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Limitations of the recombinant protein expression system Cell-free translation systems Improvement of the conventional wheat germ cell-free translation system to meet the proteomics demand Publications Brochure (PDF 5.30 MB)	Wheat Germ Cell-Free Protein Synthesis System Established by CSTRC Limitations of the recombinant protein expression system Currently, three strategies are being used for protein production: chemical synthesis, in vivo expression, and cell-free protein synthesis. While chemical synthesis is not practical for the synthesis of longer peptides, and in vivo expression systems have several inherent limitations. Although E. coli recombinant system seems to be the best option in terms of economy and ease of protein production, eukaryotic proteins expressed often get precipitated and following time consuming post treatment is inevitable to solubilize and refolding of those proteins. Mammalian cell expression systems, can be effective tool, but are plagued by difficulties in purifying recombinant proteins, limitations on the size of the recombinant protein expressed, expression time and unstable protein expression (3-5). Another common and even more serious disadvantage of in vivo expression system is that they are limited to produce the proteins that do not interfere with host cell physiology (1-2). Overall, protein production by using in vivo systems have got limitations either in terms of producing high-throughput production of proteins, the functionally active proteins, low yield, economy and/or ethical issues. Cell-free translation systems Cell-free translation systems, can synthesize proteins with high speed and accuracy, approaching in vivo rates (3-4), and they can express proteins that seriously interfere with cell physiology. However, these systems are generally unstable and thus inefficient (5). The productivity of the E.coli cell-free translation system has been well acknowledged, however due to its prokaryotic nature of the translation mechanism, proper protein folding remains a substantial challenge, especially for the synthesis of multi-domain proteins from eukaryotic genome. Though attempts are being made to synthesize the eukaryotic proteins by E.coli cell-free system after optimizing the systems with the addition of external additives and molecular chaperones (6), complexity with these additives increase when multiple protein synthesized paralally. On the other hand, rabbit reticulocyte lysate cell-free expression system, has not been adapted to provide preparative amounts of synthesized proteins due to its low efficiency, unstability, high ribonuclease content, high content of endogenous globin and ethical issues. Many of these problems can be circumvented by using wheat germ cell-free translation system. As the system is derived from eukaryotic plant source, can be more convenient and promising cell-free translation system, and it would have been especially powerful to synthesize eukaryotic multi-domain proteins in folded state on a preparative scale. However the conventional wheat germ system was plagued with the short life. We have been actively involved in improving the wheat germ cell-free translation system by understanding the system from fundamental as well as technological view. Improvement of the conventional wheat germ cell-free translation system to meet the proteomics demand In order to potentially improve the wheat germ cell-free translation to meet the high-throughput needs of proteomics, we addressed the several critical parameters and solved them as follows. 1. Understanding the cause for the instability of wheat germ cell-free translation system over the time. During our early investigations, we found that plants contain endogenous inhibitors of translation (7) and demonstrated that elimination of these inhibitors led to an extraordinarily stable and efficient translation system (8). 2. To improve the performance of the system further, several critical design parameters considered (9): (a) elimination of the 5'-7mGpppG (cap) and poly(A)-tail (pA), by optimizing the 5'- and 3'- UTRs of mRNA, thereby increasing translation initiation; (b) designing the split-primers for PCR to generate transcription templates directly from E. coli cells carrying cDNAs, thus bypassing the time-consuming cloning steps; (c) construction of an expression vector, specialized for the massive production of proteins. 3. Alternative to Spirin's continuous-flow cell-free protein syntheis system method of cell-free translation by using innovative bilayer (10) method without using any membrane and, 4. Assembling of the potentially improved wheat germ cell-free translation system into robotic automation (11). The resulting system exhibits following attractive features: (i) It is stable over long periods of time and with the new expression vector it can produce proteins in amounts of several milligrams per ml of reaction volume (8-9, 12). (ii) It is amenable to high-throughput parallel protein synthesis (9,11). (iii) Both functional and structural tests on some of the protein products suggest that the system can produce proteins that fold into their natural states (13-14). These features may prove to be essential for the systematic study of protein structure-function relations, and a series of applications delineating the scope of modern proteomics. References 1. Henrich, B. et al. (1982) Mol. Gen. Genet. 185, 493-497. 2. Golf, S. A. et al. (1987) J. Biol. Chem. 262, 4508-4515. 3. Chrunyk, B. A. et al. (1993) J. Biol Chem. 268, 18053-18061. 4. Kurland, C. G. (1982) Cell 28, 201-202. 5. Roberts, B. E. et al. (1973) Pro. Natl. Acad. Sci. U.S.A. 70, 2330-2234. 6. Ryabova, L. A. et al. (1997) Nat Biotechnol 15, 79-84. 7. Ogasawara, T. et al. (1999) EMBO J. 18, 6522-6531. 8. Madin, K. et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 559-564. 9. Sawasaki, T. et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14652-14657. 10. Sawasaki, T. et al. (2002) FEBS Lett. 514, 102-105. 11. Endo, Y. et al.,. J. Struct. Funct. Genomics. In press (2004) 12.Madin, K. et al. (2004) FEBS Lett. 562, 155-159. 13.Morita, E. H. et al. (2003) Protein Science, 12, 1216-1221. 14. Vinarov. D. A. et al. (2004) Nature Methods, 1, 1-5.
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