- Generates Increased Levels of Soluble, Heterologously Expressed Proteins E. coli Containing the T7Lac Promoter
- Works on Insoluble Proteins
- Enables Expressed Proteins to Fold Properly and Remain Soluble
HyperINDUCE™ is an innovative alternative to IPTG that increases the level of soluble, heterologously expressed protein in E. coli containing the T7lac promoter. By decreasing the rate of expression , HyperINDUCE enables expressed protein to fold properly and remain soluble thereby reducing the formation of insoluble inclusion bodies.
The simplest method for improving yield of soluble protein in E. coli is to reduce the rate of expression. Altering incubation temperature and IPTG concentrations helps, but only marginally. A better alternative for reducing expression rates is to use HyperINDUCE with its lower affinity for the laci repressor compared with IPTG. Therefore the rates of expression are slower with HyperINDUCE resulting in an increased level of soluble protein.
The HyperINDUCE is provided at a concentration of 2 mg/mL and a 10X concentrate. A concentration that favors the formation of soluble protein must be determined experimentally, and usually ranges from 2 to 6 g/L.
Determining HyperINDUCE concentration for optimal protein solubility: Pick individual bacteria colonies into 4-6 mL LB or NYZ medium plus the appropriate antibiotic. Incubate in a shaker (250 RPM) at 37°C overnight or until the culture obtains an O.D.=0.6-0.8 is obtained. Cool the culture on ice to between 15-20°C then split the volume equally into three flasks. Add the HyperINDUCE to a final concentration ranging from 1.0-6.0 g/L. Also add the same amount of antibiotic to the culture that was used initially. Incubate in a shaker (RPM 125) at room temperature for at least 12 hours or overnight. After incubation, lyse cells and isolate the soluble and insoluble fractions according to standard protocols. Determine the amount of soluble verses insoluble protein by visualizing whole cell lysate fractions using denaturing gel electrophoresis and the appropriate staining methods.