RTS 500 E. coli Disulfide Kit

he RTS 500 E. coli Disulfide Kit is the only in vitro protein expression system that can express properly folded disulfidebonded proteins that cannot be produced under reducing conditions. It provides an optimized combination of lysate, reagents, chaperones, and buffer systems to produce maximum yields of active multiple-disulfide-bonded protein in a CECF (Continuous-Exchange Cell-Free) system.
The kit employs several innovative technologies:

• Coupled transcription/translation system; no mRNA isolation necessary
• Optimized high-yield (HY) E. coli lysate
• Redox buffer to maintain the system under oxidizing conditions, which enhances disulfide-bond formation
• Disulfideisomerase for rearrangement of disulfide bonds, which enhances yields of correctly folded proteins
• Increased amounts of GroE chaperones, which produce higher yields of soluble, properly folded proteins

It utilizes Continuous-Exchange Cell-Free (CECF) technology, which prolongs the reaction time for at least 24 hours and therefore enhances the yield of produced proteins. The kit contains reagents for 5 reactions (1 ml each) that are run separately. Transcription, translation, and disulfide-bond formation take place in the 1 ml reaction compartment of the reaction device. Substrates and energy components needed for a sustained reaction are continuously supplied via a semipermeable membrane. At the same time, potentially inhibitory reaction by-products are diluted via diffusion through the same membrane into the 10 ml feeding chamber (see figure). Depending on the particular protein, expression of up to several milligrams of protein per 1 ml reaction can be obtained (e.g., >2 mg/ml of active urokinase).