Welqut Protease, His

What is WELQut Protease and what makes it unique among proteases?

WELQut Protease is a highly specific, recombinant serine protease derived from Staphylococcus aureus. Its uniqueness lies in its ability to recognize and precisely cleave recombinant proteins containing the engineered recognition sequence W-E-L-Q↓X (where X can be any amino acid). Unlike many other proteases, WELQut cleaves outside the recognition sequence, which means it doesn't leave additional amino acids bound to the target protein, resulting in a cleaner final product with native N-terminus . The protease is also notable for its activity across a broad range of conditions (temperature 4-30°C and pH 6.5-9.0) without requiring specific buffers, making it versatile for various experimental setups .

What is the molecular structure and properties of WELQut Protease?

WELQut Protease is a single, non-glycosylated polypeptide chain containing approximately 210 amino acids with a molecular mass of 22 kDa . The commercially available enzyme is typically fused to a 6 amino acid His-tag at the C-terminus and is purified by proprietary chromatographic techniques . The protease is derived from the SpIB protease of Staphylococcus aureus, which is naturally activated by proteolytic cleavage of an N-terminal signal peptide, allowing the formation of a characteristic hydrogen bond network essential for its activity . This structural arrangement contributes to its high specificity for the WELQ recognition motif.

How is WELQut Protease activity defined and measured?

The activity of WELQut Protease is defined in units, where one unit is the amount of enzyme required to cleave ≥99% of 100 μg of a control protein in 16 hours at 20°C . This activity measurement is typically assayed in 100 μl of 100 mM Tris-HCl buffer at pH 8.0 . Researchers can monitor cleavage efficiency through SDS-PAGE analysis, where the appearance of cleaved products indicates successful protease activity. When used with GFP fusion proteins, cleavage can be conveniently monitored by visualizing fluorescent bands after electrophoretic separation, as demonstrated in bacteriocin expression studies .

What are the optimal buffer conditions for WELQut Protease activity?

WELQut Protease exhibits remarkable flexibility regarding buffer conditions, functioning effectively across a pH range of 6.5 to 9.0 . While the enzyme is typically assayed in 100 mM Tris-HCl (pH 8.0), it does not strictly require this specific buffer for activity . The protease is provided in a storage buffer containing 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 140 mM NaCl, 2.7 mM KCl, and 50% glycerol at pH 7.3 . When designing cleavage experiments, researchers should consider that the protease maintains activity in most common biological buffers, allowing for direct cleavage in chromatography elution buffers or other purification steps without buffer exchange.

How should time and temperature parameters be optimized for WELQut cleavage reactions?

WELQut Protease maintains activity across a temperature range of 4-30°C, providing flexibility in experimental design . For standard cleavage reactions, incubation at 20-25°C for 16 hours is typical, though the time can be adjusted based on the specific fusion protein and required efficiency . When optimizing cleavage conditions, it is advisable to conduct time-course experiments (e.g., sampling at 2, 4, 8, and 16 hours) and vary the enzyme-to-substrate ratio to determine optimal conditions for your specific protein . A study on bacteriocin fusion proteins demonstrated that maximal activity was achieved after 16 hours of incubation at 28°C, with WELQut-to-sample ratios of 1:10 and 1:25 (μL:μL) for different constructs .

What factors might affect WELQut Protease cleavage efficiency?

Several factors can influence WELQut cleavage efficiency. Research has shown that certain fusion proteins may interact with the protease in ways that reduce its effectiveness . In studies with bacteriocin fusion proteins, researchers observed that liberated peptides might interact with the N-terminus of the protease, potentially affecting its substrate recognition and hydrolysis capabilities . Structural elements that obstruct access to the cleavage site, such as secondary structure formation or protein aggregation, can also reduce efficiency. Additionally, the presence of denaturants, detergents, or high salt concentrations may impact enzyme activity. When troubleshooting poor cleavage, consider adjusting reaction conditions (temperature, time, enzyme concentration) and ensuring the recognition sequence is fully accessible.

How can WELQut Protease be integrated into on-column cleavage protocols?

WELQut Protease is particularly well-suited for on-column proteolysis reactions, providing a streamlined approach to purification workflows . To implement on-column cleavage:

• Immobilize the His-tagged fusion protein on a Ni-NTA or similar affinity column

• Wash the column thoroughly to remove non-specific contaminants

• Apply WELQut Protease diluted in an appropriate buffer directly to the column

• Incubate the column at 20-25°C for 4-16 hours (optimize time as needed)

• Elute the cleaved target protein while the His-tagged fusion partner and His-tagged WELQut remain bound to the column

• Collect and analyze the eluted fractions for cleavage efficiency

This approach offers the advantage of simultaneous cleavage and initial purification, reducing the number of processing steps and potentially improving yield and purity of the target protein.

What methods can be used to monitor WELQut Protease cleavage efficiency in real-time?

Monitoring WELQut cleavage efficiency in real-time can be achieved through several approaches:

• Fluorescence-based monitoring: When using GFP fusion proteins, the maintenance of fluorescent properties after SDS-PAGE allows visual confirmation of cleavage progress. Researchers have observed distinct fluorescent bands for GFP-fusion proteins before and after cleavage .

• Chromatographic approaches: Size-exclusion chromatography or reverse-phase HPLC can be used to separate and quantify cleaved and uncleaved products during the reaction.

• Enzyme activity assays: If the target protein has measurable enzymatic activity that is affected by the presence of the fusion tag, monitoring this activity can provide insights into cleavage progress.

• Western blotting: Using antibodies specific to either the tag or the target protein allows quantitative assessment of cleavage efficiency at various time points.

In research with bacteriocin fusions, scientists used both the fluorescent properties of GFP and correlation with stained SDS-PAGE gels to evaluate optimal cleavage conditions .

How does WELQut Protease compare with other tag-removal proteases in terms of specificity and efficiency?

What strategies can improve WELQut cleavage efficiency when working with difficult protein substrates?

When working with difficult protein substrates that show poor WELQut cleavage efficiency, several strategies can be implemented:

• Linker modification: Introducing a flexible linker (such as Gly-Ser repeats) between the fusion partner and WELQ recognition sequence can improve accessibility of the cleavage site.

• Denaturation-refolding approach: For proteins where the cleavage site might be sterically hindered, mild denaturation followed by refolding during the cleavage reaction may improve access to the recognition sequence.

• Temperature cycling: Alternating between different temperatures within the active range (4-30°C) during the cleavage reaction may help overcome kinetic barriers to enzyme-substrate interaction.

• Additive screening: Testing various additives such as low concentrations of non-ionic detergents or stabilizing agents (e.g., glycerol, sucrose) may improve reaction efficiency.

• Extended reaction time: Increasing the incubation time beyond the standard 16 hours may improve cleavage yield for difficult substrates, as long as protein stability permits.

• Increased enzyme concentration: Using higher enzyme-to-substrate ratios can overcome inefficient cleavage, though this should be balanced against the potential for non-specific activity at very high enzyme concentrations.

Research with bacteriocin fusion proteins demonstrated that optimizing the WELQut-to-sample ratio significantly improved cleavage efficiency, suggesting this parameter should be prioritized when troubleshooting .

How can WELQut Protease be effectively used in the production of antimicrobial peptides and other bioactive compounds?

WELQut Protease has proven valuable in the production of antimicrobial peptides, particularly bacteriocins, as demonstrated in research with plantaricin 423 and mundticin ST4SA . To effectively use WELQut in such applications:

• Design fusion constructs with accessible cleavage sites: When designing expression vectors, include the WELQ recognition sequence between the fusion partner (e.g., GFP, MBP) and the antimicrobial peptide, ensuring it is accessible for cleavage .

• Optimize cleavage conditions: For bacteriocin production, researchers found that specific WELQut-to-sample ratios (1:10 and 1:25) with 16-hour incubation at 28°C yielded maximal antimicrobial activity .

• Monitor both cleavage and bioactivity: Assess not only the physical cleavage (via SDS-PAGE) but also the biological activity of the released peptide. In bacteriocin research, antilisterial activity assays confirmed successful production of active compounds .

• Address complex formation issues: Research has observed that WELQut may form complexes with certain cleaved peptides, potentially reducing efficiency. This might require additional purification steps or condition optimization .

• Incorporate visual markers: Using fluorescent fusion partners like GFP allows for visual confirmation of cleavage through the observation of fluorescent bands after electrophoretic separation .

By adapting these strategies, researchers can effectively employ WELQut Protease in the production pipeline for various bioactive compounds that require precise removal of fusion tags without leaving residual amino acids that might interfere with biological activity.

What are the optimal storage conditions for maintaining WELQut Protease activity?

WELQut Protease should be stored according to both immediate use plans and long-term preservation needs. For short-term storage (2-4 weeks), the enzyme can be kept at 4°C in its supplied buffer . For longer-term storage, it should be kept frozen at -20°C . The enzyme is typically supplied in a stabilizing buffer containing 10 mM Na₂HPO₄, 50% glycerol, 1.8 mM KH₂PO₄, 140 mM NaCl, and 2.7 mM KCl at pH 7.3 . For extended storage periods, it is recommended to add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) to enhance stability . Multiple freeze-thaw cycles should be strictly avoided as they can significantly reduce enzyme activity . Aliquoting the enzyme into single-use volumes before freezing is a recommended practice to maintain consistent activity throughout your research project.

How can researchers validate the activity of stored WELQut Protease before critical experiments?

Before using WELQut Protease in critical experiments, especially after storage, researchers should validate its activity using the following approaches:

• Control protein cleavage assay: Cleave a well-characterized control protein containing the WELQ recognition sequence at standard conditions (100 mM Tris-HCl, pH 8.0, 20°C, 16 hours) and analyze by SDS-PAGE to verify expected cleavage patterns.

• Enzyme titration: Perform a series of cleavage reactions with varying enzyme concentrations to establish the minimum amount needed for complete cleavage, comparing this to previous results or manufacturer specifications.

• Time-course analysis: Conduct a small-scale cleavage reaction with sampling at multiple time points (2, 4, 8, and 16 hours) to verify that cleavage kinetics match expected patterns.

• Activity unit calculation: Quantitatively assess the percentage of substrate cleaved under standardized conditions to calculate actual units of activity, which can be compared to the expected value (defined as amount of enzyme needed to cleave ≥99% of 100 μg control protein in 16 hours at 20°C) .

This validation is particularly important when working with valuable or difficult-to-produce recombinant proteins, as it ensures experimental success and reliable results.