How to Design Optimal Construct Boundaries: Step-by-Step Workflow

In this article, we'll show you exactly how to design optimal boundaries before you clone.

The paradigm shift: Stop guessing. Start predicting.

Traditional approach: Clone full-length → Fail → Guess truncations → Fail → Test 10-15 constructs → Maybe succeed

Modern AI-driven approach: Analyze structure → Predict boundaries → Design 2-3 rational constructs → Test → Succeed

This guide provides the complete workflow, from sequence to optimized construct.

Key Takeaways

• Design workflow: 6 systematic steps (sequence → boundaries → constructs → validation)

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

• Time saved: 3-6 months per project (vs trial-and-error)

• Tools: Free tools work (AlphaFold, IUPred), but Orbion reduces design time from 4-6 hours to 15 minutes

• Fusion proteins: Use when native boundaries fail (GPCRs, unstable proteins)

• Validation: Always predict Tm, check secondary structure integrity

The 6-Step Design Workflow

Step 1: Get Your Structure Prediction (5 minutes)

Free approach:

1. Go to AlphaFold Database

2. Search by UniProt ID or sequence

3. Download structure (PDB file) and pLDDT JSON

Orbion approach:

1. Upload sequence to Orbion

2. Automatic structure prediction with integrated boundary analysis

3. Get annotated boundaries with confidence scores

What to look for:

• pLDDT coloring: Blue/green = ordered, orange/red = disordered

• Overall fold: Is protein single-domain or multi-domain?

• Termini location: Buried or surface-exposed?

Red flags:

• Long disordered termini (>20 residues, pLDDT <50)

• Internal disordered loops (>15 residues, pLDDT <70)

• Multi-domain with flexible linkers

Step 2: Predict Disorder and Flexibility (10 minutes)

Free tools:

IUPred3: https://iupred.elte.hu/

• Paste sequence

• Select "Long disorder" option

• Score >0.5 = disordered

• Identify continuous stretches >15 residues

DISOPRED3: http://bioinf.cs.ucl.ac.uk/psipred/

• Paste sequence

• Binary prediction (ordered/disordered)

• Cross-check with IUPred

What to record:

• N-terminal disorder: Residues X-Y (length, score)

• C-terminal disorder: Residues X-Y (length, score)

• Internal loops: Position, length, score

Example output:

Protein: 1-380
N-terminal disorder: 1-22 (22 residues, IUPred >0.6)
Structured core: 23-355 (pLDDT >85)
C-terminal disorder: 356-380 (25 residues, IUPred >0.5)

Step 3: Analyze Domain Architecture (10 minutes)

Free tools:

Pfam: https://pfam.xfam.org/

• Paste sequence

• Identifies conserved domains

• Shows boundaries for each domain

InterPro: https://www.ebi.ac.uk/interpro/

• Comprehensive domain annotation

• Includes functional sites

Critical rules:

1. Don't truncate within a Pfam domain (unless you know what you're doing)

2. Keep complete secondary structure elements (don't cut helices/strands)

3. Truncate in loops between domains or elements

Example:

Full-length: 1-520
Pfam domains:
  - Kinase domain: 45-280
  - SH2 domain: 311-490
  - Disordered linker: 281-310
  - Disordered tail: 491-520

Valid constructs:
  ✓ 45-280 (kinase only)
  ✓ 311-490 (SH2 only)
  ✓ 45-490 (both domains, remove tail)
  ✗ 45-250 (truncates within kinase) → Unstable
  ✗ 100-490 (removes part of kinase) → Inactive

Step 4: Compare with Homolog Structures (15 minutes)

Why it matters: Successful structures in PDB show what boundaries actually work.

How to do it:

1. Find homologs:

• Go to BLAST

• Search against PDB database

• Find homologs with >30% identity

2. Check boundaries used:

• Open PDB entries

• Note which residues were crystallized

• Compare to full-length sequence

3. Identify consensus truncations:

• If 3+ structures truncate N-terminus at residue ~25 → likely essential

• If none include C-terminal 30 residues → likely disordered

Example: Bacterial enzyme homologs

Consensus: All truncate N-terminus (~25-30), all truncate C-terminus (~350), none include 1-25 or 355+

Your construct: 25-355 (high confidence)

Step 5: Design Your Construct Set (30 minutes)

Strategy: Design 2-3 constructs with different risk levels

Construct A: Conservative (highest stability, lowest risk)

• Remove only clearly disordered regions (pLDDT <50, IUPred >0.5)

• Keep all secondary structure elements

• Based on homolog consensus

Construct B: Optimal (balance stability and crystallizability)

• Remove all disordered regions (pLDDT <70)

• Truncate flexible loops if >20 residues

• Most likely to succeed

Construct C: Aggressive (highest crystallizability, higher risk)

• Minimal boundaries (only structured core)

• May sacrifice some stability

• Use if Construct B fails

Example design:

Target: Novel kinase (1-420)

• AlphaFold pLDDT:

  • 1-18: pLDDT 35-50 (disordered)

  • 19-380: pLDDT 85-95 (structured)

  • 381-420: pLDDT 40-60 (disordered)

• Pfam: Kinase domain 25-375

• Homologs: Truncate at ~20 and ~380

Construct set:

Construct A (Conservative): 19-385
  - Removes clear disorder (pLDDT <50)
  - Keeps some questionable C-terminus (pLDDT 50-60)
  - Risk: May not crystallize (some disorder remains)

Construct B (Optimal): 25-375
  - Aligns with Pfam domain
  - pLDDT >85 throughout
  - Matches homolog consensus
  - Risk: Lowest (recommended first)

Construct C (Aggressive): 30-370
  - Removes 5 extra residues each end
  - Maximum order (pLDDT >90)
  - Risk: May reduce stability

Which to test first: Construct B (optimal balance)

Step 6: Validate Your Design (10 minutes)

Before you order primers, check:

1. Secondary structure integrity:

• Load AlphaFold structure in PyMOL or ChimeraX

• Check N-terminal boundary: Does it cut through helix/strand?

• Check C-terminal boundary: Same check

• Rule: Truncate in loops, not secondary structure

2. Termini accessibility:

• Are N- and C-termini surface-exposed?

• If buried → fusion tag will cause problems

• Plan tag placement accordingly

3. Active site check:

• Visualize active site (if known)

• Ensure your boundaries include all catalytic residues

• Ensure fusion tag won't block substrate access

4. Predicted stability (Orbion only):

• Orbion predicts Tm for each construct

• Compare Construct A vs B vs C

• If ΔTm >10°C → choose more stable construct

Free Tools vs Orbion: When to Use What

Free Tools Approach (Total: 4-6 hours)

Advantages:

• Zero cost

• Good for single constructs

• Educational (you learn the details)

Workflow:

1. AlphaFold Database (5 min) → Structure

2. IUPred3 (10 min) → Disorder

3. Pfam (10 min) → Domains

4. BLAST + manual PDB inspection (30 min) → Homologs

5. Manual analysis in PyMOL (60 min) → Boundary design

6. Repeat for each construct variant (30 min each)

Total time: 4-6 hours for 3 constructs

Best for:

• Students, academic labs

• Single-protein projects

• Learning structural biology principles

Orbion Approach (Total: 15 minutes)

Advantages:

• Automated workflow

• Integrated analysis (disorder + domains + homologs)

• Predicted Tm for stability comparison

• Batch processing (multiple constructs simultaneously)

• Confidence scores for each boundary recommendation

Workflow:

1. Upload sequence (1 min)

2. Automatic boundary analysis (5 min)

3. Review 2-3 suggested constructs with Tm predictions (5 min)

4. Select optimal construct (2 min)

5. Export to ordering (2 min)

Total time: 15 minutes for full analysis

What Orbion adds:

• AstraSTASIS: Predicts Tm for each construct (know stability before expressing)

• Homolog mining: Automatic BLAST + PDB analysis

• Boundary confidence: Statistical scoring based on multiple predictors

• Tag optimization: Suggests N- vs C-terminal placement

• Batch mode: Analyze 10+ constructs in parallel

Best for:

• Biotech, pharma (time = money)

• Multi-construct screening

• High-throughput projects (>5 proteins/month)

• When first-construct success is critical

Cost-benefit:

• Saves 4-5 hours per protein × $100/hour scientist = $400-500 saved

• Orbion cost: Starts at $99/month (unlimited analyses)

• Break-even: 1 protein per month

When to Use Fusion Proteins

Sometimes native boundaries aren't enough. Fusion proteins can rescue difficult targets.

Fusion Type 1: Solubility Tags (MBP, GST, SUMO)

When to use:

• Protein expresses but insoluble

• Even with optimized boundaries

Common tags:

MBP (Maltose Binding Protein, 42 kDa):

• Best for: Solubilizing aggregation-prone proteins

• Placement: N-terminal

• Cleavage: TEV protease site

• Success rate: 70-80% of insoluble proteins become soluble

GST (Glutathione S-Transferase, 26 kDa):

• Best for: Small proteins (<20 kDa)

• Placement: N-terminal

• Cleavage: PreScission protease

• Bonus: Forms dimer (stabilizes small proteins)

SUMO (Small Ubiquitin-like Modifier, 12 kDa):

• Best for: Eukaryotic proteins

• Placement: N-terminal

• Cleavage: SUMO protease (leaves no extra residues)

• Bonus: Enhances expression in E. coli

Design rule:

• Always include protease cleavage site

• Remove tag after purification for crystallography/cryo-EM

• Test activity with and without tag

Fusion Type 2: Crystallization Helpers (T4L, BRIL)

When to use:

• Protein soluble but won't crystallize

• Especially for membrane proteins (GPCRs)

T4 Lysozyme (T4L, 18 kDa):

• Famous use: β2-adrenergic receptor (2007 Nobel Prize)

• Strategy: Replace flexible ICL3 with T4L

• Why it works:

  • Rigid, well-behaved protein

  • Provides crystal contacts

  • Acts as fiducial marker for cryo-EM alignment

BRIL (Thermostable apocytochrome b562, 11 kDa):

• Use: GPCR crystallization

• Advantage: Smaller than T4L (less disruption)

• Placement: Replace ICL3 or insert at N-terminus

Design strategy:

GPCR (1-350):
  - TM1-TM7: Residues 25-320
  - ICL3 (flexible): Residues 230-270 (40 residues)

Fusion construct:
  - Residues 25-229 (TM1-TM5)
  - T4L insertion (replaces ICL3)
  - Residues 271-320 (TM6-TM7)
  - Remove C-terminal tail (321-350)

Result: Rigid, crystallizable GPCR

Fusion Type 3: Nanobodies/DARPins (Conformational Stabilization)

When to use:

• Protein is dynamic (multiple conformations)

• Need to lock specific conformation for structure

How it works:

• Co-express protein + nanobody

• Nanobody binds, stabilizes one conformation

• Reduces conformational heterogeneity

• Easier to crystallize or freeze for cryo-EM

Example: G protein-coupled receptor

• GPCR has active and inactive states

• Nanobody binds active state

• Locks conformation

• Enables structure determination of active state

Orbion advantage:

• AstraBIND: Predicts nanobody epitopes

• Design nanobodies targeting specific conformations

Case Study: Multi-Domain Protein Optimization

Target: Novel bacterial enzyme with regulatory domain

Full-length: 1-520

• N-terminal tail: 1-44 (disordered)

• Catalytic domain: 45-280 (structured)

• Linker: 281-310 (flexible)

• Regulatory domain: 311-490 (structured)

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

Traditional Approach (Failed)

Attempt 1: Full-length (1-520)

• Expression: Low yield

• Purification: Multiple degradation bands

• Crystallization: No crystals after 300 conditions

• Result: Failure (disordered tails cause problems)

Attempt 2: Remove tails (45-490)

• Expression: Good

• Purification: Clean, but some degradation

• Crystallization: No crystals

• Analysis: Limited proteolysis shows cleavage in linker (281-310)

• Result: Partial success (linker still flexible)

Total time: 8 months, 2 failed attempts

AI-Driven Approach (Success)

Step 1: AlphaFold analysis

• N-terminal 1-44: pLDDT 30-50 (remove)

• Catalytic domain: pLDDT 90-95 (keep)

• Linker: pLDDT 50-70 (problem area)

• Regulatory domain: pLDDT 85-92 (keep)

• C-terminal 491-520: pLDDT 35-50 (remove)

Step 2: Homolog check

• Found 5 homologs in PDB

• 3 structures: Catalytic domain only (45-280)

• 2 structures: Regulatory domain only (311-490)

• None: Full two-domain construct

• Conclusion: Domains likely independent, linker prevents co-crystallization

Step 3: Construct design

Construct A: 45-280 (catalytic domain only)
  - Remove N-terminal tail, linker, regulatory domain, C-terminal tail
  - Pfam: Complete catalytic domain
  - Risk: May lose regulatory function

Construct B: 311-490 (regulatory domain only)
  - For understanding regulation mechanism separately

Construct C: 45-490 + linker replacement
  - Replace flexible linker (281-310) with rigid GGGGS linker
  - Risk: May disrupt domain-domain communication

Step 4: Testing

Construct A (45-280):

• Expression: Excellent (50 mg/L)

• Purification: Clean, monodisperse

• Crystallization: Success (2.1 Å structure in 2 weeks)

• Activity: 60% of full-length (good enough for structural studies)

• Result: Success

Construct B (311-490):

• Expression: Good

• Crystallization: Success (2.5 Å)

• Result: Second structure (regulatory mechanism revealed)

Total time: 6 weeks for 2 structures (vs 8 months with traditional)

Key lesson: Sometimes splitting multi-domain proteins is better than trying to crystallize them together.

Membrane Proteins: Special Workflow

Membrane proteins require additional considerations.

Extra Steps for Membrane Proteins

1. Predict transmembrane helices:

DeepTMHMM: https://dtu.biolib.com/DeepTMHMM

• Predicts TM helix positions

• Identifies inside/outside orientation

Output:

Protein: 1-450
Signal peptide: 1-25 (remove)
TM1: 35-55
TM2: 70-90
TM3: 110-130
...
TM7: 310-330
C-terminal tail: 331-450 (cytoplasmic, disordered)

2. Define TM boundaries:

Rules:

• Include complete TM helices (don't truncate in middle)

• Start boundary 5-10 residues before first TM helix

• End boundary 5-10 residues after last TM helix

• Remove signal peptide (not needed for recombinant expression)

• Truncate long intracellular/extracellular loops

3. Handle flexible loops:

GPCRs: Intracellular loop 3 (ICL3) is usually 20-80 residues, highly flexible

Options:

• Option A: Remove ICL3 entirely (if not functionally critical)

• Option B: Replace with short linker (GGGGS)

• Option C: Replace with T4 lysozyme (gold standard for crystallization)

Example construct:

GPCR (1-350):
Native: TM1-ICL1-TM2-ECL1-TM3-ICL2-TM4-ECL2-TM5-ICL3-TM6-ECL3-TM7

Optimized construct:
  - Remove signal peptide (1-25)
  - TM1-TM5: 26-229
  - Replace ICL3 with T4L
  - TM6-TM7: 271-320
  - Remove C-terminal tail (321-350)

Final: 26-229-T4L-271-320

Pre-Cloning Checklist

Before you order primers, verify:

Boundary Checklist

• [ ] Disordered termini removed: pLDDT <50 regions truncated

• [ ] Secondary structure intact: No cuts through helices/strands

• [ ] Domain boundaries respected: Pfam domains complete

• [ ] Homolog consensus: Boundaries match successful structures

• [ ] Flexible loops assessed: Internal disorder identified

• [ ] Active site included: All catalytic residues present

• [ ] Termini accessible: N/C-termini surface-exposed (for tags)

Tag Design Checklist

• [ ] Tag placement chosen: N- or C-terminal (based on structure)

• [ ] Cleavage site included: TEV, PreScission, or SUMO protease

• [ ] Tag won't block active site: Checked in structure viewer

• [ ] Tag won't block oligomerization: Checked dimer interface

Expression Design Checklist

• [ ] Codon optimized: For expression host (E. coli, insect, mammalian)

• [ ] Rare codons avoided: Check codon usage

• [ ] Signal peptide handled: Removed (if not needed) or kept (if required)

• [ ] PTMs considered: Express in appropriate system (mammalian for glycosylation)

Construct Set Checklist

• [ ] 2-3 variants designed: Conservative, optimal, aggressive

• [ ] Boundaries differ by 5-10 residues: Small variations to test

• [ ] Stability estimated: Predicted Tm for each (if using Orbion)

• [ ] Testing order decided: Start with optimal construct

Common Mistakes to Avoid

Mistake 1: Not Checking Homologs

Problem: You design boundaries based only on disorder prediction, ignore PDB

Result: Your boundaries don't match proven successful constructs

Fix: Always BLAST against PDB, check what worked for homologs

Mistake 2: Cutting Through Secondary Structure

Problem: You truncate at residue 250 because it's "round number"

Result: You cut through C-terminal helix, protein unstable

Fix: Always visualize structure, truncate in loops only

Mistake 3: Removing Too Much

Problem: AlphaFold shows pLDDT 60-70 at termini, you remove it

Result: Protein less stable (removed stabilizing element)

Fix: Be conservative with "gray zone" (pLDDT 60-80), test longer construct first

Mistake 4: Ignoring Tag Placement

Problem: You always put His-tag at N-terminus (habit)

Result: Tag blocks active site, protein appears inactive

Fix: Check structure, place tag away from functional sites

Mistake 5: Not Testing Multiple Constructs

Problem: You design one "perfect" construct

Result: It fails, you don't have backup

Fix: Always design 2-3 variants, test in parallel

Advanced: When Standard Boundaries Fail

Problem: All Constructs Aggregate

You've tried:

• Optimal boundaries (removed disorder)

• Conservative boundaries (kept more)

• Aggressive boundaries (minimal)

• All aggregate during expression or purification

Solutions:

1. Try fusion tags (MBP, GST, SUMO):

• MBP most effective for aggregation

• Express as MBP-fusion, cleave after purification

2. Change expression system:

• If E. coli fails → Try insect cells (Sf9/High Five)

• If insect fails → Try mammalian (HEK293, CHO)

3. Lower expression temperature:

• Express at 18°C instead of 37°C

• Slower folding, less aggregation

4. Add chaperone co-expression:

• GroEL/GroES for E. coli

• Helps proper folding

5. Screen for stabilizing mutations (Orbion):

• AstraSTASIS predicts stabilizing point mutations

• Increase Tm by 5-15°C

• More stable = less aggregation

Problem: Protein Soluble But Inactive

You've truncated boundaries, protein expresses and purifies, but has no activity

Diagnosis:

1. Check what you removed:

• Did you remove part of active site?

• Did you remove regulatory domain needed for activity?

• Did you remove cofactor binding site?

2. Compare with full-length:

• Express longer construct (more conservative boundaries)

• Test activity

• If active → you removed something essential

Solutions:

1. Extend boundaries:

• Add back 10-20 residues at each terminus

• Test which end matters

2. Include regulatory domain:

• If multi-domain, include both catalytic + regulatory

• Accept that you may need fusion approach for crystallization

3. Add back cofactor:

• Some proteins need metal ions (Zn²⁺, Mg²⁺) or organic cofactors

• Supplement during expression/purification

Key Takeaway

Construct boundary design is systematic engineering, not guesswork:

The 6-step workflow:

1. Get structure prediction (AlphaFold)

2. Predict disorder (IUPred, DISOPRED)

3. Analyze domains (Pfam, InterPro)

4. Compare homologs (BLAST + PDB)

5. Design 2-3 constructs (conservative/optimal/aggressive)

6. Validate before cloning (check secondary structure, termini)

Tools:

• Free: 4-6 hours, good for learning

• Orbion: 15 minutes, integrated workflow, Tm prediction

When to use fusions:

• Solubility issues → MBP, GST, SUMO

• Crystallization issues → T4L, BRIL

• Conformational heterogeneity → Nanobodies

Success rate:

• Traditional (trial-and-error): 15-25% first-construct success

• AI-driven (predicted boundaries): 60-80% first-construct success

The paradigm shift is here. Stop guessing. Start predicting.