Wheat Germ Extract Plus is a highly efficient, single-tube system designed to express proteins in the range of 10–80µg/ml using a batch mode. In a dialysis format yields can be as high as 250µg/ml. The extract contains all the cellular components necessary for protein synthesis (tRNA, ribosomes, amino acids, and initiation, elongation and termination factors). Potassium acetate is also included at a concentration to enhance translation for a wide range of mRNAs. Protein synthesis is initiated by addition of the appropriate mRNA template and the reaction is incubated for 2 hours at 25°C. Synthesized proteins can be analyzed by SDS-polyacryamide gel electrophoresis or used directly in numerous applications.
The pF3A WG (BYDV) and pF3K WG (BYDV) Flexi® Vectors are specially designed for in vitro expression of protein in wheat germ extract. These vectors contain two rare-cutting restriction enzyme sites for Sgf I and Pme I that facilitate directional cloning and transfer of protein-coding sequences between a variety of Flexi Vectors. Sequences from the barley yellow dwarf virus (BYDV), an RNA plant virus, are inserted upstream and downstream of the protein coding region of interest. These BYDV upstream and downstream elements interact with each other, form a closed loop and act synergistically to stimulate translation in wheat germ extracts, bypassing mRNA cap and polyadenylation dependencies.
Wheat Germ Extract Plus (Technical Manual TM066)
Figure 1. Wheat Germ Extract Plus protocol overview.
Figure 2. Comparison of Wheat Germ Extract Plus and Wheat Extract reactions.
Figure 3. Procaspase-3 was expressed in Wheat Germ Extract Plus using either batch or dialysis mode. After activating the procaspase-3 enzyme with caspase-8 the extracts were assayed using the Caspase-Glo® 3/7 Assay (Cat.# G8091). Assuming equivalent activities of the recombinant caspase-3 standard and the proenzyme, the batch mode produces 34µg/ml and the dialysis mode produces 120µg/ml. 1. Batch mode: Wheat Germ Extract Plus expressed procaspase-3; activated with caspase-8. 2. 7hr Dialysis: Wheat Germ Extract Plus expressed procaspase-3; activated with caspase-8. 3. Overnight Dialysis: Wheat Germ Extract Plus expressed procaspase-3; activated with caspase-8.
Figure 4. Solubility of proteins expressed in cell-free extracts. Solubility of the expressed protein was evaluated by comparing the labeled protein band on a SDS-PAGE before and after centrifugation. The reactions were centrifuged at 14,000rpm. Lanes 13, 14, and 15 refer to samples before centrifugation. Lanes 13*, 14*, and 15* refer to samples after centrifugation. Panel A. Proteins expressed using E. coli T7 S30 Extract (Cat.# L1130). Panel B. Proteins expressed using Wheat Germ Extract Plus (Cat.# L3250).
Wheat Germ Extract Plus (Cat.# L3250 & L3251)
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