Barrick Lab :: ProtocolsLargeScaleProteinExpressionEColi

---++ 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
Barrick Lab > ProtocolList > ProtocolsLargeScaleProteinExpressionEColi
Contributors to this topic
ColinBrown
Topic revision: r1 - 2016-06-28 - 15:22:23 - Main.ColinBrown