Enterokinase Bovine His

What is the role of Enterokinase Bovine His in recombinant protein purification?

Enterokinase Bovine His cleaves fusion tags at the DDDDK↓ recognition site, enabling the isolation of target proteins with native N-termini. Methodologically:

• Step 1: Incubate the enzyme with the fusion protein at a 1:100 to 1:1000 molar ratio in 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 2 mM CaCl₂ .

• Step 2: Use immobilized metal affinity chromatography (IMAC) to remove the His-tagged enzyme post-cleavage .

• Critical Parameter: Calcium ions stabilize enzymatic activity; EDTA-containing buffers reduce efficiency by ≥70% .

Table 1: Activity Comparison Across Expression Systems

How does the His-tag enhance Enterokinase Bovine His utility?

The C-terminal His₅ tag simplifies post-cleavage purification:

• Protocol: Post-digestion, apply the mixture to a Ni²⁺-charged resin. The enzyme binds via its His-tag, while the target protein flows through .

• Efficiency: This step achieves >95% removal of enterokinase, confirmed by SDS-PAGE and mass spectrometry .

• Caution: Avoid imidazole concentrations >20 mM during cleavage, as they interfere with tag binding .

What conditions optimize Enterokinase Bovine His cleavage efficiency?

Optimal parameters derive from structural studies of the serine protease domain :

• Buffer: 50 mM Tris-HCl, 250 mM NaCl, 2 mM CaCl₂ (pH 8.0)

• Time-Temperature Tradeoff:

  • 16 hours at 25°C (standard)

  • 4 hours at 37°C (accelerated, but risk aggregation)

• Denaturant Compatibility: ≤2 M urea or 0.1% SDS maintains ≥50% activity .

How to troubleshoot incomplete cleavage of fusion proteins?

Incomplete cleavage often stems from:

• Cause 1: Occluded cleavage sites. Solution: Insert a 5–10 aa linker (e.g., GGGGS) between the tag and target .

• Cause 2: Suboptimal Ca²⁺. Validation: Measure activity via chromogenic substrates like Bz-PFR-pNA; ΔA₄₀₅/min should exceed 0.05 .

• Cause 3: Glycosylation variants. Mitigation: Use PNGase F pretreatment if expressing in eukaryotic systems .

Table 2: Troubleshooting Guide

Why does Enterokinase Bovine His exhibit residual activity in denaturing conditions?

The enzyme’s stability under denaturants arises from:

• Structural Insight: Two disulfide bonds (Cys58–Cys158, Cys122–Cys189) maintain the catalytic triad (His71, Asp115, Ser217) .

• Experimental Evidence: 40% activity persists in 2 M guanidine HCl, enabling in situ cleavage during refolding .

• Application: Cleave inclusion body proteins during solubilization, minimizing purification steps .

How to validate Enterokinase Bovine His specificity in complex mixtures?

Use orthogonal assays:

• Fluorogenic Assay: Monitor cleavage of DDDDK-AMC substrate (λₑₓ/λₑₘ = 380/460 nm) .

• Western Blot: Probe with anti-His antibodies to confirm enzyme removal .

• Mass Spectrometry: Detect residual cleavage (e.g., DDDDK fragments) at ≤1% threshold .

Can Enterokinase Bovine His cleave non-canonical sequences?

While designed for DDDDK, promiscuity occurs at:

• P1 Position: Lys → Arg reduces efficiency by 90% (kₐₜ/Kₘ = 1.2 × 10³ vs. 1.4 × 10⁴ M⁻¹s⁻¹) .

• P2–P4 Positions: Asp → Glu decreases activity 4-fold; substitutions beyond P4 have minimal impact .

• Workaround: For non-native sites, fuse a TEV protease site upstream of DDDDK .

Methodological Recommendations

• Storage: Aliquot in 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 50% glycerol; avoid >3 freeze-thaw cycles .

• Negative Control: Always include a no-enzyme reaction to detect spontaneous proteolysis.

• Kinetic Analysis: Calculate specific activity using $v_0 = \frac{ΔA_{405}}{ε \cdot t}$, where ε = 9.6 mM⁻¹cm⁻¹ for p-nitroaniline .