Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Enhanced In Vitro Protein Synthesis Through Optimal Design of PCR Primers

  • Ahn Jin-Ho (Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University) ;
  • Son Jeong-Mi (School of Chemical and Biological Engineering, College of Engineering, Seoul National University) ;
  • Hwang Mi-Yeon (School of Chemical and Biological Engineering, College of Engineering, Seoul National University) ;
  • Kim Tae-Wan (School of Chemical and Biological Engineering, College of Engineering, Seoul National University) ;
  • Park Chang-Kil (Department of Fine Chemical Engineering and Chemistry, Chungnam National University) ;
  • Choi Cha-Yong (Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University, School of Chemical and Biological Engineering, College of Engineering, Seoul National University) ;
  • Kim Dong-Myung (Department of Fine Chemical Engineering and Chemistry, Chungnam National University)
  • Published : 2006.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

The functional stability of mRNA is one of the crucial factors affecting the efficiency of in vitro translation. As the rapid degradation of mRNA in the cell extract (S30 extract) causes early termination of the translational reactions, extending the mRNA half-life will improve the productivity of the in vitro protein synthesis. Thus, a simple PCR-based method is introduced to increase the stability of mRNA in an S30 extract. The target genes are PCR-amplified with primers designed to make the ends of the transcribed mRNA molecule anneal to each other. When compared with normal mRNA, the mRNA with the annealing sequences resulted in an approximately 2-fold increase of protein synthesis in an in vitro translation reaction. In addition, sequential transcription and translation reactions in a single tube enabled direct protein expression from the PCR-amplified genes without any separate purification of the mRNA.

Keywords

References

  1. Betton, J. M. 2004. High throughput cloning and expression strategies for protein production. Biochimie 86: 601-605 https://doi.org/10.1016/j.biochi.2004.07.004
  2. Braun, P. and J. LaBaer. 2003. High throughput protein production for functional proteomics. Trends Biotechnol. 21: 383-388 https://doi.org/10.1016/S0167-7799(03)00189-6
  3. Davanloo, P., A. H. Rosenberg, J. J. Dunn, and F. W. Studier. 1984. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81: 2035- 2039
  4. DiTursi, M. K. W., J. H. Chung, M. R. Newman, and J. S. Dordick. 2004. Simultaneous in vitro protein synthesis using solid-phase DNA template. Biotechnol. Prog. 20: 1705- 1709 https://doi.org/10.1021/bp049772f
  5. Heo, M. S., J. H. Kim, S. H. Park, G. J. Woo, and H. Y. Kim. 2004. Detection of genetically modified maize by multiplex PCR method. J. Microbiol. Biotechnol. 14: 1150-1156
  6. Kang, T. J., H. K. Song, J. H. Ahn, C. Y. Choi, and H. Joo. 2003. Optimization of programmed suppression in a cellfree protein synthesis system with unnatural amino acid S- (2-nitrobenzyl)cysteine. J. Microbiol. Biotechnol. 13: 344- 347
  7. Katzen, F., G. Chang, and W. Kudlicki. 2005. The past, present and future of cell-free protein synthesis. Trends Biotechnol. 23: 150-156 https://doi.org/10.1016/j.tibtech.2005.01.003
  8. Kim, D. M., T. Kigawa, C. Y. Choi, and S. Yokoyama. 1996. A highly efficient cell-free protein synthesis system from Escherichia coli. Eur. J. Biochem. 239: 881-886 https://doi.org/10.1111/j.1432-1033.1996.0881u.x
  9. Kim, T. K., H. D. Shin, M. C. Seo, J. N. Lee, and Y. H. Lee. 2003. Molecular structure of PCR cloned PHA synthase genes of Pseudomonas putida KT2440 and its utilization for medium-chain length polyhydroxyalkanoate production. J. Microbiol. Biotechnol. 13: 182-190
  10. Lee, K. and S. N. Cohen. 2001. Effects of 3' terminus modifications on mRNA functional decay during in vitro protein synthesis. J. Biol. Chem. 276: 23268-23274 https://doi.org/10.1074/jbc.M102408200
  11. Matzura, O. and A. Wennborg. 1996. RNAdraw: An integrated program for RNA secondary structure calculation and analysis under 32-bit Microsoft Windows. Comput. Appl. Biosci. 12: 247-249
  12. Mergulhao, F. J. M., G. A. Monterio, J. M. S. Cabral, and M. A. Taipa. 2004. Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. J. Microbiol. Biotechnol. 14: 1-14 https://doi.org/10.1159/000076921
  13. Nierlich, D. P. and G. J. Murakawa. 1996. The decay of bacterial messenger RNA. Prog. Nucleic Acid Res. Mol. Biol. 52: 153-216 https://doi.org/10.1016/S0079-6603(08)60967-8
  14. Rungpragayphan, S., H. Nakano, and T. Yamane. 2003. PCR-linked in vitro expression: A novel system for highthroughput construction and screening of protein libraries. FEBS Lett. 540: 147-150 https://doi.org/10.1016/S0014-5793(03)00251-5
  15. Shaw, W. V. 1975. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Meth. Enzymol. 43: 737-755
  16. Tang, J. Y., J. Temsamani, and S. Agrawal. 1993. Selfstabilized antisense oligodeoxynucleotide phosphorothioates: Properties and anti-HIV activity. Nucleic Acids Res. 21: 2729-2735 https://doi.org/10.1093/nar/21.11.2729
  17. Tohda, H., N. Chikazumi, T. Ueda, K. Nishikawa, and K. Watanabe. 1994. Efficient expression of E. coli dihydrofolate reductase gene by an in vitro translation system using phosphorothioate mRNA. J. Biotechnol. 34: 61-69 https://doi.org/10.1016/0168-1656(94)90166-X
  18. Verheijen, J. C., A. M. van Roon, N. J. Meeuwenoord, H. R. Stuivenberg, S. F. Bayly, L. Chen, G. A. van der Marel, P. F. Torrence, et al. 2000. Incorporation of a 4-hydroxy-Nacetylprolinol nucleotide analogue improves the 3'-exonuclease stability of 2'-5'-oligoadenylate-antisense conjugates. Bioorg. Med. Chem. Lett. 10: 801-804 https://doi.org/10.1016/S0960-894X(00)00100-1
  19. Yoshizawa, S., T. Ueda, Y. Ishido, K. Miura, K. Watanabe, and I. Hirao. 1994. Nuclease resistance of an extraordinary thermostable mini-hairpin DNA fragment, d(GCGAAGG), and its application to in vitro protein synthesis. Nucleic Acids Res. 22: 2217-2221 https://doi.org/10.1093/nar/22.12.2217