insect-cell

name: insect-cell description: Baculovirus expression system for recombinant protein production. Use when the user asks about insect cell expression, baculovirus, V0/V1 production, Sf9/Hi5/Sf21 cells, DH10αEMBacY transfection, or large-scale protein expression.

Insect Cell Expression Skill

Baculovirus expression system for recombinant protein production.

Cell Lines

Media: ESF-921 (serum-free, Expression Systems)

All lines can be grown as suspension or adherent cultures.

Baculovirus System Overview

Virus: AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus)

• ~130 kb dsDNA genome
• Cannot infect human cells
• Modified into bacmid for easy gene insertion

Bacmid host: DH10αEMBacY

• Contains AcMNPV bacmid + Tn7 transposase helper plasmid
• YFP reporter under PolH promoter (tracks infection)
• Selection: kanamycin (bacmid), tetracycline (helper)

Vector system: 438 series (MacroLab, UC Berkeley)

• LIC cloning, multiple tagging options
• Tn7-mediated integration into bacmid
• Gentamycin resistance after integration
• PolH promoter drives expression

Workflow Overview

438 vector with GOI
       ↓
Electroporate into DH10αEMBacY
       ↓
Blue/white selection (gent + X-Gal + IPTG)
       ↓
Isolate bacmid DNA (alkaline lysis)
       ↓
Transfect adherent Sf9 → V0 (2-4 days)
       ↓
Infect suspension Sf9/Sf21 → V1 (DPA + 48-72 hrs)
       ↓
Large-scale expression (300-600 mL Hi5/Sf9/Sf21)
       ↓
Harvest & purify protein

DH10αEMBacY Recombination

Electroporation

1. Add 0.25-1 µg plasmid to 100 µL electrocompetent DH10αEMBacY
   • DNA must be in water (not EB buffer) — salt causes arcing
2. Incubate on ice 10 min
3. Transfer to chilled 0.1 cm cuvette
4. Pulse: 25 µF, 1.8 kV (E. coli program 1)
   • If "arc" warning → too much salt, use less DNA
5. Add 1 mL LB, transfer to culture tube
6. Shake 5 hrs to overnight at 37°C

Selection

1. Plate 25-150 µL on LB agar + gentamycin + X-Gal (150 µg/mL) + IPTG (1 mM)
2. Incubate 1.5 days at 37°C
3. Pick white colonies (integration disrupts LacZ → white)
   • Blue = no integration (negative control)
4. Streak white colonies on fresh plate (gent + X-Gal + IPTG)
5. Inoculate same colony into 5 mL LB-gent

Bacmid Isolation

⚠️ Do NOT use commercial miniprep kits — they shear the large (~140 kb) bacmid DNA. Bacmid must remain supercoiled for efficient transfection.

Alkaline Lysis Protocol

1. Pellet entire 5 mL culture
2. Resuspend in 250 µL resuspension buffer
3. Add 250 µL lysis buffer, invert 3-5×
4. Add 350 µL neutralization buffer, invert 3-5×
5. Spin 15,000×g for 10 min at RT
6. Transfer supernatant to fresh tube
7. Spin again 15,000×g for 10 min (remove remaining precipitate)
8. Add 700 µL isopropanol (-20°C or RT)
9. Invert 3-5×, incubate at -20°C for 1 hr (or -80°C for 1-2 hrs)
10. Spin 15,000×g for 30 min at 4°C
11. Remove supernatant carefully
12. Wash with 500 µL 70% EtOH
13. Spin 15,000×g for 10 min at 4°C
14. Remove EtOH, add 30 µL 70% EtOH to cover pellet
15. Store at -20°C until transfection

Buffers

See Notion page for buffer recipes: <YOUR_NOTION_PAGE_ID>

Transfection (V0 Production)

Materials

• Bacmid DNA (from above)
• Sf9 cells at 1×10⁶ cells/mL
• ESF-921 medium
• ESF Transfection Medium
• X-tremeGene 9 transfection reagent
• 6-well plate

Protocol

1. Air-dry bacmid pellet in TC hood (remove EtOH)
2. Dissolve in 20 µL sterile water (5-10 min)
3. Transfer 3 µL bacmid to new tube (store remainder at -20°C)
4. Plate 1 mL Sf9 (1×10⁶/mL) per well, incubate 27°C for 30 min
5. Prepare transfection mix:
   • Mastermix: 100 µL ESF Transfection Medium + 6 µL X-tremeGene 9 (per construct)
   • Add 100 µL ESF Transfection Medium to 3 µL bacmid, incubate 5 min
   • Add 100 µL mastermix to bacmid, incubate RT 30 min
6. Add 800 µL ESF-921 to transfection mix
7. Gently remove media from cells
8. Add 1 mL transfection mix to cells
9. Incubate 27°C for 4 hrs to overnight
10. Add 2 mL ESF-921 to prevent drying
11. Check YFP fluorescence after 2-4 days
12. Harvest V0: gently remove media, transfer to 15 mL falcon

V1 Production

Protocol

1. Add 0.15-3 mL V0 to 25 mL Sf9 or Sf21 at 1×10⁶ cells/mL
2. Check after 24 hrs:
   • Cells should divide once then stop
   • If no division → V0 too strong, use less next time
   • Dilute to 1×10⁶/mL if needed
3. Note day of proliferation arrest (DPA)
4. Grow additional 48-72 hrs after DPA
5. Monitor: cell viability, cell count, YFP fluorescence
6. Harvest:
   • Centrifuge 238×g for 15 min
   • Transfer supernatant (V1) to fresh 50 mL falcon
   • Store V1 at 4°C (stable ~1-1.5 years)
7. Test pellet for expression (SDS-PAGE pull-down)

Large-Scale Expression

Setup

• Volume: 300-600 mL per construct
• Cell lines: Hi5, Sf9, or Sf21
• Cell density: 1-2×10⁶ cells/mL at infection

Protocol

1. Infect with V1 virus (amount determined from V1 test expression)
2. Grow 2-5 days post-infection
3. Monitor cell viability and YFP
4. Harvest when viability drops to ~80% or YFP peaks
5. Centrifuge, discard supernatant
6. Flash-freeze pellet or proceed directly to purification

Cell Line Selection

Troubleshooting

Glycerol Stocks

Make glycerol stocks of positive DH10αEMBacY clones for future bacmid isolation:

• 500 µL culture + 500 µL 50% glycerol
• Store at -80°C

Notion Reference

Comprehensive protocol with images: <YOUR_NOTION_PAGE_ID>

Last updated: 2026-01-17