Translation initiation in eukaryotes is a complex process
 
There are two mechanisms of translation initiation:

• Cap-dependent
• Cap-independent

_{The 40 S ribosomal subunit, together with certain eukaryotic initiation factors (eIF‘s) binds to the 5‘ terminal m7GpppN and scans the untranslated region (UTR) until the AUG (protein synthesis initiation) codon is reached. The joining of the 60 S subunit, results in the formation of the 80 S initiation complex, which includes the initiator tRNA.}
 
Translation initiation in RTS Wheat Germ is a cap-independent process
 
In order to improve initiation of protein synthesis, RTS Wheat Germ uses a newly developed IRES- like element, which is, though non-viral, as efficient as the strongest known translational enhancers.

_{Certain mRNAs reveal internal ribosomal entry sites (IRES), usually in the 5‘- UTR. These IRES containing mRNAs are not subject to some of the complex regulatory mechanisms involved in cap-dependent translation. IRES mediated translation initiation is typically found in mRNAs translated under conditions of cellular stress. (F stands for additional protein factor(s), binding to IRES, involved in internal initiation.) For further reading we recommend: Kapp, L.D. & Lorsch, J.R.:The molecular mechanism of eukaryotic translation. Ann. Rev. in Biochem. 73, 657, 2004.}

Transcription and translation in eukaryotic lysates have very different Mg ²⁺ optima. Highly efficient protein synthesis therefore normally requires the separate preparation of mRNA ( at high [Mg ²⁺] ) prior to the translation ( at low [Mg ²⁺] ). Consequently, eukaryotic in vitro coupled transcription-translation can normally not be brought to preparative yields in one and the same reaction.
The RTS Wheat Germ Kits overcome this hurdle by using the Continuous Exchange Cell-Free technology, which allows to go from high to low [Mg ²⁺] in the course of the same reaction via dialysis. As a consequence, yields of up to 1mg per ml are reached. (see Fig. Protein Synthesis Level).
The environment provided by the wheat germ lysate has been shown to be much better suited for the expression of eukaryotic proteins in terms of reaction kinetics and polypeptide folding, leading to much higher success rates and solubilities than those found when using E. coli lysates.

Important notes:

• Like RTS E. coli, RTS Wheat Germ in the absence of further modifications, cannot perform the following post-translational modifications: glycosylation; formation of disulfide bonds; cleavage of signal sequences.
  
  
• In contrast to RTS 100 E. coli HY Kit, it is not recommended to run RTS 100 Wheat Germ reactions in batch (=non-CECF) mode without the RTS ProteMaster Instrument, because under these conditions the expression reaction will stop after a few hours and final protein yields will be significantly lower. Batch mode could only be used with radiolabeled amino acids.

Accelerate protein expression

• The Wheat Germ System does not require rational gene design (ProteoExpert), because of higher initial success rates.
  
  
• Rapidly generate expression templates by PCR with the RTS Wheat Germ Linear Template Generation Set, His₆-tag, and then express them with the RTS 100 Wheat Germ CECF kit and get micrograms of expressed protein per reaction in just one day.
  
  
• Start with circular or linear DNA templates (or with mRNA - ideally capped - *) as described in the Workflow Overview.
  *If the gene of interest is cloned in other T7 promotor containing vectors than RTS pIVEX Wheat Germ, we strongly recommend to generate capped mRNA first, with the T7 Cap Scribe Kit (Cat.No. 1581066), and to add the resulting mRNA exogeneously to the RTS Wheat Germ reaction mix.

Workflow Overview  

Perform high-throughput screening

• Quickly and easily screen many sequences with especially designed array-like CECF devices:

The RTS Wheat Germ reaction kits use CECF technology to reach unprecedented yields in 24 hour reactions.
A two-chamber reaction device allows production of up to 1mg protein per ml. Transcription and translation take place simultaneously in the 50 µl (RTS 100 WG CECF Kit), respectively 1 ml (RTS 500 WG CECF Kit) reaction compartments 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 since they diffuse through the same membrane into the 1ml (RTS 100 WG CECF Kit) or 10 ml (RTS 500 WG CECF Kit) feeding compartments. In contrast to traditional batch systems, CECF conditions allow protein synthesis to continue for 24 hours. Yields may reach up to several hundred µgs of newly synthesised protein, in reactions with PCR-generated linear template. If vectors or exogenously added capped mRNA templates are applied, yields can reach a milligram.

The RTS 100 Wheat Germ CECF Kit - for use in the RTS Proteomaster Instrument to express up to 1 mg of protein /ml.

The RTS 500 Wheat Germ CECF Kit - for use in the RTS Proteomaster Instrument to express up to 1 mg of protein /ml.
RTS 500 Wheat Germ reactions are performed in RTS 500 ProteoMaster devices: