KLK7 Human, sf9

What is KLK7 and how is it produced in the Sf9 expression system?

KLK7, also known as Kallikrein-Related Peptidase 7, is a serine protease that belongs to the kallikrein family. When expressed in Sf9 baculovirus cells, it is produced as a single, glycosylated polypeptide chain containing 190 amino acids (residues 1-181 of the native sequence) with a molecular mass of 20.9 kDa, although it typically migrates at 28-40 kDa on SDS-PAGE under reducing conditions due to glycosylation . The baculovirus expression system is preferred for KLK7 production because it enables proper folding and post-translational modifications, particularly glycosylation, which are crucial for the enzyme's stability and function.

For research purposes, recombinant KLK7 is often expressed with a 6-amino acid His-tag at the C-terminus to facilitate purification using affinity chromatography techniques . The typical amino acid sequence of the recombinant protein includes the native KLK7 sequence plus the histidine tag: "ADPMNEYTVH LGSDTLGDRR AQRIKASKSF RHPGYSTQTH VNDLMLVKLN SQARLSSMVK KVRLPSRCEP PGTTCTVSGW GTTTSPDVTF PSDLMCVDVK LISPQDCTKV YKDLLENSML CAGIPDSKKN ACNGDSGGPL VCRGTLQGLV SWGTFPCGQP NDPGVYTQVC KFTKWINDTM KKHRHHHHHH" .

What are the primary physiological functions of KLK7?

KLK7 serves multiple physiological functions that make it a significant target for research. In the skin, it catalyzes the degradation of intercellular cohesive structures in the cornified layer, facilitating the continuous shedding of cells from the skin surface in a process known as desquamation . This role in skin homeostasis is critical for maintaining the protective barrier function of the epidermis.

Beyond skin physiology, KLK7 demonstrates involvement in inflammatory processes by activating precursors to inflammatory cytokines . Recent research has also identified KLK7 as an astrocyte-derived amyloid-β (Aβ) degrading enzyme with potential implications in Alzheimer's disease (AD) pathogenesis . The expression of KLK7 mRNA is significantly decreased in the brains of AD patients, and experimental evidence suggests that loss of KLK7 can exacerbate amyloid pathology in AD model mice .

KLK7 shows specificity for cleaving amino acid residues with aromatic side chains in the P1 position. For example, it cleaves insulin B chain at specific sites: '6-Leu-Cys-7', '16-Tyr-Leu-17', '25-Phe-Tyr-26', and '26-Tyr-Thr-27' . This substrate specificity is a critical characteristic that determines its biological functions and potential therapeutic applications.

What are the optimal storage and handling conditions for KLK7 Human, sf9?

For optimal preservation of KLK7 activity and stability, researchers should follow specific storage and handling guidelines. If the entire vial will be used within 2-4 weeks, the protein solution can be stored at 4°C . For longer-term storage, it is recommended to store the protein frozen at -20°C. To enhance stability during storage, especially for prolonged periods, adding a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) is advisable .

Multiple freeze-thaw cycles significantly reduce enzyme activity and should be avoided. The standard formulation of commercially available KLK7 protein solution (0.5mg/ml) contains phosphate-buffered saline (pH 7.4) and 10% glycerol . The glycerol component helps prevent freeze damage and maintains protein stability. For experimental use, it's important to work with the protein on ice when thawed and to use sterile technique to prevent contamination.

When designing experiments, researchers should consider that KLK7 activity is optimal under specific buffer conditions, typically at physiological pH (around 7.4), although activity testing across a range of pH values may be necessary depending on the research question.

How can the enzymatic activity of KLK7 be accurately measured?

Accurate measurement of KLK7 enzymatic activity is crucial for various research applications. The enzyme exhibits chymotryptic-like cleavage preferences, which guides the selection of appropriate substrates for activity assays . Several methodological approaches can be employed:

• Synthetic peptide substrates: Fluorogenic or chromogenic peptide substrates containing KLK7's preferred cleavage sites (typically with aromatic residues at the P1 position) can be used. Cleavage results in the release of a detectable signal that can be measured using spectrophotometric or fluorometric methods.

• Natural protein substrates: Insulin B chain is a well-established natural substrate for KLK7 and can be used to assess activity by analyzing the cleavage products using techniques such as high-performance liquid chromatography (HPLC) or mass spectrometry.

• PICS (Proteomic Identification of protease Cleavage Sites): This mass spectrometry-based approach utilizes human proteome-derived peptide libraries of varying lengths to determine subsite preferences of KLK7 in a global setting . The method involves:

  • Generating peptide libraries from cellular proteomes

  • Treating these libraries with purified KLK7

  • Identifying cleavage products using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)

  • Bioinformatic analysis to determine subsite preferences

The PICS approach has confirmed that KLK7 exhibits chymotryptic-like cleavage preferences and has characterized its subsite preferences in the P2-P2′ region, demonstrating a preference for hydrophobic residues in the non-prime and hydrophilic residues in the prime subsites .

What controls should be used when studying KLK7 enzymatic activity?

When studying KLK7 enzymatic activity, appropriate controls are essential for result validation. Interestingly, research has shown that the conventional single catalytic triad mutant KLK7 (mKLK7; S195A) still displays residual catalytic activity (kcat/KM = 7.93 × 102 s−1M−1) . This finding has important implications for experimental design, as it suggests that this common negative control may not be completely inactive.

For studies requiring a completely inactive KLK7 control, especially when using highly sensitive MS-based approaches, a double catalytic triad mutant with an additional D102N mutation is recommended . This double mutation effectively abolishes the residual activity observed with the single S195A mutation.

Additional controls that should be considered include:

• Specific inhibitors: Chemical inhibitors specific to serine proteases or, more ideally, to KLK7 specifically.

• Heat-inactivated enzyme: KLK7 subjected to heat denaturation.

• Enzyme-free buffer controls: To account for any non-enzymatic degradation of substrates.

• Other proteases with different specificities: To confirm the specificity of observed cleavage patterns.

Implementing these controls ensures that observed effects can be attributed specifically to KLK7 activity, enhancing the reliability and reproducibility of research findings.

How does KLK7 contribute to Alzheimer's disease pathology?

Recent research has identified KLK7 as an important component in Alzheimer's disease (AD) pathology, particularly in relation to amyloid-β (Aβ) metabolism. KLK7 has been identified as an astrocyte-derived Aβ degrading enzyme, with expression levels significantly decreased in the brains of AD patients . This reduction in KLK7 expression correlates well with Braak NFT stage, indicating a potential relationship with disease progression .

In experimental models, ablation of the Klk7 gene in AD model mice (App NL-G-F/NL-G-F) exacerbated thioflavin S-positive Aβ pathology, leading to:

• Increased levels of Aβ40 and Aβ42 in the brain

• Enhanced accumulation of insoluble Aβ (SDS-soluble and formic acid-soluble)

• A dramatic 5.6-fold increase in brain amyloid deposition

• Accelerated phosphorylation of murine endogenous tau

• Formation of BACE1-accumulated dystrophic neurites

• Increased GFAP-positive gliosis around the plaques

These findings suggest that KLK7 is a crucial component of brain Aβ economy and plays a significant role in attenuating brain amyloid pathology. The magnitude of increase in endogenous Aβ levels observed in Klk7−/− mice is comparable to effects seen with genetic deletion of other established Aβ-degrading enzymes like neprilysin or insulin-degrading enzyme .

Mechanistically, KLK7 can cleave the hydrophobic core motif of Aβ fibrils, reducing their cell toxicity. This indicates that KLK7 may be particularly important in the clearance of aggregated, oligomeric forms of Aβ in vivo .

How is KLK7 expression regulated in astrocytes?

The regulation of KLK7 expression in astrocytes represents an intriguing area of research with potential therapeutic implications. Several key regulatory mechanisms have been identified:

• Aβ-dependent regulation: Treatment of primary astrocytes with Aβ42 significantly increases the expression of Klk7, suggesting a homeostatic response mechanism . Interestingly, this effect appears to be selective, as lipopolysaccharide treatment does not produce the same upregulation.

• Age-dependent regulation: In AD model mice (App NL-G-F/NL-G-F), mRNA expression of Klk7 is augmented in an age-dependent manner, unlike other Aβ-degrading enzymes such as matrix metalloproteases, neprilysin, and insulin-degrading enzyme .

• Glutamate signaling: Glutamate and N-methyl-d-aspartate treatment significantly reduce Klk7 mRNA levels, suggesting that astrocytic glutamate signaling may be involved in regulating Klk7 expression .

• Memantine effects: The FDA-approved anti-dementia drug memantine can selectively increase the expression of Klk7 in astrocytes and enhance Aβ degradation activity . This effect is abolished in astrocytes from Klk7-knockout mice, confirming the specificity of this mechanism.

• Epigenetic regulation: In cancer cell lines, expression of KLK7 mRNA is regulated by methylation of histones, suggesting potential epigenetic mechanisms that might also be relevant in the central nervous system .

These regulatory mechanisms provide potential targets for therapeutic interventions aimed at increasing KLK7 expression to enhance Aβ clearance in AD.

How might KLK7 be targeted for Alzheimer's disease therapy?

The identification of KLK7 as a key component in Aβ metabolism opens potential avenues for AD therapeutic development. Several approaches could be explored:

• Upregulation of KLK7 expression: Given that KLK7 expression is decreased in AD patients and that its loss exacerbates amyloid pathology, strategies to increase its expression could be beneficial. The finding that memantine, an FDA-approved anti-dementia drug, selectively increases Klk7 expression in astrocytes is particularly promising . Memantine could potentially be used in combination therapy to facilitate amyloid clearance, either alongside immunotherapy or as monotherapy after successful amyloid removal by immunotherapy.

• Targeting astrocytic responses: Since KLK7 is primarily expressed in astrocytes, modulating astrocytic responses to increase KLK7 expression represents a novel therapeutic approach to reduce Aβ deposition . Understanding the mechanisms underlying the selective Klk7 induction by memantine could pave the way for developing astrocyte-targeted AD therapeutics.

• Epigenetic regulation: Given that KLK7 expression can be regulated by histone methylation in cancer cell lines, exploring epigenetic approaches to increase its expression in astrocytes could be a viable strategy .

• Recombinant KLK7 delivery: Direct delivery of purified KLK7 to areas of amyloid deposition might be explored, although challenges related to delivery, stability, and potential off-target effects would need to be addressed.

A significant challenge in developing KLK7-based therapies is understanding why KLK7 expression is decreased in AD patients despite Aβ deposition, which normally induces its expression in experimental models. One possibility is that prolonged inflammatory responses to Aβ deposition might alter astrocyte phenotypes, reducing KLK7 expression . Understanding this pathological phenotype would be crucial for developing effective therapeutic strategies.

What are the potential advantages and limitations of using recombinant KLK7 in research?

Recombinant KLK7 produced in Sf9 baculovirus cells offers several advantages for research applications, but also presents certain limitations that researchers should consider:

Advantages:

• Post-translational modifications: The Sf9 expression system allows for glycosylation and other modifications that may be important for KLK7's stability and function .

• Purification efficiency: The addition of a His-tag enables efficient purification using affinity chromatography techniques, resulting in preparations with greater than 95.0% purity as determined by SDS-PAGE .

• Defined composition: Recombinant production provides a well-defined protein with consistent structure and properties, facilitating reproducible research.

• Avoidance of contaminants: Unlike KLK7 isolated from biological samples, recombinant production minimizes the risk of contamination with other proteases or inhibitors that might confound experimental results.

Limitations:

• Potential differences from native enzyme: Despite the advantages of the Sf9 system, the recombinant protein may still differ from the native human enzyme in terms of glycosylation patterns or other post-translational modifications, potentially affecting its activity or specificity.

• Storage and stability challenges: Recombinant KLK7 requires careful storage conditions to maintain activity, including avoidance of multiple freeze-thaw cycles and potential addition of carrier proteins for long-term storage .

• Residual activity in mutant controls: As noted earlier, even the conventional single catalytic triad mutant (S195A) retains residual activity, necessitating the use of double mutants for complete inactivation in certain experimental contexts .

• Context-dependent activity: The activity of KLK7 in simplified in vitro systems may not fully recapitulate its behavior in complex biological environments where multiple substrates, inhibitors, and regulatory factors are present.

Understanding these advantages and limitations is essential for designing rigorous experiments and correctly interpreting research findings involving recombinant KLK7.

What are the key considerations for designing experiments with KLK7 Human, sf9?

Designing robust experiments with KLK7 Human, sf9 requires careful consideration of multiple factors to ensure reliable and reproducible results. Key considerations include:

• Proper storage and handling: Store at 4°C for short-term use, at -20°C with carrier protein (0.1% HSA or BSA) for long-term storage, and avoid multiple freeze-thaw cycles .

• Appropriate controls: Use double catalytic triad mutants (S195A and D102N) rather than single mutants as negative controls, particularly for sensitive assays, as single mutants retain residual activity .

• Substrate selection: Choose substrates that reflect KLK7's preference for cleaving at sites with aromatic residues in the P1 position and consider its extended subsite preferences in the P2-P2′ region .

• Assay conditions: Optimize buffer composition, pH, temperature, and substrate concentration to ensure optimal enzyme activity while maintaining physiological relevance.

• Detection methods: Select methods with appropriate sensitivity and specificity for the research question, ranging from simple spectrophotometric assays to sophisticated MS-based approaches like PICS .

• Biological context: Consider the biological environment in which KLK7 naturally functions, including potential interaction partners, inhibitors, and regulatory factors.

• Translation to in vivo settings: When extrapolating from in vitro findings to in vivo situations, consider the complexities of the biological environment and potential differences between recombinant and native KLK7.

By carefully addressing these considerations, researchers can design experiments that yield meaningful insights into KLK7's structure, function, and potential therapeutic applications, particularly in contexts such as Alzheimer's disease where KLK7 appears to play a significant role in amyloid metabolism .