KLK2 Human, sf9

What is KLK2 and what is its biological significance?

KLK2 (Human kallikrein-related peptidase 2) is a trypsin-like serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, which is a major component of seminal fluid . It belongs to the glandular kallikrein protein family whose members engage in diverse biological functions . KLK2 shares the highest homology (78-80% at amino acid and DNA levels) with KLK3 (better known as PSA) .

Biologically, KLK2 is most likely involved in activating and complementing chymotryptic KLK3 in cleaving seminal clotting proteins, resulting in sperm liquefaction . It selectively cleaves at arginine residues and is responsible for cleaving pro-prostate-specific antigen into its enzymatically active form . Recent research indicates that KLK2 might be involved in carcinogenesis and tumor metastasis of prostate cancer, suggesting its potential as both a prognostic marker and therapeutic target .

What are the structural and functional characteristics of KLK2 protein?

KLK2 Human recombinant protein produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 246 amino acids (residues 25-261) with a molecular mass of 27.2 kDa, though it migrates at 28-40 kDa on SDS-PAGE under reducing conditions . When expressed recombinantly, it typically includes a 6 amino acid His tag at the C-terminus to facilitate purification .

Structurally, KLK2 contains several notable features:

• The 99-loop (also called kallikrein loop): A distinctive feature shared with the "classical" KLKs 1-3, this extended loop near the non-primed substrate binding site acts as a master regulator of activity

• Active site interface: Involved in substrate recognition and catalysis

• Autolysis site: Located between residues 95e and 95f in the 99-loop, responsible for autocatalytic cleavage

Functionally, KLK2 displays several biochemical peculiarities, including reversible inhibition by micromolar Zn²⁺ concentrations and permanent inactivation by autocatalytic cleavage, both regulated by the 99-loop . Crystal structures at 1.9 Å resolution have revealed discontinuous electron density for the 99-loop, indicating this region is largely disordered, which influences its substrate binding and catalytic properties .

How is recombinant KLK2 Human protein produced in Sf9 cells?

Recombinant KLK2 production in Sf9 Baculovirus cells involves a systematic approach that ensures proper protein folding and post-translational modifications. While the search results don't detail the exact production protocol, standard procedures for recombinant protein expression in this system typically involve:

• Gene cloning: The KLK2 gene (coding for amino acids 25-261 of the protein) is inserted into a baculovirus expression vector, typically with a 6-amino acid His tag at the C-terminus .

• Transfection and viral amplification: The recombinant vector is transfected into Sf9 insect cells, followed by baculovirus production and amplification.

• Protein expression: The amplified virus is used to infect fresh Sf9 cells, leading to high-level expression of the recombinant KLK2 protein.

• Purification: The expressed protein is purified using proprietary chromatographic techniques, typically including affinity chromatography leveraging the His tag .

The final product is a glycosylated polypeptide chain with over 90% purity as determined by SDS-PAGE . The protein is typically formulated in a solution containing Phosphate Buffered Saline (pH 7.4) and 10% glycerol at a concentration of 0.25 mg/ml .

What are the optimal storage and handling conditions for KLK2 Human, sf9?

Proper storage and handling are crucial for maintaining KLK2 protein stability and activity. Based on the search results, the following conditions are recommended:

For short-term storage (2-4 weeks): Store at 4°C if the entire vial will be used within this period .

For long-term storage: Store frozen at -20°C or preferably at -70°C, where recombinant proteins remain stable for up to 1 year from the date of receipt .

For extended stability: Addition of a carrier protein (0.1% HSA or BSA) is recommended for long-term storage .

Important handling considerations:

• Avoid repeated freeze-thaw cycles as these can degrade the protein and reduce activity

• For experimental use, thaw aliquots just before use

• The standard formulation (0.25mg/ml in PBS with 10% glycerol) maintains stability during routine laboratory handling

How does the 99-loop regulate KLK2 activity and what are its implications for experimental design?

The 99-loop (kallikrein loop) of KLK2 serves as a master regulator of the protein's activity through multiple mechanisms, which has profound implications for experimental design when studying this protease . Recent structural analyses at 1.9 Å resolution of KLK2-small molecule inhibitor complexes have revealed that this loop displays discontinuous electron density, indicating significant disorder that affects function .

The 99-loop regulates KLK2 activity through:

• Zinc inhibition mechanism: The loop creates a Zn²⁺ binding site at the 99-loop/active site interface, making KLK2 susceptible to reversible inhibition by micromolar concentrations of Zn²⁺ . Mutational studies of the 99-loop have demonstrated altered susceptibility to Zn²⁺, confirming this regulatory mechanism .

• Autolytic inactivation: The loop contains an autolysis site between residues 95e and 95f, whose cleavage leads to permanent inactivation of the enzyme. Eliminating this site through mutation prevents the mature enzyme from limited autolysis and irreversible inactivation .

• Conformational dynamics: Comprehensive structural comparisons have shown that the 99-loop exists in both open and closed conformations, allowing or preventing substrate access, which extends the concept of conformational selection in trypsin-related proteases .

For experimental design, researchers should consider:

• Buffer composition: Controlling Zn²⁺ concentration is critical for consistent activity measurements

• Time-dependent activity changes: Account for potential autolytic inactivation during extended incubations

• Mutation strategies: Consider 99-loop modifications to study specific aspects of regulation

• Inhibitor design: Target the 99-loop/active site interface for selective inhibition

What role does KLK2 play in prostate cancer development and progression?

KLK2 has emerged as an important factor in prostate cancer (PCa) pathophysiology, with particular significance in castration-resistant prostate cancer (CRPC) . Multiple lines of evidence illuminate its role:

Histologic analyses have demonstrated that increased KLK2 expression correlates with higher cell proliferation rates and lower cell apoptosis indices in CRPC specimens . This suggests a direct role in promoting cancer cell survival and growth.

Functional studies provide mechanistic insights:

• Adding functional KLK2 cDNA to high-passage LNCaP cells leads to increased cell growth

• Knockdown of KLK2 expression with KLK2-siRNA results in increased cell apoptosis with cell growth arrest at the G1 phase

• Both in vitro colony formation assays and in vivo xenografted PCa tissues confirm that targeting KLK2 suppresses PCa growth in the castration-resistant stage

Molecular mechanism investigations reveal that KLK2 may cooperate with the androgen receptor (AR) coregulator ARA70 to enhance AR transactivation, potentially altering PCa formation . This provides a mechanistic link between KLK2 and the androgen signaling pathway critical in prostate cancer.

Additionally, KLK2 is highly expressed in prostate tumor cells and may serve as a prognostic marker for prostate cancer risk . Unlike PSA, which lacks sensitivity for identifying tumor grade and stage in newly detected cancers, serum KLK2 levels correlate with tumor stage, degree of differentiation, and total tumor volume, enhancing its prognostic value .

How can genetic variations in KLK2 be used as prognostic markers in prostate cancer research?

Genetic variations in KLK2, particularly single nucleotide polymorphisms (SNPs), have shown potential as prognostic markers in prostate cancer research . The KLK2 c.748C>T polymorphism has been specifically studied in this context.

In a study population of 182 prostate cancer patients, the KLK2 c.748C>T genotype distribution was 48% CC, 44% CT, and 8% TT, which was in Hardy-Weinberg equilibrium . This distribution was very similar to previously described prostate cancer cohorts, suggesting consistency across populations .

When analyzing the correlation between genotype and Gleason Score (GS), researchers found a notable pattern:

• Among CC genotype patients: 14 (17%) had high GS (8-10), 61 (74%) had moderate GS (5-7), and 7 (9%) had low GS (2-4)

• Among CT/TT genotype patients: 22 (25%) had high GS (8-10), 64 (74%) had moderate GS (5-7), and only 1 (1%) had low GS (2-4)

This distribution suggests potential associations between genotype and disease aggressiveness, though the search results don't provide comprehensive statistical analysis of these associations.

For researchers studying KLK2 polymorphisms as prognostic markers, considerations should include:

• Genotyping methodologies: Ensuring accurate and consistent genotype determination

• Population stratification: Accounting for ethnic and demographic factors that may influence genotype distribution

• Clinical correlation: Integrating genotype data with other clinical parameters for comprehensive prognostic modeling

• Longitudinal follow-up: Assessing the relationship between genotype and long-term outcomes

What experimental approaches are most effective for targeting KLK2 in prostate cancer therapy research?

Based on the search results, several experimental approaches have proven effective for targeting KLK2 in prostate cancer therapy research, particularly for castration-resistant prostate cancer (CRPC) :

• RNA interference technology:

  • KLK2-siRNA has shown promise in suppressing PCa growth

  • In LNCaP cells, KLK2 knockdown resulted in increased cell apoptosis with cell growth arrest at the G1 phase

  • This approach provides specificity and can be developed as an alternative therapeutic strategy

• Small molecule inhibitors:

  • Crystal structures of KLK2-small molecule inhibitor complexes at 1.9 Å resolution provide templates for structure-based drug design

  • The 99-loop/active site interface represents a promising target for selective inhibition

  • Zinc-based inhibition mechanisms can be leveraged in designing novel compounds

• In vitro and in vivo model systems:

  • High passage LNCaP cells with KLK2 manipulation (overexpression or knockdown) provide an established in vitro model

  • Colony formation assays effectively demonstrate growth inhibition effects

  • Xenografted PCa tissues in mouse models allow for in vivo validation of therapeutic approaches

• Targeting KLK2-AR signaling axis:

  • KLK2's cooperation with the AR coregulator ARA70 suggests targeting this interaction could disrupt AR transactivation

  • Combined approaches targeting both KLK2 and AR pathways may provide synergistic effects

Methodological considerations for researchers include:

• Selection of appropriate cell lines representative of different PCa stages

• Validation of target engagement using enzymatic and cellular assays

• Assessment of off-target effects, particularly on related kallikreins

• Evaluation of combination strategies with established therapeutic agents

What are the optimal methods for studying KLK2 enzymatic activity?

Studying KLK2 enzymatic activity requires careful consideration of its unique regulatory mechanisms and biochemical properties. Based on the search results, the following methodological approaches are recommended:

• Activity assays with consideration of zinc inhibition:

  • KLK2 is reversibly inhibited by micromolar Zn²⁺ concentrations

  • Buffer composition should be carefully controlled for zinc content

  • Including zinc chelators or defined zinc concentrations ensures reproducible activity measurements

  • Comparing activity in the presence and absence of zinc can provide insights into regulatory mechanisms

• Accounting for autocatalytic inactivation:

  • The 99-loop contains an autolysis site between residues 95e and 95f that leads to irreversible inactivation

  • Time-course measurements are essential to distinguish initial rates from apparent rates affected by autolysis

  • Mutant versions with eliminated autolysis sites can be used as controls for activity stability

• Substrate selection:

  • KLK2 is a tryptic protease that selectively cleaves at arginine residues

  • Synthetic substrates with appropriate leaving groups (chromogenic or fluorogenic) enable quantitative measurements

  • Natural substrates, including pro-PSA, provide physiologically relevant activity assessment

• Structural and conformational analysis:

  • The 99-loop exists in open and closed conformations that affect substrate access

  • Techniques such as hydrogen-deuterium exchange mass spectrometry can monitor conformational dynamics

  • X-ray crystallography with substrate analogs or inhibitors reveals binding mode details

• Experimental controls:

  • Include related kallikreins (particularly KLK1 and KLK3) for specificity comparisons

  • Use site-directed mutants targeting catalytic residues as negative controls

  • Employ known inhibitors to validate assay sensitivity

How should researchers address the challenges of KLK2 stability and autolytic inactivation in experimental settings?

KLK2's susceptibility to autolytic inactivation presents significant challenges for experimental protocols requiring extended incubation periods. Based on the search results, the following strategies can address these challenges:

• Mutation-based approaches:

  • Eliminating the autolysis site between residues 95e and 95f in the 99-loop prevents the mature enzyme from limited autolysis and irreversible inactivation

  • Structure-function analyses can guide the design of stabilized KLK2 variants that retain catalytic activity

• Optimal buffer conditions:

  • Inclusion of 10% glycerol in storage buffers enhances stability

  • Phosphate-buffered saline at pH 7.4 provides an optimal environment for maintaining structural integrity

  • For long-term storage, addition of carrier proteins (0.1% HSA or BSA) is recommended

• Temperature control strategies:

  • Storage at 4°C is suitable if the entire preparation will be used within 2-4 weeks

  • For longer periods, storage at -20°C or ideally -70°C maintains stability for up to one year

  • Avoiding repeated freeze-thaw cycles by preparing single-use aliquots is crucial

• Kinetic monitoring approaches:

  • Implementing continuous assays to monitor activity in real-time allows detection of inactivation kinetics

  • Short incubation periods minimize the impact of autolysis on experimental outcomes

  • Pre-incubation under different conditions can reveal factors affecting stability

• Inhibitor utilization:

  • Reversible inhibitors can protect the active site during storage

  • Zinc at controlled concentrations can be used to stabilize the enzyme through reversible inhibition

  • Removing inhibitors immediately before activity assays ensures accurate measurements

What cellular models are most appropriate for studying KLK2 function in prostate cancer research?

Based on the search results, several cellular models have proven valuable for studying KLK2 function in prostate cancer research, each with specific advantages for different experimental questions:

• LNCaP cell line:

  • High passage LNCaP cells have been successfully used for both overexpression and knockdown studies of KLK2

  • Addition of functional KLK2 cDNA led to increased cell growth, while knockdown with KLK2-siRNA resulted in apoptosis and G1 arrest

  • These cells allow for studying the interaction between KLK2 and androgen receptor signaling, as KLK2 cooperates with the AR coregulator ARA70

• Xenograft models:

  • In vivo xenografted PCa tissues have demonstrated that targeting KLK2 suppresses prostate cancer growth in the castration-resistant stage

  • These models provide physiologically relevant environments for validating in vitro findings

• Selection criteria for appropriate models:

  • Expression levels: Choose models with detectable baseline KLK2 expression or the capacity for regulated expression

  • Androgen sensitivity: Consider both androgen-sensitive and castration-resistant models to study differential roles

  • Genetic background: Models with different AR signaling pathway components help elucidate KLK2's interaction with AR regulation

• Experimental approaches with cellular models:

  • Colony formation assays: Effective for assessing long-term growth effects of KLK2 modulation

  • Cell cycle analysis: Useful for determining the mechanism of growth inhibition (e.g., G1 arrest)

  • Apoptosis assays: Important for quantifying programmed cell death following KLK2 knockdown

  • Co-immunoprecipitation: Valuable for studying protein-protein interactions, such as KLK2-ARA70 association

• Considerations for translational relevance:

  • Patient-derived xenografts or organoids may provide higher clinical relevance than established cell lines

  • Models representing different stages of disease progression help understand KLK2's role throughout cancer development

  • Correlation with human tissue samples enhances the translational value of findings

What emerging approaches could enhance our understanding of KLK2's role in prostate cancer?

Based on the search results and current research trends, several emerging approaches show promise for advancing our understanding of KLK2's role in prostate cancer:

• Structural biology and dynamics:

  • Further exploration of the 99-loop's conformational dynamics using advanced techniques like cryo-electron microscopy and molecular dynamics simulations could reveal additional regulatory mechanisms

  • Structure-guided development of selective inhibitors targeting the 99-loop/active site interface represents a promising therapeutic approach

• Genetic profiling and personalized medicine:

  • Expanded studies of KLK2 polymorphisms, including the c.748C>T variant, could identify patient subgroups most likely to benefit from KLK2-targeted therapies

  • Integration of KLK2 genetic data with broader genomic profiles may reveal novel association patterns with disease aggressiveness and treatment response

• KLK2-AR signaling axis:

  • Deeper mechanistic investigation of how KLK2 cooperates with ARA70 to enhance AR transactivation could identify novel intervention points

  • Exploring the potential of combined therapies targeting both KLK2 and AR pathways for synergistic effects, particularly in castration-resistant disease

• Advanced therapeutic modalities:

  • Beyond traditional small molecule inhibitors, exploring aptamer-based inhibitors, antibody-drug conjugates, or proteolysis-targeting chimeras (PROTACs) targeting KLK2 could provide alternative therapeutic strategies

  • RNA interference approaches, including optimized KLK2-siRNA delivery systems, represent promising alternatives to conventional therapies

• Biomarker development:

  • Further validation of KLK2 as a prognostic biomarker, particularly in comparison and combination with PSA, could enhance clinical decision-making

  • Development of sensitive assays for detecting KLK2 in liquid biopsies might enable non-invasive monitoring of disease progression

How might advances in KLK2 research impact clinical approaches to prostate cancer management?

The search results suggest several potential clinical impacts of advances in KLK2 research:

• Enhanced prognostic stratification:

  • Unlike PSA, which lacks sensitivity for identifying tumor grade and stage in newly detected cancers, serum KLK2 levels correlate with tumor stage, degree of differentiation, and total tumor volume

  • This enhanced prognostic value could improve patient stratification for treatment decisions, particularly in identifying aggressive disease requiring more intensive intervention

• Novel therapeutic targets for CRPC:

  • KLK2 has been identified as a potential therapeutic target in castration-resistant prostate cancer (CRPC), addressing an area of significant clinical need

  • Targeting KLK2 led to suppressed growth of PCa in the castration-resistant stage in both in vitro and in vivo models

  • KLK2-siRNA approaches might be developed as alternative therapeutic strategies for CRPC patients with limited treatment options

• Precision medicine approaches:

  • KLK2 polymorphism analysis could enable more personalized treatment selection based on genetic profiles

  • The differential distribution of high Gleason scores among different KLK2 genotypes suggests potential for genotype-guided risk assessment

• Combination therapy strategies:

  • Understanding KLK2's role in enhancing AR transactivation through ARA70 cooperation suggests potential synergistic effects of combined therapies targeting both pathways

  • This could lead to more effective treatment regimens with reduced resistance development

• Monitoring and disease management:

  • Improved KLK2 assays could complement PSA testing for more accurate monitoring of disease progression and treatment response

  • The high specificity of KLK2 for prostatic tissue makes it a valuable marker for detecting recurrence or residual disease

These advances collectively suggest that KLK2-focused research has significant potential to improve clinical management of prostate cancer patients, particularly those with aggressive or treatment-resistant disease.

What are the key considerations for researchers working with KLK2 Human, sf9?

Researchers working with KLK2 Human, sf9 should consider several critical factors to ensure experimental success and valid interpretation of results:

• Structural and functional characteristics:

  • KLK2 is a tryptic serine protease with a single, glycosylated polypeptide chain of 246 amino acids (25-261 aa) and a molecular mass of 27.2 kDa

  • The 99-loop (kallikrein loop) serves as a master regulator of activity and is responsible for two key biochemical properties: zinc inhibition and autocatalytic inactivation

• Handling and stability:

  • Optimal storage conditions include 4°C for short-term (2-4 weeks) or -20°C to -70°C for long-term storage

  • Addition of carrier proteins (0.1% HSA or BSA) enhances long-term stability

  • Avoiding repeated freeze-thaw cycles is crucial for maintaining activity

• Experimental design considerations:

  • Buffer composition, particularly zinc content, significantly affects KLK2 activity

  • Time-dependent activity changes due to autolytic inactivation must be accounted for in kinetic studies

  • Appropriate cellular models, particularly LNCaP cells, provide valuable platforms for functional studies

• Relevance to prostate cancer research:

  • KLK2 shows promise as both a prognostic marker and therapeutic target in prostate cancer, especially CRPC

  • Its interaction with the androgen receptor pathway through ARA70 provides a mechanistic link to cancer progression

  • Genetic variations, including the c.748C>T polymorphism, may have prognostic value

• Methodological approaches:

  • RNA interference technologies offer effective means to study KLK2 function

  • Structure-based drug design targeting the 99-loop/active site interface represents a promising approach for inhibitor development

  • Both in vitro cellular assays and in vivo xenograft models provide complementary insights into KLK2 function