The RTS 9000 ProteoMaster E. coli HY Kit contains reagents and a Reaction Device for large-scale protein expression, and is designed for use with the Eppendorf Thermomixer* or the RTS ProteoMaster Instrument. It utilizes a biochemically enhanced high-yield (HY) E. coli lysate and aCECF Reaction Device (Figure 2), which allows an immediate ten-fold scale-up of protein production from one RTS 500 HY reaction.

Figure 1: RTS 9000 Reaction Device. The disposable device contains a 10-ml reaction compartment and a 100-ml feeding compartment. The reaction compartment is separated from the feeding compartment by a semi-permeable membrane.

Produce up to 50 mg protein per reaction with a reagent kit that contains everything you need to perform a 10 ml coupled transcription/translation reaction.
Due to the production of preparative-levels of protein, sufficient amounts for various downstream applications can be obtained using the RTS 9000 E. coli HY Kit. It is also suitable for the production of protein for NMR spectroscopy or X-ray crystallography. The kit design allows convenient and easy labeling of proteins by exchanging the provided amino acids with amino acids mixtures of your choice. Methionine (included in the kit) can be easily exchanged for seleno-methionine (not included). Other amino acids can be exchanged by preparing tailored amino-acid mixtures.
The RTS 9000 Reaction Device is designed for use with the Eppendorf Thermomixer* or theRTS ProteoMaster Instrument which provides ideal conditions for the RTS reaction and guarantees efficient protein expression by continuous agitation of the device.

Important Note: We recommend selecting suitable expression clones and optimizing expression conditions with the RTS 100 and 500 E. coli HY Kits prior to scaling-up using the RTS 9000 E. coli HY Kit. The protein production can be scaled-up from the RTS 500 HY product platform to the RTS 9000 without further optimization (Figure 2).

Figure 2: Coomassie-stained SDS-PAGE analysis of different proteins expressed using the RTS 500 and 9000 E. coli HY Kits.
a: RTS 9000 E. coli HY expression; b: RTS 500 E. coli HY expression
CAT: Chloramphenicol Acetyltransferase; β-Gal: β-Galactosidase; GFP: Green Fluorescent Protein;
RHO: Rhodanese; rPA: recombinant Plasminogen Activator; CEC: GFP-(CecoprinP1)2;
PGA: Penicillin G Amidase, β-UE; LUC: Firefly Luciferase; MBP-EPO: MBP-Erythropoietin

Description

One RTS 9000 reaction produces ten times more total protein yield than a RTS 500 reaction. This higher total yield is achieved by increasing the reaction volume by a factor of ten, while the productivity per ml lysate in the RTS 500 HY and RTS 9000 HY reactions is similar (Figure 2).
The RTS 9000 Reaction Device (Figure 1) is composed of a reaction compartment and a feeding compartment. The 10-ml reaction compartment, where the coupled transcription/translation reaction takes place, is separated from the 100-ml feeding compartment by a semipermeable membrane (cut-off 10 kD). The plasmid that contains the gene of interest serves as a template for the transcription. An optimized E. coli lysate contains tRNAs, ribosomes, enzymes, factors, and other necessary components required for highly efficient translation. The feeding compartment continuously supplies ions, energy substrates, nucleotides, and amino acids through the semipermeable membrane. Simultaneously, by-products that may inhibit the reaction are diluted through the same membrane into the feeding compartment. This continuous exchange of substrates and by-products across the two semipermeable membranes (CECF technology) allows protein synthesis to continue for up to 24 hours.

Read more about RTS E.coli System and Technology

*See instrument compatibility table and the Eppendorf ThermoMixer Quickreference

Former Roche customers please click here to see the product code cross reference table.