ISOGRO® Amplify Recombinant Protein Expression

  • Decrease lag time by as much as 60%.
  • Maximize OD and recombinant protein expression.
  • Supplement with as little as 10% ISOGRO and improve the production of difficult to express proteins in E. coli.

 

Comparing minimal media supplemented with ISOGRO to minimal media alone

Figure 1. Cardiac troponin cTnC(1-89) in pLysS. Cells grown at 37 °C in shaker flasks. Red curve is ISOGRO supplemented minimal media and black curve is minimal media alone.

SDS-PAGE cTnC(1-89) cell lysates

Figure 2. SDS-PAGE cTnC(1-89) cell lysates. M-low molecular weight marker; 0= uninduced; 1=induced minimal media; 2=induced minimal media + ISOGRO; L=Lysozyme. Data provided by Dr. Paul R. Rosevear The Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati Medical Center, Cincinnati, OH.

Procedure for 10% ISOGRO Supplementation of M9 Minimal Media:

  1. Inoculate E. coli BL21(DE3) pLysS/(cTnC 1-89) in Luria Bertani Broth (LB) containing Carbenicillin (Carb) and Chloramphenicol (Chl).

  2. Grow at 37 °C with shaking at 250 rpm until slightly turbid.

  3. Transfer 1mL to 50 mL of LB/Carb/Chl and grow at 37°C. Harvested cells at OD600 nm ~ 0.9 by centrifugation.

  4. Harvest cells at OD600 nm ~ 0.9 by centrifugation.

  5. Resuspend starter culture in 50 mL of sterile filtered minimal media (pH 7.0) supplemented with 1 g/L of ISOGRO®-13C,15N powder growth medium and containing the following:

 

7 g/L Na2H PO4 1mg/L D-pantothenate
3 g/L KH2PO4 1 mg/L biotin
2.5 g/L NaCl 50 mg/L thiamine
10.5 g/L K2HPO4 1 mg/L pyridoxal phosphate
0.5 g NaOH 50 mg/L niacin
1 g/L 15NH4Cl 1 mg/L folic acid
4 mM MgSO4 100 μg/L riboflavin
10 mM FeCl3 1 mg/L choline chloride
125 mM CaCl2 0.26 μg/L H3BO3
50 mM ZnSO4 2.4 ng/L Na2MoO4•2H2O
2 g/L D-Glucose 13C6 16 ng/L CuCl2•2H2O
107 μg/L MgCl2•6H2O 0.16 μg/L MnCl2•4H2O
20 μg/L FeCl2•4H2O  
  1. Inoculate 50 mL starter culture into 1 L of minimal media supplemented with 10% ISOGRO.

  2. Grow at 37 °C with shaking at 250 rpm.

  3. Induce at OD600 nm ~ 0.9 by the addition of isopropyl b-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM.

  4. Harvest cells eight hours post-induction.

  5. Analyze protein production by SDS-PAGE.

Diminish lag time by > 60% and the number of hours required to attain high cell density by > 45% with the inclusion of 10% ISOGRO in M9.

Materials

     
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