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.
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.