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.
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.
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:
- Inoculate E. coli BL21(DE3) pLysS/(cTnC 1-89) in Luria Bertani Broth (LB) containing Carbenicillin (Carb) and Chloramphenicol (Chl).
- Grow at 37 °C with shaking at 250 rpm until slightly turbid.
- Transfer 1mL to 50 mL of LB/Carb/Chl and grow at 37°C. Harvested cells at OD600 nm ~ 0.9 by centrifugation.
- Harvest cells at OD600 nm ~ 0.9 by centrifugation.
- 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 |
- Inoculate 50 mL starter culture into 1 L of minimal media supplemented with 10% ISOGRO.
- Grow at 37 °C with shaking at 250 rpm.
- Induce at OD600 nm ~ 0.9 by the addition of isopropyl b-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM.
- Harvest cells eight hours post-induction.
- 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.
