KLK5 Human, Sf9

What is KLK5 and what are its key characteristics when expressed in Sf9 cells?

KLK5 (Kallikrein-5) is a member of the serine protease family of proteolytic enzymes, also known as Kallikrein-like protein 2 (KLK-L2), Stratum corneum tryptic enzyme (SCTE), and KLKL2. When expressed in Sf9 baculovirus insect cells, recombinant human KLK5:

• Forms a single, glycosylated polypeptide chain containing 236 amino acids (67-293 a.a.)

• Has a molecular mass of 26.2kDa but migrates at 28-40kDa on SDS-PAGE under reducing conditions

• Is typically expressed with a 6 amino acid His tag at the C-terminus

• Demonstrates trypsin-like activity with strong preference for Arg over Lys in the P1 position

The protein is purified using proprietary chromatographic techniques and typically supplied as a sterile filtered colorless solution containing phosphate-buffered saline (pH 7.4) and 10% glycerol with purity greater than 95% as determined by SDS-PAGE .

What are the optimal storage conditions for KLK5 Human, Sf9 to maintain protein stability?

For optimal storage of KLK5 Human, Sf9 to maintain protein stability:

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

• Store frozen at -20°C for longer periods of time

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

• Avoid multiple freeze-thaw cycles which can degrade the protein

These conditions are critical for maintaining the structural integrity and enzymatic activity of KLK5. The addition of carrier proteins helps prevent non-specific binding to storage vials and loss of protein through surface adsorption.

What is the substrate specificity of KLK5, and how does it inform experimental design for enzymatic assays?

KLK5 demonstrates distinct substrate specificity that should inform experimental design for enzymatic assays:

• It exhibits trypsin-like activity with strong preference for Arg over Lys in the P1 position

• Shows high activity with fluorogenic substrates like Gly-Pro-Arg-AMC and Gly-Pro-Lys-AMC, with higher k(cat)/K(m) ratio for the Arg-containing substrate

• Effectively digests extracellular matrix components including collagens type I, II, III, and IV, fibronectin, and laminin

When designing KLK5 activity assays, researchers should consider using substrates with arginine at the P1 position for optimal detection. For competitive inhibition studies, the substrate Abz-KLRSSKQ-Eddnp has been successfully used with 5-minute pre-incubation of potential inhibitors before substrate addition . Lineweaver-Burk plot analysis can be employed to determine the mechanism of inhibition.

What are the key protease inhibitors that regulate KLK5 activity and how effective are they?

Several protease inhibitors have been characterized for their ability to regulate KLK5 activity:

These findings are crucial for designing experiments involving KLK5 inhibition and for understanding the regulation of KLK5 in physiological contexts . Engineered inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) with specific substitutions at positions P1, P2, P4, and P2′ have demonstrated improved activity against KLK5 with Ki values as low as 4.2±0.2 nM .

How can researchers effectively differentiate between the enzymatic activity of KLK5 and other closely related kallikreins in experimental settings?

Differentiating the enzymatic activity of KLK5 from other closely related kallikreins requires careful experimental design:

• Substrate selection: Use substrates with sequence preferences unique to KLK5. Sequences most favored by KLK5 include GRSR, YRSR, and GRNR, which can help distinguish it from other kallikreins .

• Specific inhibitors: Employ engineered inhibitors with selectivity for KLK5. For example, modified sunflower trypsin inhibitor-1 (SFTI-1) with substitutions at P1, P2, P4, and P2′ positions has shown 12-fold selectivity for KLK5 over the closely related KLK14 .

• Kinetic analysis: Compare kinetic constants (kcat and KM) across different kallikreins using identical substrates. KLK5 shows distinct kinetic profiles compared to other KLKs.

• pH and salt conditions: Optimize reaction conditions based on KLK5's specific pH optimum and salt sensitivity, which may differ from other kallikreins.

• Antibody-based detection: Use highly specific antibodies that recognize KLK5 but not other kallikreins in immunoassays to confirm the presence of KLK5 in complex samples .

What are the implications of KLK5 glycosylation patterns when expressed in Sf9 cells versus other expression systems?

The glycosylation patterns of KLK5 have significant implications for research:

• When expressed in Sf9 insect cells, KLK5 is glycosylated but with simpler, high-mannose glycans rather than complex mammalian-type glycans

• This leads to a molecular mass discrepancy: the theoretical mass is 26.2kDa, but the protein migrates at 28-40kDa on SDS-PAGE under reducing conditions

• Glycosylation can affect protein folding, stability, solubility, and potentially enzymatic activity

• In human samples, KLK5 has been found to be glycosylated in ovarian cancer fluids, suggesting physiological relevance of this post-translational modification

Researchers should consider these glycosylation differences when:

• Comparing results between recombinant KLK5 from different expression systems

• Extrapolating in vitro findings to in vivo contexts

• Developing antibodies against KLK5

• Studying protein-protein interactions that might be affected by glycosylation patterns

What methodological approaches are recommended for studying KLK5's role in tumor progression and angiogenesis?

Based on KLK5's established capabilities, the following methodological approaches are recommended:

• Extracellular matrix degradation assays:

  • Use fluorescently labeled collagen, fibronectin, or laminin substrates

  • Measure degradation products by SDS-PAGE or fluorescence-based assays

  • Compare degradation patterns with and without KLK5 inhibitors

• Angiogenesis models:

  • Study KLK5's ability to release angiostatin 4.5 from plasminogen

  • Use in vitro tube formation assays with endothelial cells

  • Employ chorioallantoic membrane (CAM) assays for ex vivo analysis

  • Consider zebrafish or mouse models for in vivo angiogenesis studies

• Invasion and migration assays:

  • Employ transwell invasion chambers coated with ECM components

  • Perform wound healing assays in the presence/absence of KLK5

  • Use 3D spheroid invasion models with cancer cell lines

• Molecular interaction studies:

  • Examine KLK5's effects on plasminogen activator inhibitor 1 binding to vitronectin

  • Study the release of bioactive peptides from fibrinogen and kininogen

  • Use pull-down assays or surface plasmon resonance to identify novel binding partners

These approaches will help elucidate KLK5's functional roles in cancer progression mechanisms and potentially identify new therapeutic targets.

How can researchers effectively measure and interpret changes in KLK5 enzymatic activity in different physiological and pathological conditions?

To effectively measure and interpret changes in KLK5 enzymatic activity across different conditions:

• Develop standardized activity assays:

  • Use specific fluorogenic substrates like Gly-Pro-Arg-AMC

  • Establish baseline kinetic parameters (Km, Vmax, kcat) under controlled conditions

  • Create standard curves with purified recombinant KLK5 Human, Sf9

• Account for endogenous inhibitors:

  • Measure levels of α2-antiplasmin and antithrombin in biological samples

  • Consider pre-treatment steps to remove or inactivate inhibitors when appropriate

  • Use immunodepletion to remove specific inhibitors selectively

• pH and ion considerations:

  • Maintain consistent pH across experiments (KLK5 has trypsin-like activity which is pH-dependent)

  • Control for divalent ions like Zn2+ which can inhibit KLK5 activity

  • Use chelating agents when appropriate to eliminate ion effects

• Data normalization approaches:

  • Normalize activity to total KLK5 protein levels determined by ELISA or Western blot

  • Calculate specific activity (activity units per mg protein)

  • Consider activity ratios of KLK5 to related proteases for comparative analysis

• Statistical analysis:

  • Use appropriate statistical tests considering the distribution of enzymatic activity data

  • Perform power analysis to determine adequate sample sizes

  • Consider multivariate analysis when examining activity across diverse conditions

What are the critical factors to consider when designing inhibitor specificity studies for KLK5 versus other kallikreins?

When designing inhibitor specificity studies for KLK5 versus other kallikreins, researchers should consider:

• Panel selection for specificity testing:

  • Include closely related KLKs (particularly KLK7 and KLK14) that share substrate preferences

  • Test against more distantly related serine proteases as controls

  • Include physiologically relevant proteases that might co-exist with KLK5

• Structural considerations for inhibitor design:

  • Target the substrate binding loop with sequence substitutions at P1, P2, and P4 positions

  • Fine-tune selectivity with modifications at the P2′ position

  • Consider scaffold proteins such as sunflower trypsin inhibitor-1 (SFTI-1) that have been successfully used for KLK5 inhibition

• Comprehensive inhibition parameters:

  • Determine both Ki values and inhibition mechanisms (competitive, non-competitive, etc.)

  • Use Lineweaver-Burk plots to analyze inhibition patterns

  • Calculate selectivity indices (ratio of Ki values) between KLK5 and other proteases

• Physiological relevance validation:

  • Test inhibitors in complex biological fluids where multiple proteases are present

  • Validate inhibition using cell-based assays that model relevant disease states

  • Consider the effects of pH, salt conditions, and protein concentration on inhibitor efficacy

These considerations will help researchers develop inhibitors with improved selectivity for KLK5, which is particularly important for therapeutic applications in skin diseases like Netherton syndrome and various cancers.

What are common challenges in obtaining enzymatically active KLK5 from Sf9 expression systems and how can they be addressed?

Common challenges and solutions for obtaining enzymatically active KLK5 from Sf9 expression systems include:

• Zymogen activation issues:

  • Challenge: KLK5 is produced as an inactive zymogen requiring proper processing for activation

  • Solution: Ensure correct signal peptide cleavage and pro-peptide removal strategies in expression construct design

  • Approach: Consider co-expression with appropriate processing proteases or post-purification activation steps

• Protein aggregation and inclusion body formation:

  • Challenge: Overexpression can lead to insoluble protein aggregates

  • Solution: Optimize expression conditions (temperature, induction timing, cell density)

  • Approach: Use lower induction temperatures (19-27°C) and shorter induction periods

• Incomplete glycosylation:

  • Challenge: Insect cells produce simpler glycans than mammalian cells

  • Solution: Verify that glycosylation is sufficient for proper folding and stability

  • Approach: Consider using SweetBac or similar engineered Sf9 cells for humanized glycosylation if required

• Autolysis during purification:

  • Challenge: Active KLK5 can self-degrade during purification

  • Solution: Include appropriate protease inhibitors and optimize purification conditions

  • Approach: Perform purification at lower temperatures (4°C) and consider adding reversible inhibitors

• Proper disulfide bond formation:

  • Challenge: Incorrect disulfide bonding can lead to inactive protein

  • Solution: Ensure oxidizing environment during protein folding

  • Approach: Monitor disulfide bond formation using non-reducing SDS-PAGE

Researchers can verify successful expression of enzymatically active KLK5 by performing activity assays with fluorogenic substrates like Gly-Pro-Arg-AMC, and comparing kinetic parameters with published values.

How should researchers interpret and reconcile contradictory data regarding KLK5 function across different experimental models?

When faced with contradictory data regarding KLK5 function across different experimental models, researchers should:

• Examine expression system differences:

  • Compare glycosylation patterns between Sf9-expressed and mammalian-expressed KLK5

  • Assess whether His-tags or other fusion partners might affect activity in different systems

  • Consider that KLK5 from Sf9 cells migrates at 28-40kDa on SDS-PAGE despite having a theoretical mass of 26.2kDa

• Evaluate assay condition variations:

  • Analyze pH, temperature, and buffer composition differences between contradictory studies

  • Consider the presence of divalent cations like Zn2+, which can inhibit KLK5 activity

  • Examine substrate concentration ranges and whether they span appropriate Km values

• Consider biological context complexities:

  • Assess whether contradictions arise from differences in KLK5 regulation by tissue-specific factors

  • Evaluate the presence of endogenous inhibitors like α2-antiplasmin or antithrombin in different models

  • Examine potential interactions with other proteases in complex biological systems

• Apply statistical meta-analysis approaches:

  • Perform quantitative comparisons across studies using effect sizes rather than p-values

  • Weight evidence based on methodological rigor and sample sizes

  • Identify patterns in subgroups of studies with similar methodologies

• Design reconciliation experiments:

  • Develop experiments specifically addressing contradictions using multiple approaches within the same study

  • Consider side-by-side testing of different experimental models under identical conditions

  • Use genetic approaches (knockout/knockdown) alongside biochemical methods to confirm function

What are the most effective methods for analyzing KLK5's role in complex proteolytic cascades and how should the data be interpreted?

Analyzing KLK5's role in complex proteolytic cascades requires sophisticated methodologies and careful data interpretation:

• Multi-enzyme cascade reconstitution:

  • Use purified components to rebuild proteolytic cascades in vitro

  • Systematically add or remove KLK5 to observe cascade progression changes

  • Employ selective inhibitors to block specific steps in the cascade

  • Interpret data by constructing kinetic models that account for reaction rates at each step

• Degradomics approaches:

  • Use mass spectrometry-based techniques to identify KLK5 substrates in complex mixtures

  • Apply Terminal Amine Isotopic Labeling of Substrates (TAILS) or similar technologies

  • Analyze cleavage site specificity using positional scanning libraries

  • Interpret data by looking for enriched sequence motifs and comparing to known KLK5 preferences for Arg in P1 position

• Systems biology modeling:

  • Develop computational models of proteolytic networks including KLK5

  • Incorporate known inhibitors like α2-antiplasmin and antithrombin

  • Simulate perturbations to predict system-wide effects

  • Validate model predictions experimentally to refine parameters

• Temporal analysis of cascade activation:

  • Use time-course experiments to determine the sequence of proteolytic events

  • Apply specific antibodies against cleaved substrates to track progression

  • Consider using FRET-based reporters to monitor activity in real-time

  • Interpret data by constructing timeline maps of cascade activation

• In vivo validation using genetic models:

  • Correlate in vitro cascade findings with in vivo phenotypes in KLK5 knockout or overexpression models

  • Use conditional genetic systems to manipulate KLK5 expression with temporal control

  • Apply tissue-specific promoters to examine cascade functions in relevant physiological contexts

  • Interpret data by comparing phenotypic outcomes to biochemical predictions