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

Cell Line	Species	Origin	Use
Sf9	Spodoptera frugiperda	Fall Army Worm ovary	Transfection, V0/V1 production
Sf21	Spodoptera frugiperda	Fall Army Worm ovary	V1 production, expression
Hi5	Trichoplusia ni	Cabbage Looper ovary	Large-scale expression

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

Cell Line	Advantages	Best For
Hi5	Highest yields typically	Most proteins
Sf9	Consistent, robust	Membrane proteins, difficult targets
Sf21	Good yields	General use

Troubleshooting

Problem	Cause	Solution
No white colonies	Plasmid issue, dead cells	Check plasmid, make fresh competent cells
Arc during electroporation	Salt in DNA	Re-precipitate DNA in water
Low transfection (no YFP)	Bad bacmid DNA, old reagents	Re-isolate bacmid, fresh X-tremeGene
Cells die immediately	V0 too concentrated	Use less V0 for V1
Low expression	Poor virus, wrong cell line	Re-make virus, try different cell line
Protein degraded	Harvested too late	Harvest earlier, add protease inhibitors

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>

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Last updated: 2026-01-17