KLK13 Human, sf9

What is KLK13 and what is its biological significance?

KLK13 (Kallikrein-related peptidase 13) is a member of the kallikrein family of serine proteases, encoded by the KLK13 gene located on chromosome 19q13.3-13.4. It belongs to a 15-member family of homologous secreted serine proteases (KLK1-15) that constitute the largest contiguous cluster of protease genes in the human genome . KLK13 was previously known as KLK-L4 or Kallikrein-Like Protein 4 .

Biologically, KLK13 functions as a trypsin-like serine peptidase, though its precise physiological roles are still being elucidated. Expression analyses have shown that KLK13 is present in various human tissues including breast, testis, prostate, and salivary glands . Its expression is regulated by steroid hormones, suggesting hormone-dependent functions .

Why is KLK13 produced in Sf9 cells for research purposes?

Sf9 cells (derived from Spodoptera frugiperda pupal ovarian tissue) are used for KLK13 production because they offer several advantages for recombinant protein expression:

• Post-translational modifications: Sf9 cells can perform many eukaryotic post-translational modifications, including glycosylation. This is crucial for KLK13, which is naturally a glycosylated polypeptide .

• Correct protein folding: The baculovirus expression system in Sf9 cells facilitates proper protein folding of complex mammalian proteins like KLK13, which is essential for maintaining enzymatic activity .

• High protein yield: The baculovirus-Sf9 system allows for high-level expression of recombinant proteins, enabling production of sufficient quantities for research applications .

• Functional integrity: KLK13 produced in Sf9 cells maintains its functional characteristics, including catalytic activity, making it suitable for enzymatic assays and other functional studies .

• Scalability: The system can be scaled up relatively easily when larger quantities of protein are required for extensive experimental work .

This expression system delivers a KLK13 product that closely resembles the native human protein in structure and function, making it valuable for investigating KLK13's biochemical properties and potential roles in pathophysiological processes.

How can KLK13 Human, sf9 be used in cancer research?

KLK13 has significant applications in cancer research, particularly in the following areas:

A multivariate analysis study demonstrated that KLK13 expression is a significant predictor of DFS and OS with hazard ratios of 0.41 and 0.46 (p<0.001 and p=0.009, respectively), suggesting approximately 55-60% reduction in either risk of relapse or death in patients with KLK13-positive tumors .

What are the recommended storage and handling conditions for KLK13 Human, sf9?

Proper storage and handling of KLK13 Human, sf9 is critical for maintaining its structural integrity and enzymatic activity. Based on manufacturer recommendations, researchers should follow these guidelines:

• Short-term storage: Store at 4°C if the entire vial will be used within 2-4 weeks .

• Long-term storage: For periods exceeding 4 weeks, store frozen at -20°C .

• Protein stabilization: For long-term storage, it is recommended to add a carrier protein (0.1% human serum albumin or bovine serum albumin) to prevent protein degradation and maintain activity .

• Avoid freeze-thaw cycles: Multiple freeze-thaw cycles can significantly reduce protein activity and should be avoided. Aliquoting the protein solution before freezing is recommended to minimize the number of freeze-thaw cycles .

• Working concentration: The protein is typically supplied at a concentration of 0.5 mg/mL in phosphate-buffered saline (pH 7.4) containing 10% glycerol .

• Temperature sensitivity: As an enzyme, KLK13 is temperature-sensitive. Keep the protein on ice during experiments and avoid prolonged exposure to room temperature.

• pH considerations: KLK13 activity is pH-dependent. Maintain appropriate pH conditions (typically physiological pH) during experimental procedures to ensure optimal activity.

• Sterility: The product is supplied as a sterile filtered colorless solution . Maintain sterile conditions when handling to prevent microbial contamination.

Adherence to these storage and handling recommendations will help ensure consistent experimental results when working with KLK13 Human, sf9.

What assays are commonly used to measure KLK13 activity and specificity?

Several assay formats have been developed to measure KLK13 enzymatic activity and substrate specificity:

• Fluorogenic peptide substrate assays: These are among the most commonly used methods for measuring KLK13 activity. Researchers have designed internally quenched fluorogenic peptide substrates based on KLK13's preferred cleavage sites. Upon cleavage by KLK13, these substrates release a fluorescent signal that can be measured to quantify enzymatic activity .

• Combinatorial substrate libraries: These have been utilized to determine the specificity of substrate binding subsites of KLK13 in both prime and non-prime regions. For example, studies have identified that KLK13 has a preference for substrates with the sequence motif ABZ-Val-Arg-Phe-Arg-ANB-NH2 .

• Activity-based probe assays: Selective activity-based probes targeting KLK13 have been developed by incorporating a chloromethylketone warhead and biotin bait into optimized substrate sequences. These probes can be used to detect active KLK13 in diverse biological samples, including cell lysates and saliva .

• Cleavage of recombinant protein substrates: KLK13's ability to cleave specific proteins can be assessed by incubating the enzyme with recombinant substrates and analyzing the cleavage products by SDS-PAGE and Western blotting. This approach has been used to demonstrate KLK13's ability to cleave the S1/S2 site of the HCoV-HKU1 spike protein .

• N-terminal Edman degradation: This technique can be used to sequence the products of KLK13-mediated cleavage, allowing precise identification of cleavage sites. For example, this method confirmed that KLK13 cleaves the S1/S2 region of the HCoV-HKU1 spike protein at the R↓SISA site .

These assays not only help characterize KLK13's enzymatic properties but also provide insights into its potential physiological and pathological functions. When designing experiments to measure KLK13 activity, researchers should include appropriate controls, such as heat-inactivated enzyme or specific inhibitors, to confirm the specificity of the observed proteolytic activity.

How does KLK13 contribute to cancer progression or suppression mechanisms?

The role of KLK13 in cancer appears to be complex and context-dependent, with evidence suggesting both tumor-suppressive and potentially tumor-promoting functions:

• Favorable prognostic marker: Multiple studies have established KLK13 as a favorable prognostic marker in several cancer types. In breast cancer, higher KLK13 expression correlates with a 55-60% reduction in the risk of relapse or death . Similar positive prognostic associations have been observed in non-small cell lung cancer and gastric cancer .

• Hormone regulation: KLK13 expression is regulated by steroid hormones, particularly in hormone-dependent cancers like breast cancer . This hormone responsiveness suggests potential involvement in hormone-regulated cancer pathways. Patients with KLK13-positive tumors are more likely to have estrogen receptor-positive status, indicating complex interactions with hormone signaling networks .

• Proteolytic cascades: As a serine protease, KLK13 likely participates in proteolytic cascades that can influence tumor microenvironment. These may include:

  • Activation of other proteases involved in extracellular matrix remodeling

  • Processing of growth factors or cytokines that regulate cancer cell behavior

  • Modification of cell surface receptors that mediate cancer cell-microenvironment interactions

• Age-related expression patterns: Interestingly, higher KLK13 positivity has been found in older breast cancer patients , suggesting age-related mechanisms that may influence its role in cancer progression.

• Potential therapeutic implications: The strong association between KLK13 expression and improved survival suggests that KLK13 could potentially identify patients likely to benefit from specific treatments, particularly hormonal therapies in breast cancer .

The seemingly paradoxical role of a protease serving as a favorable prognostic marker warrants further investigation into the specific molecular mechanisms through which KLK13 influences cancer cell behavior and patient outcomes.

What is the role of KLK13 in viral infections, particularly in coronavirus pathogenesis?

Recent research has uncovered a potentially significant role for KLK13 in viral infections, particularly in the context of coronavirus pathogenesis:

• Priming protease for viral entry: KLK13 has been identified as a priming protease for the human coronavirus HKU1 (HCoV-HKU1). Studies have demonstrated that KLK13 facilitates viral entry by cleaving the viral spike (S) protein .

• S protein cleavage specificity: KLK13 specifically targets the S1/S2 cleavage site of the HCoV-HKU1 spike protein. Experimental evidence shows that when the S1/S2 region was exposed to 500 nM KLK13, the protein was degraded, while the S2′ site remained intact. N-terminal Edman degradation confirmed that KLK13 cleaves at the R↓SISA sequence, which corresponds to the S1/S2 site .

• Functional validation through genetic approaches: The critical role of KLK13 in HCoV-HKU1 infection was validated through both loss-of-function and gain-of-function experiments:

  • Knockout of KLK13 in human airway epithelial cells blocked their infection by HCoV-HKU1

  • Overexpression of KLK13 in non-permissive cells enabled their infection by the virus

• Potential broad implications for respiratory viruses: Given that KLK13 is expressed in airway tissues, its ability to cleave viral proteins may have implications for other respiratory viruses that require proteolytic activation for entry.

• Research tool development: The identification of KLK13 as a priming protease for HCoV-HKU1 may help establish cell lines that facilitate further investigation of viral pathogenesis mechanisms .

These findings highlight KLK13 as a potential target for antiviral strategies, particularly against coronaviruses. Inhibitors of KLK13 might represent a novel approach to preventing viral entry and replication. Additionally, variation in KLK13 expression or activity among individuals could potentially contribute to differences in susceptibility to certain viral infections.

How can activity-based probes be designed to monitor KLK13 activity in biological samples?

Designing effective activity-based probes (ABPs) for monitoring KLK13 activity in biological samples requires a systematic approach based on understanding the enzyme's substrate specificity and catalytic mechanism:

These ABPs represent powerful tools for studying KLK13 biology, as they specifically detect the active form of the enzyme rather than just its presence. This capability is particularly important for understanding KLK13's functional roles in physiological and pathological processes, including cancer and viral infections. The development of such selective activity-based probes for KLK13 represents a significant advance in the field, as it provides the first tools to analyze the presence of the active enzyme in biological samples .

What challenges are encountered in purifying active KLK13 and how can they be addressed?

Purifying active KLK13 presents several technical challenges that researchers must address to obtain high-quality, functional protein:

By addressing these challenges through careful optimization of expression, purification, and storage conditions, researchers can obtain high-quality KLK13 preparations suitable for a wide range of experimental applications.

How can researchers ensure specificity when studying KLK13 in complex biological systems?

Ensuring specificity when studying KLK13 in complex biological systems requires a multifaceted approach that addresses the challenges of distinguishing KLK13 activity and function from those of other proteases, particularly other kallikrein family members:

• Selective substrate identification and validation:

  • Use combinatorial peptide libraries to identify highly selective substrates for KLK13

  • Validate substrate selectivity by testing against related proteases, particularly other kallikreins

  • The sequence ABZ-Val-Arg-Phe-Arg-ANB-NH2 has been identified as having good selectivity for KLK13

• Development and use of specific activity-based probes:

  • Incorporate selective substrate sequences into activity-based probes

  • Include appropriate controls when using these probes, such as pre-treatment with known inhibitors

  • Verify probe selectivity across a panel of related proteases

• Genetic approaches:

  • Use KLK13 knockout cell lines or tissues as negative controls

  • Employ KLK13 overexpression systems to confirm phenotypes

  • Utilize RNA interference approaches with careful validation of knockdown specificity and efficiency

• Antibody validation for immunological detection:

  • Validate antibody specificity using KLK13 knockout samples and recombinant protein

  • Test for cross-reactivity with other kallikrein family members

  • Consider using multiple antibodies targeting different epitopes for confirmation

• Functional validation strategies:

  • Rescue experiments in KLK13-deficient systems using wild-type and catalytically inactive mutants

  • Pharmacological approach using selective inhibitors when available

  • Correlation of observed effects with KLK13 expression and activity levels

• Consideration of biological context:

  • Account for the expression of other proteases in the system under study

  • Consider the presence of endogenous inhibitors that may regulate KLK13 activity

  • Evaluate potential compensatory mechanisms in knockout or knockdown systems

• Analytical controls:

  • Include positive controls using recombinant KLK13 Human, sf9

  • Employ negative controls such as heat-inactivated enzyme

  • Use parallel experiments with other kallikreins to assess specificity of observed effects

By implementing these strategies, researchers can enhance confidence in the specificity of their findings regarding KLK13 function in complex biological systems, leading to more reliable and interpretable results.

What experimental controls are essential when designing KLK13 functional studies?

Designing rigorous KLK13 functional studies requires careful consideration of appropriate experimental controls to ensure valid and interpretable results:

• Enzyme activity controls:

  • Positive control: Include purified, active KLK13 Human, sf9 at known concentrations to establish baseline activity levels

  • Negative control: Use heat-inactivated KLK13 (e.g., 95°C for 10 minutes) to confirm that observed effects are due to enzymatic activity

  • Concentration gradient: Employ multiple KLK13 concentrations to establish dose-dependent relationships and enzyme kinetics

• Specificity controls:

  • Substrate specificity: Test multiple substrates, including non-preferred sequences, to confirm enzymatic specificity

  • Inhibitor controls: Use broad-spectrum serine protease inhibitors (e.g., PMSF) and, when available, more selective KLK13 inhibitors

  • Related proteases: Include other kallikrein family members to distinguish KLK13-specific effects from general kallikrein effects

• Genetic manipulation controls:

  • Knockout validation: When using KLK13 knockout models, verify complete absence of KLK13 at both mRNA and protein levels

  • Overexpression controls: Include both wild-type KLK13 and catalytically inactive mutants (e.g., serine to alanine mutation in the active site)

  • Vector controls: Use empty vector transfections to control for transfection effects in overexpression studies

• Sample preparation controls:

  • Storage conditions: Verify enzyme activity retention after storage under recommended conditions

  • Buffer compatibility: Test activity in experimental buffers to ensure they don't interfere with enzymatic function

  • Contaminant exclusion: Include controls to detect potential contaminants that might affect KLK13 activity

• Analytical controls:

  • Standard curves: Establish standard curves using recombinant KLK13 for quantitative analyses

  • Technical replicates: Perform at least triplicate measurements to assess technical variability

  • Biological replicates: Use multiple independent biological samples to assess biological variability

• Application-specific controls:

  • Cancer studies: Include controls for hormone status when studying hormone-regulated effects of KLK13

  • Viral infection studies: Use viral strains with mutations at KLK13 cleavage sites as controls when studying viral processing

  • Activity-based probe studies: Include competition controls with excess unlabeled substrates to confirm binding specificity

• Time-dependent controls:

  • Enzyme stability: Monitor KLK13 activity over the experimental time course to account for potential activity loss

  • Kinetic measurements: Take multiple time points to establish reaction kinetics rather than single endpoint measurements

What are the emerging applications of KLK13 in precision medicine?

KLK13 shows promising potential for several applications in precision medicine, particularly in oncology and infectious disease fields:

• Prognostic stratification in multiple cancers:

  • KLK13 has demonstrated significant value as a prognostic biomarker in breast cancer, with higher expression correlating with approximately 55-60% reduction in risk of relapse or death

  • Similar prognostic value has been observed in non-small cell lung cancer and gastric cancer

  • Future research should focus on developing standardized clinical assays for KLK13 detection that could be incorporated into prognostic algorithms

• Prediction of treatment response:

  • Evidence suggests KLK13 may identify patients likely to benefit from hormonal treatment in breast cancer

  • Research is needed to determine if KLK13 status can guide therapeutic decisions across various cancer types

  • Correlation studies between KLK13 expression/activity and response to specific therapy regimens could lead to personalized treatment protocols

• Therapeutic target development:

  • Given KLK13's role in cancer progression and viral infections, developing specific inhibitors could have therapeutic potential

  • Structure-based drug design targeting KLK13's active site could yield novel therapeutic compounds

  • The availability of recombinant KLK13 Human, sf9 facilitates high-throughput screening of potential inhibitors

• Viral infection risk assessment and treatment:

  • KLK13's role as a priming protease for HCoV-HKU1 suggests potential applications in coronavirus infection management

  • Individual variations in KLK13 expression or activity might influence susceptibility to certain viral infections

  • Inhibitors of KLK13 could potentially serve as broad-spectrum antivirals against viruses requiring similar activation mechanisms

• Biomarker panel development:

  • Integration of KLK13 with other biomarkers could enhance diagnostic and prognostic accuracy

  • Multi-kallikrein panels including KLK13 might provide more comprehensive disease assessment than single markers

  • Activity-based probes for KLK13 could enable functional assessment rather than mere expression level measurement

• Non-invasive diagnostics:

  • Detection of KLK13 in saliva and other accessible biological fluids using activity-based probes opens possibilities for non-invasive testing approaches

  • Development of point-of-care tests for active KLK13 could facilitate rapid assessment in clinical settings

As research progresses, translating these potential applications into clinical practice will require rigorous validation studies, standardization of detection methods, and integration into existing clinical decision-making frameworks. The continued availability of high-quality recombinant KLK13 Human, sf9 will be essential for these research efforts.

How might KLK13 research contribute to understanding protease networks in disease?

KLK13 research offers a valuable window into the complex protease networks that underlie various diseases, potentially yielding broader insights into proteolytic cascades and their dysregulation:

• Kallikrein activation cascades:

  • Kallikreins can activate each other in enzymatic cascades similar to the coagulation and complement systems

  • KLK13 may participate in these cascades, either as an activator of other KLKs or as a substrate

  • Research using KLK13 Human, sf9 could map these interactions and identify key regulatory nodes in the kallikrein network

• Integration with other protease systems:

  • KLK13 may interact with non-kallikrein proteases, forming bridges between distinct proteolytic systems

  • Understanding these cross-system interactions could reveal how discrete protease networks coordinate in health and disease

  • The development of activity-based probes for KLK13 provides tools to investigate these networks in complex biological samples

• Substrate identification and biological consequences:

  • Comprehensive identification of KLK13 substrates would illuminate its biological roles

  • Beyond viral spike proteins , KLK13 likely cleaves endogenous proteins involved in various physiological processes

  • Connecting specific cleavage events to biological outcomes would enhance understanding of how protease networks drive disease progression

• Inhibitor networks and dysregulation:

  • Endogenous inhibitors likely regulate KLK13 activity in vivo

  • Dysregulation of these inhibitory mechanisms could contribute to disease pathogenesis

  • Investigation of inhibitor-KLK13 interactions could reveal therapeutic opportunities

• Hormone regulation of protease networks:

  • KLK13's regulation by steroid hormones points to hormone-dependent control of proteolytic networks

  • This connection may explain sex-specific differences in disease prevalence and progression

  • Understanding how hormones orchestrate protease expression and activity could inform sex-specific therapeutic approaches

• Evolutionary perspectives on protease function:

  • Comparative studies of KLK13 across species could reveal evolutionary conservation of protease networks

  • The role of KLK13 in viral infections suggests evolutionary pressures that may have shaped kallikrein function

  • Ancient host-pathogen interactions may have influenced the development of complex protease networks

• Systems biology approaches:

  • Integration of KLK13 research into systems-level analyses of protease networks

  • Computational modeling of protease cascades including KLK13 could predict network behavior under various conditions

  • These models could identify critical control points that might serve as therapeutic targets

The availability of well-characterized recombinant KLK13 Human, sf9 and specific activity-based probes will be instrumental in advancing these research directions, ultimately contributing to a more comprehensive understanding of protease networks and their roles in disease pathogenesis.

What technological advances are needed to enhance KLK13 research capabilities?

Advancing KLK13 research requires technological innovations across multiple domains, from protein production to detection and functional analysis:

• Enhanced protein production systems:

  • Development of expression systems that yield higher amounts of fully active KLK13

  • Engineered cell lines with reduced proteolytic background for cleaner KLK13 production

  • Systems capable of producing KLK13 with tissue-specific post-translational modifications to better reflect in vivo forms

• Improved activity-based probes and sensors:

  • Next-generation KLK13-specific activity-based probes with enhanced selectivity and sensitivity

  • Development of FRET-based sensors for real-time monitoring of KLK13 activity in living cells

  • Multiplex probes capable of simultaneously tracking multiple kallikreins to understand network dynamics

• Advanced structural biology approaches:

  • High-resolution crystal structures of KLK13 in complex with substrates and inhibitors

  • Cryo-EM studies of KLK13 within larger protein complexes

  • Dynamic structural analyses using hydrogen-deuterium exchange mass spectrometry to understand conformational changes during activation and catalysis

• Single-cell analysis technologies:

  • Methods for detecting KLK13 expression and activity at the single-cell level

  • Spatial proteomics approaches to map KLK13 localization within tissues with subcellular resolution

  • Integration of KLK13 data with single-cell transcriptomics to correlate activity with gene expression patterns

• In vivo imaging capabilities:

  • Development of non-invasive imaging probes for visualizing KLK13 activity in living organisms

  • PET tracers based on KLK13 inhibitors for potential diagnostic applications

  • Contrast agents activated by KLK13 for disease-specific imaging

• High-throughput functional screening platforms:

  • Microfluidic systems for rapid assessment of KLK13 activity against large substrate libraries

  • CRISPR-based functional genomic screens to identify genes that modulate KLK13 activity

  • Drug discovery platforms optimized for identifying selective KLK13 inhibitors

• Computational tools and resources:

  • Improved algorithms for predicting KLK13 substrates based on structural and sequence features

  • Systems biology frameworks for modeling KLK13 within broader protease networks

  • Databases integrating diverse KLK13 datasets from expression to activity and clinical correlations

• Standardized clinical assays:

  • Development of robust, standardized assays for measuring KLK13 expression and activity in clinical samples

  • Point-of-care testing platforms for rapid KLK13 detection

  • Reference standards and quality control materials for ensuring reproducibility across laboratories

These technological advances would significantly enhance researchers' ability to study KLK13 biology, potentially accelerating the translation of basic science findings into clinical applications. The continued refinement of recombinant KLK13 Human, sf9 production methods will provide researchers with the high-quality tools needed to leverage these emerging technologies effectively.

What are the key takeaways for researchers working with KLK13 Human, sf9?

Researchers working with KLK13 Human, sf9 should consider several key points to optimize their experimental approaches and interpretations: