Construct Boundary Design: The 4 Problems Killing Your Protein Expression

You clone the full-length gene. Express it. Inclusion bodies. You try with tags. Still insoluble. You try insect cells. It expresses, but aggregates during purification. You truncate the C-terminus—guess where. The protein is now soluble but completely inactive.

Six months gone, and you still don't have a working construct.

The problem isn't your expression system or purification protocol. It's your construct boundaries. Defining where a protein starts and ends—what to include, what to truncate—is the single most important decision in structural biology.

Key Takeaways

• 80-90% of construct failures are due to wrong boundaries

• Main problems: Disordered termini (40%), flexible internal loops (20%), wrong domain boundaries (30%), bad tag placement (10%)

• Average constructs tested: 5-15 per successful structure (traditional trial-and-error)

• Cost of poor boundaries: $50-100K wasted per failed construct (6-12 months)

• Modern solution: AI-driven boundary prediction reduces constructs tested from 10+ to 1-3

• Success rate improvement: 60-80% first-construct success (vs 15-25% traditional)

What Are Construct Boundaries?

Construct boundaries define which residues you express and purify:

• N-terminal boundary: Where does your protein start?

• C-terminal boundary: Where does it end?

• Internal boundaries: Which loops or domains do you include, truncate, or replace?

Example:

• Full-length: Residues 1-450

• Your construct: Residues 28-412 (N-terminal truncation of 27 residues, C-terminal truncation of 38 residues)

Why Boundaries Matter: The Goldilocks Principle

Include too much (disordered regions):

• Flexible termini prevent crystallization

• Disordered loops cause aggregation

• PTMs create heterogeneity (blocks cryo-EM)

• Result: Expresses but won't crystallize/freeze

Include too little (remove essential domains):

• Protein loses stability (unfolds, aggregates)

• Loses function (active site disrupted)

• Result: Doesn't express, or is insoluble/inactive

Just right (structured core, minimal disorder):

• Protein is stable, monodisperse

• Crystallizes or forms good cryo-EM grids

• Retains function

• Result: Structure determination succeeds

Problem 1: Disordered N- and C-Terminal Tails (40% of Failures)

What it means: Your construct includes long (>15 residues) disordered regions at the termini.

Why It's a Problem

For crystallization:

• Disordered residues adopt multiple conformations

• Cannot form regular crystal lattice (requires identical packing)

• Act as "entropy shields" (prevent crystal nucleation)

For cryo-EM:

• Flexible termini create heterogeneity

• 2D class averages become fuzzy

• Reduces resolution

For biochemistry:

• Disordered termini contain protease cleavage sites

• Protein degrades during purification

• Stability decreases (lower Tm)

How to Diagnose

1. AlphaFold pLDDT (per-residue confidence):

• Blue (pLDDT >90): Well-ordered

• Green (pLDDT 70-90): Likely ordered

• Orange (pLDDT 50-70): Questionable

• Red (pLDDT <50): Disordered

Rule of thumb: If >15 consecutive residues have pLDDT <50 at N- or C-terminus → Truncate

2. Disorder prediction tools:

• IUPred: Score >0.5 = disordered

• PrDOS: Probability >0.5 = disordered

• DISOPRED3: Binary prediction

3. Sequence composition:

• Disordered regions enriched in: Gly, Ser, Pro, Glu, Lys, Gln

• Structured regions enriched in: Trp, Phe, Tyr, Ile, Val, Leu, Cys

• Calculate composition: >40% disorder-promoting → likely disordered

4. Homolog comparison:

• Find crystal structures of homologs in PDB

• Check what boundaries they used

• Disordered termini often truncated in successful structures

Example: Bacterial Enzyme

Full-length (1-380):

• AlphaFold pLDDT:

  • Residues 1-22: pLDDT 30-50 (disordered)

  • Residues 23-355: pLDDT 85-95 (structured)

  • Residues 356-380: pLDDT 40-60 (disordered)

Test constructs:

• Construct 1 (1-380, full-length): Expresses, won't crystallize

• Construct 2 (23-355, truncate both): Expresses, crystallizes, 2.3 Å

• Construct 3 (30-350, aggressive): Expresses, lower yield/stability

Winner: Construct 2 (remove disorder, keep structured core)

Problem 2: Flexible Internal Loops (20% of Failures)

What it means: Long (>15 residues) flexible loops connecting structured domains.

Why It's a Problem

For crystallization:

• Flexible loops adopt multiple conformations

• Prevent ordered crystal packing

• Often proteolytically cleaved during crystallization

For cryo-EM:

• Loops create local disorder

• Reduce resolution in those regions

• May cause preferred orientation

Common Locations

GPCRs:

• Intracellular loop 3 (ICL3): 20-80 residues, highly flexible

• Extracellular loop 2 (ECL2): Can be long and flexible

Multi-domain proteins:

• Linkers between domains (5-50 residues)

• Hinge regions (allow domain movement)

Enzymes:

• Active site loops (flexible when substrate-free)

• Regulatory loops

How to Diagnose

1. AlphaFold pLDDT analysis:

• Internal loops with pLDDT <70 are likely flexible

• Long loops (>20 residues) with pLDDT 70-85 may still cause problems

2. B-factors in homolog structures:

• High B-factors (>80 Ų) indicate flexibility

• These regions will be problematic

3. Proteolysis sensitivity:

• Limited proteolysis (trypsin 1:1000, 30 min at RT)

• Flexible loops cleaved preferentially

• Mass spec identifies cleavage sites

The GPCR ICL3 Problem

Famous example: β2-adrenergic receptor

• ICL3 (residues ~230-270): 40 residues, highly flexible

• Wild-type: Cannot crystallize

• Solution: Replace ICL3 with T4 lysozyme

• Construct: Residues 1-229 + T4L + 271-350

• Result: First GPCR crystal structure (2007, Nobel Prize 2012)

Why it worked:

• T4L is rigid, well-behaved

• Provides crystal contacts

• Acts as "fiducial marker" for alignment

Problem 3: Wrong Domain Boundaries (30% of Failures)

What it means: You've truncated too aggressively and removed essential structural elements.

Why It's a Problem

Protein destabilization:

• Removing C-terminal helix exposes hydrophobic core

• Protein unfolds, aggregates

• Lower Tm, reduced stability

Loss of function:

• Active site disrupted

• Cofactor binding site incomplete

• Regulatory domain missing

How to Diagnose

1. AlphaFold structure inspection:

• Check if truncation cuts through secondary structure

• Bad: Truncate in middle of helix or β-strand

• Good: Truncate in loop between elements

2. Domain architecture prediction:

• Pfam: Identifies conserved domains

• InterPro: Comprehensive domain annotation

• Rule: Don't truncate within a Pfam domain

3. Homolog comparison:

• Align with PDB homologs

• Check if successful structures include the region you're removing

4. Stability testing:

• Express truncated construct, measure Tm

• Compare to longer construct

• If ΔTm < -5°C → removed something important

Case Study: Removing Essential Helix

Target: Novel bacterial enzyme

Attempt 1:

• Construct: Residues 1-320 (removed C-terminal 30 residues)

• Expression: Inclusion bodies (completely insoluble)

Analysis:

• AlphaFold structure: Residues 310-330 form amphipathic helix

• Helix packs against core (stabilizes hydrophobic pocket)

• Removing it exposes core → aggregation

Attempt 2:

• Construct: Residues 1-340 (keep helix, remove last 10 disordered)

• Expression: Soluble, stable

• Crystallization: Success, 2.1 Å structure

Lesson: Even one helix can be critical. Respect secondary structure boundaries.

Problem 4: Wrong Fusion/Tag Placement (10% of Failures)

What it means: Affinity tag (His-tag, GST, MBP) or fusion protein placed where it disrupts folding or function.

Why It's a Problem

Fusion blocks active site:

• Tag near substrate binding site

• Protein cannot bind substrate

• Appears inactive

Fusion disrupts oligomerization:

• Protein normally forms dimer

• Tag at dimerization interface prevents assembly

• Monomeric protein is unstable

Fusion prevents membrane insertion:

• For membrane proteins, N-terminal signal peptide required

• Tag at N-terminus blocks signal peptide

• Protein not inserted, aggregates

How to Diagnose

1. Check AlphaFold structure:

• Visualize where N- and C-termini are located

• Are they buried or surface-exposed?

• Are they near active site or oligomerization interface?

2. Homolog comparison:

• Where are termini in crystal structures?

• Are they accessible?

3. Test both termini:

• Construct A: His-tag at N-terminus

• Construct B: His-tag at C-terminus

• Test expression, solubility, activity

Tag Placement Rules

Rule 1: Surface-exposed termini are best

• If AlphaFold shows N-terminus on surface → N-terminal tag okay

• If buried → try C-terminal tag

Rule 2: Avoid active sites

• Check where substrate binds

• Place tag on opposite side

Rule 3: Avoid oligomerization interfaces

• If protein forms dimer, check interface

• Place tag away from contact surface

Rule 4: Use cleavable tags

• For crystallography/cryo-EM: Remove tag after purification

• Use TEV protease site, PreScission site, or SUMO

Multi-Domain Proteins: Special Considerations

Example: Full-length (1-520)

• Domain 1 (catalytic): Residues 45-280

• Linker (flexible): Residues 281-310

• Domain 2 (regulatory): Residues 311-490

• C-terminal tail (disordered): Residues 491-520

Good construct options:

• Construct A: 45-280 (catalytic domain only)

• Construct B: 45-490 (both domains, no disordered tail)

• Construct C: 311-490 (regulatory domain only)

Bad construct options:

• Construct D: 45-250 (truncates within catalytic domain) → Unstable

• Construct E: 100-490 (removes part of catalytic domain) → Inactive

Key principle: Keep entire domains, remove linkers between them.

Membrane Proteins: Transmembrane Boundaries

The Challenge

• Need to include entire transmembrane (TM) helices

• Truncating in middle of TM helix → unstable, aggregates

• Including too much intracellular/extracellular domain → flexible, prevents crystallization

How to Define TM Boundaries

1. Predict TM helices:

• TMHMM: Classic tool, reliable

• DeepTMHMM: Deep learning-based, more accurate

• Orbion: Integrates TM prediction with structure

2. AlphaFold structure:

• TM helices have high pLDDT (usually >90)

• TM regions form distinct bundle

3. Hydropathy plot:

• Kyte-Doolittle plot identifies hydrophobic regions (TM helices)

Boundary Rules for Membrane Proteins

Rule 1: Include complete TM helices

• Don't truncate in middle of helix

• Start/end in loop regions

Rule 2: Truncate flexible loops

• Especially long loops (>20 residues)

• GPCRs: ICL3 is classic target

Rule 3: Remove signal peptide

• Signal peptide directs to ER

• Not needed for recombinant expression

• Predict with SignalP

Understanding Your Problem: Quick Diagnostic

Key Takeaway

Construct boundary design isn't guesswork. It's a systematic engineering problem with predictable failure modes:

1. Disordered termini (40%): Include flexible tails that prevent crystallization

2. Flexible loops (20%): Internal disorder blocks structure determination

3. Wrong domain boundaries (30%): Remove essential structural elements

4. Bad tag placement (10%): Tags disrupt function or folding

Understanding your boundary problem is the first step to fixing it.