Abstract
In this study, we describe a cell-free protein synthesis consolidated with polymerase chain reaction (PCR)-based synthetic gene assembly that allows for streamlined translation of genetic information. In silico-designed fragments of target genes were PCR-assembled and directly expressed in a cell-free synthesis system to generate functional proteins. This method bypasses the procedures required in conventional cell-based gene expression methods, integrates gene synthesis and cell-free protein synthesis, shortens the time to protein production, and allows for facile regulation of gene expression by manipulating the oligomer sequences used for gene synthesis. The strategy proposed herein expands the flexibility and throughput of the protein synthesis process, a fundamental component in the construction of synthetic biological systems.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Tan, C., P. Marguet, and L. You (2009) Emergent bistability by a growth-modulating positive feedback circuit. Nat. Chem. Biol. 5: 842–848.
Kwok, R. (2010) Five hard truth for synthetic biology. Nature 463: 288–290.
Seo, S. W., S. C. Kim, and G. Y. Jung (2012) Synthetic regulatory tools for microbial engineering. Biotechnol. Bioproc. Eng. 17: 1–7.
Carlson, R. (2009) The changing economics of DNA synthesis. Nat. Biotechnol. 27: 1091–1094.
Tian, J., H. Gong, N. Sheng, X. Zhou, E. Gulari, X. Gao, and G. Church (2004) Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432: 1050–1054.
Leprince, A., M. W. van Passel, and V. A. dos Santos (2012) Streamlining genomes: Toward the generation of simplified and stabilized microbial systems. Curr. Opin. Biotechnol. 23: 651–658.
Matzas, M., P. F. Stahler, N. Kefer, N. Siebelt, V. Boisguerin, J. T. Leonard, A. Keller, C. F. Stahler, P. Haberle, B. Gharizadeh, F. Babrzadeh, and G. M. Church (2010) High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing. Nat. Biotechnol. 28: 1291–1294.
Pennisi, E. (2010) Genomics. Synthetic genome brings new life to bacterium. Sci. 328: 958–959.
Ahn, J. H., H. S. Chu, T. W. Kim, I. S. Oh, C. Y. Choi, G. H. Hahn, C. G. Park, and D. M. Kim (2005) Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions. Biochem. Biophys. Res. Commun. 338: 1346–1352.
Kim, H. C., Y. C. Kwon, K. H. Lee, and D. M. Kim (2011) Multihour translation of mRNA in a cell-free system. Biotechnol. Bioproc. Eng. 6: 1152–1156.
Richardson, S. M., S. J. Wheelan, R. M. Yarrington, and J. D. Boeke (2006) GeneDesign: Rapid, automated design of multikilobase synthetic genes. Genome Res. 16: 550–556.
Stemmer, W. P., A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164: 49–53.
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.
Johe, K. K., T. G. Hazel, T. Muller, M. M. Dugich-Djordjevic, and R. D. McKay (1996) Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev. 10: 3129–3140.
Ahn, J. H., M. Y. Hwang, K. H. Lee, C. Y. Choi, and D. M. Kim (2007) Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts. Nucleic Acids Res. 35: e21.
Son, J. M., J. H. Ahn, M. Y. Hwang, C. G. Park, C. Y. Choi, and D. M. Kim (2006) Enhancing the efficiency of cell-free protein synthesis through the polymerase-chain-reaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues. Anal. Biochem. 351: 187–192.
Isaacs, F. J., D. J. Dwyer, and J. J. Collins (2006) RNA synthetic biology. Nat. Biotechnol. 24: 545–554.
Mukherji, S. and A. van Oudenaarden (2009) Synthetic biology: Understanding biological design from synthetic circuits. Nat. Rev. Genet. 10: 859–871.
Pfleger, B. F., D. J. Pitera, C. D. Smolke, and J. D. Keasling (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 24: 1027–1032.
Keum, J. W., J. H. Ahn, T. J. Kang, and D. M. Kim (2009) Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system. Biotechnol. Bioeng. 102: 577–582.
Dias, N. and C. A. Stein (2002) Antisense oligonucleotides: Basic concepts and mechanisms. Mol. Cancer Ther. 1: 347–355.
Lee, L. K. and C. M. Roth (2003) Antisense technology in molecular and cellular bioengineering. Curr. Opin. Biotechnol. 14: 505–511.
Shao, Y., Y. Wu, C. Y. Chan, K. McDonough, and Y. Ding (2006) Rational design and rapid screening of antisense oligonucleotides for prokaryotic gene modulation. Nucleic Acids Res. 34: 5660–5669.
Ahn, J. H., T. J. Kang, and D. M. Kim (2008) Tuning the expression level of recombinant proteins by modulating mRNA stability in a cell-free protein synthesis system. Biotechnol. Bioeng. 101: 422–427.
Stenstrom, C. M., E. Holmgren, and L. A. Isaksson (2001) Cooperative effects by the initiation codon and its flanking regions on translation initiation. Gene 273: 259–265.
Stenstrom, C. M., H. N. Jin, L. L. Major, W. P. Tate, and L. A. Isaksson (2001) Codon bias at the 3′-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli. Gene 263: 273–284.
Kim, D. M., J. H. Ahn, M. Y. Hwang, C. G. Park, C. Y. Choi, and D. M. Kim (2006) Enhancing the efficiency of cell-free protein synthesis through the polymerase-chain-reaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues. Anal. Biochem. 351: 187–192.
Ahn, J. H., J. W. Keum, and D. M. Kim (2008) High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins. J. Proteome Res. 7: 2107–2113.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ahn, JH., Lee, KH., Shim, JW. et al. Streamlined cell-free protein synthesis from sequence information. Biotechnol Bioproc E 18, 1101–1108 (2013). https://doi.org/10.1007/s12257-013-0303-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12257-013-0303-7