How to Interpret AI Feasibility Check Results

Understanding expressibility scores and protein expression predictions

The AI Feasibility Check tool analyzes protein sequences to predict expressibility, solubility, and potential expression risks. This guide will help you understand the results and use them to improve your protein expression experiments.

Understanding the Expressibility Score

The Expressibility Score (0-100) is a composite metric that combines multiple factors:

  • Solubility Score: Based on hydrophobicity, charge, and aggregation potential
  • Complexity Score: Low-complexity regions can cause expression issues
  • Codon Score: For DNA sequences, rare codons affect expression
  • Transmembrane Penalty: TM domains reduce expressibility

Score Categories

  • Good (70-100): High likelihood of successful expression
  • Moderate (40-69): Expression possible but may require optimization
  • Poor (<40): Expression likely to be difficult, significant optimization needed

Key Parameters Explained

1. Mean Hydrophobicity

Calculated using the Kyte-Doolittle scale. Values:

  • Negative values: Hydrophilic (good for solubility)
  • Positive values: Hydrophobic (may cause aggregation)
  • Optimal range: -0.5 to 0.5

High hydrophobicity (>1.0): Protein may form inclusion bodies, reduce solubility.

2. Charge per Residue

Net charge divided by sequence length. Affects:

  • Protein solubility
  • Isoelectric point (pI)
  • Electrostatic interactions

Optimal range: -0.1 to +0.1 charge per residue for most expression systems.

3. Disorder Proxy

Fraction of disorder-promoting amino acids (P, E, D, K, Q, S). High disorder:

  • May indicate flexible regions
  • Can affect protein stability
  • May require fusion tags for expression

4. Low-Complexity Regions

Regions with repetitive sequences. Problems:

  • Can cause expression difficulties
  • May form aggregates
  • Can interfere with protein folding

Warning: If detected, consider removing or modifying these regions.

5. Transmembrane Segments

Hydrophobic regions that span membranes. Issues:

  • Difficult to express in soluble form
  • Require special expression systems
  • May need membrane mimetics

Solution: Express only extracellular or intracellular domains separately.

6. Aggregation Clusters

Regions with ≥5 consecutive hydrophobic residues (I, L, V, F, W, Y). These:

  • Promote protein aggregation
  • Reduce solubility
  • Can cause inclusion body formation

7. Signal Peptide

N-terminal sequence for protein secretion. If detected:

  • May need to be removed for intracellular expression
  • Can affect expression levels
  • Important for secreted proteins

8. Rare Codon Fraction (for DNA sequences)

Fraction of rare codons in E.coli. High rare codon usage:

  • Reduces expression efficiency
  • Can cause translation stalling
  • May lead to truncated proteins

Solution: Use codon optimization tools to replace rare codons.

Interpreting Warnings

The tool provides warnings for potential issues:

  • High hydrophobicity: Consider adding solubility tags or using refolding protocols
  • Aggregation clusters: May need to mutate hydrophobic residues
  • Transmembrane domains: Consider expressing domains separately
  • Low-complexity regions: May need to modify or remove
  • Rare codons: Optimize codon usage for expression host

Recommendations for Improvement

For Low Expressibility Scores (<50)

  1. Add solubility tags: GST, MBP, or SUMO tags can improve solubility
  2. Optimize codon usage: Replace rare codons with preferred ones
  3. Reduce hydrophobicity: Mutate key hydrophobic residues
  4. Remove problematic regions: Delete low-complexity or aggregation-prone regions
  5. Use fusion partners: Express as fusion protein initially

For Moderate Scores (50-70)

  1. Optimize expression conditions: Temperature, IPTG concentration, time
  2. Try different expression hosts: E.coli, yeast, insect cells
  3. Use chaperones: Co-express folding helpers
  4. Consider refolding: Express as inclusion bodies and refold

Practical Example

Example Analysis Results:

Expressibility Score: 65 (Moderate)
Mean Hydrophobicity: 0.8 (slightly high)
Charge per Residue: -0.05 (good)
Disorder Proxy: 0.25 (moderate)
Low-Complexity Regions: None detected
Transmembrane Segments: None detected
Aggregation Clusters: 1 detected (positions 45-50)
Rare Codon Fraction: 0.15 (moderate)

Warnings:
- Slightly high hydrophobicity may reduce solubility
- Aggregation cluster detected at positions 45-50

Recommendations:
- Consider adding solubility tag (GST or MBP)
- Mutate hydrophobic residues in aggregation cluster
- Optimize rare codons for better expression
- Try lower expression temperature (18-25°C)

Best Practices

  • Always check multiple parameters: Don't rely on score alone
  • Address warnings: Each warning indicates a potential issue
  • Compare variants: Test multiple sequence variants
  • Validate experimentally: Predictions are guides, not guarantees
  • Consider expression system: Different hosts have different requirements

Related Tools

Use these tools together for comprehensive analysis:

Conclusion

Understanding AI Feasibility Check results helps you optimize protein expression experiments. By interpreting the expressibility score, individual parameters, and warnings, you can make informed decisions about sequence modifications and expression strategies. Use our free AI Feasibility Check tool to analyze your sequences and improve expression success rates.