The utilization of wheat germ extracts in cell-free protein expression traces back to 1973 when Roberts and Paterson introduced an S-30 extract that efficiently translated RNA from various sources. Despite early limitations such as RNase activity and batch variations, wheat germ extracts proved invaluable in protein research. Over the last 15 years, a highly potent wheat germ protein synthesis system has been developed, overcoming these limitations and becoming a go-to method for various applications, including antigen production, protein structure determination, and functional assays.
Related Products
The Wheat Germ System
Researchers from the Endo group at Ehime University in Japan were pioneers in investigating the instability of wheat germ extracts. Their observation that contaminants like RNA N-glycosidase tritin, thionin, ribonucleases, deoxyribonucleases, and proteases mainly originated from the endosperm led to the development of a protocol for removing endosperm contaminations from wheat germs. Through extensive washing of wheat embryos, stable extracts with high translation activity were obtained. These wheat germ extracts were then applied to establish a general cell-free protein expression system, particularly for high-throughput proteomics.
The system introduced an optimal expression vector (pEU) and a special primer set for direct template preparation by PCR, using the natural omega (Ω) translational enhancer from tobacco mosaic virus. This approach facilitated the preparation of RNA transcripts that didn't require 5'-capping and a poly(A) tail for efficient translation. The translation efficiency was found to be dependent on the length of the 3'-untranslated region, protecting RNA against degradation by 3′ to 5′ exonucleases.
Fig. 1 Wheat germ protein expression system (Harbers M., 2014).
The wheat germ system employs two "linked" reactions, separating RNA preparation and protein translation. Despite requiring more setup time, this approach allows for optimal reaction conditions, complex translation reactions, and the use of additives without interfering with transcription. The "bilayer" reaction format, where substrate buffer forms two separate layers on top of the translation mixture, enables a diffusion-controlled translation process, yielding substantially higher protein amounts compared to batch reactions. This format is flexible, suitable for screening different additives, and can be automated for effective protein synthesis and template screening. For preparative protein preparations, various methods, such as dialysis or repeated "re-feeding" using an automated device, have been developed to supply the translation reaction effectively with substrates and RNA templates over an extended period, removing inhibitory byproducts.
Applications of Wheat Germ System
Human Proteins
The wheat germ system emerges as a powerful platform for expressing human proteins, surpassing traditional methods in solubility rates. Studies comparing E. coli systems with the wheat germ approach demonstrated a significantly higher success rate for human protein expression in the latter. The "Human Protein Factory" project in Japan exemplifies this, generating a vast collection of human entry clones and achieving high success rates and solubility for expressed proteins. The wheat germ system's capacity to express large numbers of human proteins has applications in structural studies, autoantibody characterization, and protein interaction studies.
Arabidopsis
The wheat germ system has demonstrated its value in plant protein analysis, leveraging cDNA clones from the RIKEN Arabidopsis collection. This system has proven instrumental in expressing and analyzing proteins, notably kinases and transcription factors. It offers insights into autophosphorylation activity, calcium-dependent kinase function, and DNA binding. The system's advantages for structural studies on Arabidopsis proteins establish it as a preferred choice, especially in NMR-based structural proteomics studies.
Malaria Research and Vaccine Development
In malaria research, vaccine development is crucial for global public health. The wheat germ system's success in expressing soluble and biologically active forms of malaria proteins offers a breakthrough. This allows the analysis of numerous proteins for vaccine and therapeutic development, enabling proteome-wide antibody profiling to study malaria protein antigenicity. The system has also been used at the Seattle Structural Genomics Center for Infectious Diseases (SSGCID) to express proteins from diverse organisms, overcoming limitations in E. coli expression. Examples include the production of hemagglutinin-neuraminidase for a nasal vaccine against human parainfluenza virus type 3 and functional non-structural proteins NS3 and NS5 from dengue virus. The wheat germ system, in combination with advancements in virus-like particle preparation, holds promise for enhancing vaccine stability and antigenicity, contributing to future vaccine development efforts.
In summary, the wheat germ cell-free expression system has expanded beyond its traditional use, finding application in diverse research areas, from genome-wide studies to vaccine development. Its efficiency, high success rates, and flexibility in expression conditions contribute to its significance in advancing scientific endeavors.
References
- Harbers M. Wheat germ systems for cell-free protein expression. FEBS Letters. 2014, 588(17): 2762-2773.
- Endo Y., Sawasaki T. High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system. Biotechnology Advances. 2003, 21(8): 695-713.
.
