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
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Blue/white selection (gent + X-Gal + IPTG)
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Isolate bacmid DNA (alkaline lysis)
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Transfect adherent Sf9 → V0 (2-4 days)
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Infect suspension Sf9/Sf21 → V1 (DPA + 48-72 hrs)
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Large-scale expression (300-600 mL Hi5/Sf9/Sf21)
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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