Large-scale Protein Expression in E. Coli:

Notes:

• This can be applied to either soluble proteins (for a downstream prep in native conditions) or insoluble proteins in inclusion bodies (downstream denaturing prep)
• Various expression systems can be used (IPTG, Arabinose, etc.?), the basic process is the same
• Good resources for additional information: *The QiaExpressionist ([LINK])

Supplies:

• 1L of appropriate media (LB can be used, 2xYT is richer and gives better growth; no experience with minimal media)
• Agar plates (LB or 2xYT) w/ appropriate antibiotics
  • can add 0.2% glucose to cut down on leakiness of IPTG-inducible expression systems
• Expression construct in an appropriate strain
  • ALWAYS good practice to get full insert sequence before starting experiment
  • Make sure host strain contains appropriate components for chosen expression system (e.g. T7 polymerase for T7-driven constructs)
  • If expressing a protein for the first time, good practice to do smaller-scale test expressions first (see protocol [XXX]) to determine toxicity & solubility of protein
• 2L Baffle Flask or 4L Standard
  • Want to aim for 1/4 - 1/5 volume of the flask to get good aeration; can go up to 1/2 volume with baffle flasks
• Shaking incubator, appropriately-sized flask clamps
• 100mM IPTG (or other as appropriate for expression system)

DAY 1:

1. Revive culture from frozen stock on a 2xYT plate w/ appropriate antibiotics
   • I usually add 0.2% glucose to the plates; can help prevent growth problems, esp. w/ toxic proteins
2. Grow overnight at 37degC

DAY 2:

1. Pick a single colony from the overnight plate, and innoculate a 60mL (1/20 vol + 10mL) culture of the expression media + antibiotics
   • I usually add 0.2% glucose to these as well
2. Grow overnight (~16h) at 37degC *(optional) Put (sterile) growth media in 37C (non-shaking) incubator to warm overnight

DAY 3:

1. Add antibiotic(s) to pre-warmed media, and innoculate with 50mL (1/20 vol) of the overnight starter culture
   • Save 1mL samples of pre-innoculated (blank) and 0h (just after innoculation) for OD600 measurements (should be 0.1-0.3)
2. Put flask in 37degC incubator, shaking at 275 RPM
3. Measure OD600 every 30-60 minutes
4. When OD600 reaches 0.4-0.6, add 10mL (1/100 vol) IPTG to a final concentration of 1mM
   • Before adding IPTG, make sure to save 50uL of the culture for the pre-induction lane on the gel later. Add SDS-PAGE sample buffer directly to this sample (i.e. don't spin down).
5. Continue growth at 37degC, 275RPM for 4h
6. Remove flask, and spin down cells in 500mL centrifuge bottle
   • save 50uL of culture for the post-induction on the gel.
   • can centrifuge in two batches to get all cells in the same bottle, or resuspend in Saline / PBS and transfer to another container
7. Freeze cell pellet:
   • For soluble proteins:
     • Freeze cell pellet in liquid nitrogen & store at -80C
   • For insoluble proteins / inclusion bodies:
     • Store bottle / pellet at -20 or -80degC
8. Run pre- and post-induction samples on an SDS-PAGE gel (see [Protocol]); expressed protein should be visible as a heavy band of the appropriate MW present only in the post-induction sample