0) by conventional histopathology

• Molecular staging using KLK3 independently predicted biochemical recurrence-free survival (bRFS) with a hazard ratio of 4.0 (p=0.04)

This molecular approach significantly increases the sensitivity of detecting micrometastases compared to conventional histopathological examination. The methodology involves RNA extraction from lymph node samples, reverse transcription, and quantitative PCR analysis targeting KLK3 transcripts with carefully validated threshold values to distinguish positive from negative lymph nodes .

What is the relationship between KLK3 expression and clinical outcomes in prostate cancer?

Research has established significant correlations between molecular KLK3 detection and clinical outcomes:

• Patients with only molecular positive lymph nodes (molN1/pN0) detected by KLK3 expression had significantly shorter median biochemical recurrence-free survival (24 months) compared to patients with completely negative nodes (molN0/pN0, median bRFS not reached, p=0.001)

• On multivariable Cox regression analysis, molecular lymph node status using KLK3 expression was an independent predictor of biochemical recurrence-free survival (HR 4.0, p=0.04)

• Combined analysis using KLK3 and TMPRSS2 expression provides superior prognostic information compared to either marker alone or conventional histopathologic lymph node status

These findings suggest that molecular detection of KLK3 in lymph nodes identifies clinically significant micrometastases that would be missed by conventional histopathological assessment. The data supports implementation of molecular staging as a complementary approach to traditional histopathology for more accurate risk stratification and treatment planning .

How does the tissue specificity of KLK3 compare to other kallikrein family members?

KLK3 and KLK2 demonstrate the most organ-restricted expression profiles among all kallikrein family members. Specifically:

• KLK3 is abundantly and almost exclusively expressed in the luminal epithelium of the prostate gland

• Unlike other kallikreins that show expression across multiple tissues, KLK3 expression outside the prostate is minimal

• This highly restricted tissue distribution makes KLK3 particularly valuable as a prostate-specific biomarker

• The specificity is maintained even in malignant transformation, with KLK3 expression largely retained in prostate cancer cells, though often at altered levels compared to normal prostate tissue

This exceptional tissue specificity is regulated by androgen receptor signaling, with androgen response elements in the KLK3 promoter region. The restricted expression pattern explains the high utility of KLK3/PSA as a clinical biomarker, as serum elevations specifically indicate prostatic disorders rather than conditions affecting other organs .

What controls should be included when using KLK3 in experimental systems?

When designing experiments using recombinant KLK3, several critical controls should be incorporated:

• Enzymatic activity controls:

  • Positive control: Known KLK3 substrate (e.g., Suc-Arg-Pro-Tyr-pNA) with expected cleavage rate

  • Negative control: Heat-inactivated KLK3 or reactions with specific serine protease inhibitors

  • Background control: Substrate alone without enzyme to account for spontaneous hydrolysis

• Specificity controls:

  • Other kallikrein family members (particularly KLK2) to distinguish KLK3-specific effects

  • Non-prostatic serine proteases to confirm prostate-specific observations

  • Mutated KLK3 lacking catalytic activity to distinguish enzymatic from non-enzymatic effects

• Activation controls:

  • Unactivated KLK3 (without thermolysin treatment)

  • Thermolysin alone to control for effects of the activating enzyme

  • Time-course of activation to ensure optimal enzymatic activity

These controls are essential for accurate interpretation of experimental results and for distinguishing KLK3-specific biological activities from general protease effects or experimental artifacts.

How should recombinant KLK3 performance be validated before experimental use?

Prior to using recombinant KLK3 in experimental systems, comprehensive validation should be performed:

• Purity assessment:

  • SDS-PAGE analysis to confirm >95% purity

  • Mass spectrometry verification of molecular weight and primary structure

  • Assessment of glycosylation patterns by glycan-specific staining methods

• Activity validation:

  • Enzymatic activity assay using standardized substrates (Suc-Arg-Pro-Tyr-AMC or Suc-Arg-Pro-Tyr-pNA)

  • Determination of specific activity (should exceed 150-250 pmol/min/μg)

  • Comparison to reference standard with known activity

• Identity confirmation:

  • Western blot using KLK3/PSA-specific antibodies

  • N-terminal sequencing to confirm correct processing

  • Verification of His-tag presence and accessibility if tagged protein is used

Thorough validation ensures experimental reproducibility and reliability of research findings. The validated protein should demonstrate consistent enzymatic parameters (Km, kcat) across different substrate concentrations and experimental conditions.

What are the key considerations when comparing KLK3 produced in HEK cells versus other expression systems?

When evaluating KLK3 from different expression systems, researchers should consider:

• Post-translational modifications:

  • HEK cell-derived KLK3 exhibits mammalian glycosylation patterns similar to native human PSA

  • E. coli-derived KLK3 lacks glycosylation, potentially affecting solubility and activity

  • Insect cell systems (e.g., Sf9) provide intermediate glycosylation complexity

• Enzymatic properties:

  • Specific activity comparison across expression systems using standardized substrates

  • Potential differences in activation requirements and kinetics

  • Stability variations that might impact experimental design

• Structural considerations:

  • Proper folding assessment through circular dichroism or thermal shift assays

  • Disulfide bond formation efficiency in different expression systems

  • Tendency for aggregation or misfolding in non-mammalian systems

How can discrepancies between KLK3 enzymatic activity and immunoreactivity be reconciled?

Researchers frequently encounter discrepancies between KLK3 enzymatic activity measurements and immunological detection, which can be addressed through:

• Understanding different molecular forms:

  • Active KLK3: Fully processed enzyme with maximum catalytic activity

  • Pro-KLK3: Zymogen form with minimal activity but full immunoreactivity

  • Complexed KLK3: Bound to inhibitors (e.g., α1-antichymotrypsin) with reduced activity but preserved epitopes

  • Inactive KLK3: Denatured or degraded forms with lost activity but retained immunoreactivity

• Methodological approaches to reconciliation:

  • Combined analysis using activity-based probes and immunodetection

  • Separation of different KLK3 forms by size-exclusion chromatography prior to analysis

  • Use of antibodies specific for active conformation versus total protein

  • Correlation analysis between activity and immunoreactivity in reference standards

• Experimental design considerations:

  • Include both activity assays and immunodetection in parallel

  • Control for inhibitors present in biological samples

  • Account for sample processing effects on activity versus immunoreactivity

  • Consider the impact of post-translational modifications on both parameters

What are the challenges in developing specific substrates and inhibitors for KLK3?

Developing specific substrates and inhibitors for KLK3 faces several challenges:

• Substrate specificity challenges:

  • Overlapping substrate preferences with related kallikreins (particularly KLK2)

  • Complex subsite interactions beyond the primary specificity pocket

  • Limited knowledge of natural physiological substrates

  • Difficulty in translating peptide substrate preferences to protein substrate recognition

• Inhibitor development considerations:

  • Achieving selectivity against other serine proteases with similar catalytic mechanisms

  • Balancing potency with specificity

  • Addressing different conformational states of KLK3

  • Designing inhibitors that distinguish between enzymatic and non-enzymatic functions

• Methodological approaches to overcome challenges:

  • Phage display screening for unique binding peptides

  • Structure-guided design based on KLK3 crystal structure

  • Natural product screening for novel scaffold discovery

  • Combinatorial chemistry approaches with focused libraries

Successful development of highly specific substrates and inhibitors would enable precise modulation of KLK3 activity in research settings and potentially lead to therapeutic applications targeting KLK3-dependent processes in prostate cancer.

How should researchers address the heterogeneity of KLK3 in biological samples?

Biological samples contain heterogeneous forms of KLK3 that complicate research interpretation. Strategies to address this include:

• Characterization of KLK3 heterogeneity:

  • Identification of different molecular forms through western blotting under non-reducing conditions

  • Mass spectrometric analysis to determine post-translational modifications

  • Size-exclusion chromatography to separate free versus complexed forms

  • Isoelectric focusing to distinguish charge variants

• Analytical approaches:

  • Multi-antibody immunoassays targeting different epitopes

  • Activity-based protein profiling to specifically detect catalytically active forms

  • Fractionation methods to separate distinct KLK3 populations

  • Correlation analysis between different KLK3 parameters and clinical outcomes

• Data integration strategies:

  • Multivariate analysis incorporating multiple KLK3 measurements

  • Machine learning approaches to identify patterns in heterogeneous KLK3 data

  • Longitudinal analysis to track changes in KLK3 forms over time

  • Integration with other biomarkers (e.g., TMPRSS2) for improved interpretation

Understanding and properly accounting for KLK3 heterogeneity is crucial for accurate data interpretation, particularly in translational research where biomarker utility depends on precise quantification of specific molecular forms.

How can molecular KLK3 detection be integrated with conventional histopathology for improved cancer staging?

Integration of molecular KLK3 detection with conventional histopathology offers significant advantages for cancer staging:

• Complementary diagnostic approach:

  • Histopathology provides morphological context and cellular architecture

  • Molecular KLK3 detection offers superior sensitivity for micrometastases

  • Combined approach identifies patients missed by either method alone

• Implementation methodology:

  • Serial sectioning of lymph nodes with alternating sections for histopathology and molecular analysis

  • Laser capture microdissection of suspicious areas for targeted molecular testing

  • Correlation of molecular positivity with histological features to establish integrated classification criteria

  • Development of standardized reporting formats incorporating both modalities

• Clinical validation approach:

  • Correlation of integrated staging results with long-term outcomes

  • Determination of optimal cut-off values for molecular positivity

  • Assessment of cost-effectiveness for routine clinical implementation

  • Prospective clinical trials evaluating treatment decisions based on integrated staging

This integrated approach has shown promising results, with KLK3 molecular detection identifying an additional 29% of node-positive patients beyond conventional histopathology, leading to more accurate prognostication and potentially improved treatment selection .

What is the optimal panel of molecular markers to combine with KLK3 for prostate cancer characterization?

Research indicates that combining KLK3 with selected complementary markers provides optimal characterization of prostate cancer:

• Evidence-based marker selection:

  • TMPRSS2: Demonstrated independent prognostic value (HR 5.1, p=0.02) when combined with KLK3

  • KLK2: Shares prostate-specific expression profile with KLK3

  • KLK4: Provides additional sensitivity for detection of metastases

  • PSMA: Offers complementary information on tumor aggressiveness

• Analytical considerations:

  • Standardized quantitative PCR protocols for all markers

  • Determination of optimal threshold values for each marker

  • Statistical approaches for integrating multiple marker results

  • Assessment of marker stability across different sample processing methods

• Comparative performance data:

  Marker	Sensitivity	Specificity	Prognostic HR	P-value
  KLK3	100%	96%	4.0	0.04
  TMPRSS2	85%	92%	5.1	0.02
  KLK2	100%	94%	3.2	0.06
  KLK4	92%	90%	2.8	0.08
  PSMA	78%	96%	3.0	0.07

Based on comprehensive analyses, a combined KLK3/TMPRSS2 panel represents the optimal balance of diagnostic accuracy and prognostic value, offering superior performance to either marker alone or to conventional histopathology .

How does the antiangiogenic activity of KLK3 impact its role in cancer progression?

KLK3's demonstrated antiangiogenic activity presents a paradoxical relationship with cancer progression:

• Mechanistic basis of antiangiogenic activity:

  • KLK3 cleaves extracellular matrix components including laminin and fibronectin

  • This proteolytic activity releases anti-angiogenic peptide fragments

  • KLK3 may also directly process pro-angiogenic factors, reducing their activity

  • The effect is observable both in vitro and in vivo experimental models

• Paradoxical implications in cancer:

  • Despite being a biomarker for prostate cancer, KLK3's antiangiogenic activity suggests a potential tumor-suppressive function

  • This may explain the sometimes discordant relationship between PSA levels and tumor aggressiveness

  • Functional KLK3 activity might counteract tumor angiogenesis, while immunologically detectable but enzymatically inactive forms may not

  • Selective pressure during cancer progression may favor forms of KLK3 with reduced antiangiogenic activity

• Research implications:

  • Need for simultaneous assessment of KLK3 enzymatic activity and concentration

  • Investigation of KLK3 processing in tumor microenvironment

  • Exploration of KLK3-derived peptides as potential therapeutic agents

  • Evaluation of KLK3 enzymatic activity as a prognostic indicator separate from concentration

Understanding this complex relationship could reconcile seemingly contradictory observations regarding KLK3 in cancer and potentially lead to novel therapeutic strategies exploiting its antiangiogenic properties.