Maximize recombinant protein yields with ISOGRO®

  • Substantially increase recombinant protein expression levels using ISOGRO as a stand-alone media versus M9 media. 
  • Save time and money by using ISOGRO growth media to shorten production time.
  • Stable isotope label sufficient amounts of protein to permit the achievement of high quality NMR spectra of difficult to express proteins.
  • As a standard quality control measure, the suitability of each batch of ISOGRO as a culture medium is determined by comparison with a LB growth curve.
        

 A 39 μM sample of p38 alpha was produced from 50 mL of culture as seen below:

SDSPAGE of p38 growth

Figure 1. SDSPAGE of p38 growth. Left to right – Molecular weight marker, uninduced minimal media, induced minimal media, uninduced ISOGRO, induced ISOGRO; black arrow – p38 alpha.

1H-15N-TROSY-spectrumof-13C-15N-p38-alpha

Figure 2. 1H-15N TROSY spectrum of [13C,15N] p38 alpha collected at 700 MHz. Data provided by Dr. Jeffrey W. Peng, Dept. of Chem/Biochemistry, Univ. of Notre Dame, Notre Dame, Indiana.

Procedure for Growing E. coli using ISOGRO® Powder p38 alpha expression

  1. Dissolve 0.5 g of ISOGRO powder into 45 mL of filtered, sterilized water.
  2. Make stock solutions of the following salts and use the listed quantities in the preparation:
  Stock Amount
K2HPO4
100g/L
0.9 mL
KH2PO4
50g/L
1.4 mL
MgSO4
50g/L
1.0 mL
CaCl2
37g/L
15.0 uL
  1. Adjust the pH to 7.0 with NaOH and bring the solution up to 50 mL.
  2. Add 2.5 mg ampicillin to the solution.
  3. In the positive flow hood, pass the solution through  a 0.22 um filter and transfer the filtrate to a sterilized 500 mL flask. (Used 2 filters and 2 syringes).
  4. Inoculate the culture with a loop of E. coli.
  5. Shake at 37 ºC at 200 rpm.
  6. OD600 nm= 0.36 at 6.5 hours.
  7. OD600 nm= 1.2 at 8 hours.
  8. Save an aliquot for gel: 0.4 mL of 1:10 dilution from OD, spin down and save pellet in -20 ºC.
  9. Change the shaker temperature to 20 ºC, let the cells equilibrate for 30 minutes.
  10. Induce using 25 uL 1M IPTG and incubate overnight at 20 ºC at 225 rpm.
  11. OD600 nm =2.0 at 14 hours after induction.
  12. Save an aliquot: 1:10 dilution from OD, spin down, and save pellet in -20 ºC.

ISOGRO usage results in the production of micromolar amounts of protein from 50 mL of culture.

Materials

     
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