Bioinformatics Analysis and Optimization of Cell-Free Protein Synthesis

  • Alexander A. Tokmakov
  • Atsushi Kurotani
  • Mikako Shirouzu
  • Yasuo Fukami
  • Shigeyuki Yokoyama
Part of the Methods in Molecular Biology book series (MIMB, volume 1118)


Cell-free protein synthesis offers substantial advantages over cell-based expression, allowing direct access to the protein synthetic reaction and meticulous control over the reaction conditions. Recently, we identified a number of statistically significant correlations between calculated and predicted properties of amino acid sequences and their amenability to heterologous cell-free expression. These correlations can be of practical use for predicting expression success and optimizing cell-free protein synthesis. In this chapter, we describe our approach and demonstrate how computational and predictive bioinformatics can be used to analyze and optimize cell-free protein expression.

Key words

Cell-free protein synthesis Heterologous expression Rationalization Optimization Physicochemical and structural protein properties Bioinformatics analysis 


  1. 1.
    Yokoyama S (2003) Protein expression systems for structural genomics and proteomics. Curr Opin Chem Biol 7:39–43PubMedCrossRefGoogle Scholar
  2. 2.
    Marsden RL, Orengo CA (2008) Target selection for structural genomics: an overview. Methods Mol Biol 426:3–25PubMedCrossRefGoogle Scholar
  3. 3.
    Farokki N, Hrmova M, Burton RA et al (2009) Heterologous and cell-free protein expression systems. Methods Mol Biol 513:175–198CrossRefGoogle Scholar
  4. 4.
    Spirin AS (2004) High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol 22:538–545PubMedCrossRefGoogle Scholar
  5. 5.
    Katzen F, Chang G, Kudlicki W (2005) The past, present and future of cell-free protein synthesis. Trends Biotechnol 23:150–156PubMedCrossRefGoogle Scholar
  6. 6.
    He M (2008) Cell-free protein synthesis: applications in proteomics and biotechnology. Nat Biotechnol 23:126–132Google Scholar
  7. 7.
    Goh CS et al (2004) Mining the structural genomics pipeline: identification of protein properties that effect high-throughput experimental analysis. J Mol Biol 336:115–130PubMedCrossRefGoogle Scholar
  8. 8.
    Bertone P, Kluger Y, Lan N et al (2001) SPINE: an integrated tracking database and data mining approach for identifying feasible targets in high-throughput structural proteomics. Nucleic Acids Res 29:2884–2898PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Dyson MR, Shadbolt SP, Vincent KJ et al (2004) Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol 4:32PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Idicula-Thomas S, Balaji P (2005) Understanding the relationships between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coli. Protein Sci 14:582–592PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Kurotani A, Takagi T, Toyama M et al (2010) Comprehensive bioinformatics analysis of cell-free protein synthesis: identification of multiple protein properties that correlate with successful expression. FASEB J 24:1095–1104PubMedCrossRefGoogle Scholar
  12. 12.
    Tokmakov AA, Kurotani A, Takagi T et al (2012) Multiple post-translational modifications affect heterologous protein synthesis. J Biol Chem 287:27106–27116PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Kigawa T, Yabuki T, Yoshida Y et al (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett 442:15–19PubMedCrossRefGoogle Scholar
  14. 14.
    Kigawa T, Yokoyama S (1991) A continuous cell-free protein synthesis system for coupled transcription-translation. J Biochem 110:166–168PubMedGoogle Scholar
  15. 15.
    Kudlicki W, Kramer G, Hardesty B (1992) High efficiency cell-free synthesis of proteins: refinement of the coupled transcription/translation system. Anal Biochem 206:389–393PubMedCrossRefGoogle Scholar
  16. 16.
    Yokoyama S, Hirota H, Kigawa T et al (2000) Structural genomics projects in Japan. Nat Struct Biol 7(Suppl):943–945PubMedCrossRefGoogle Scholar
  17. 17.
    Yokoyama S (2005) Large-scale structural proteomics project at RIKEN: present and future. Tanpakushitsu Kakusan Koso 50:836–845PubMedGoogle Scholar
  18. 18.
    Yokoyama S, Kigawa T, Shirouzu M et al (2008) RIKEN structural genomics/proteomics initiative. Tanpakushitsu Kakusan Koso 53:632–637PubMedGoogle Scholar
  19. 19.
    Yabuki T, Motoda Y, Hanada K et al (2007) A robust two-step PCR method of template DNA production for high-throughput cell-free protein synthesis. J Struct Funct Genom 8:173–191CrossRefGoogle Scholar
  20. 20.
    Kigawa T, Matsuda T, Yabuki T et al (2008) Bacterial cell-free system for highly efficient protein synthesis. In: Spirin AS, Swartz JR (eds) Cell-free protein synthesis. Wiley, Weinheim, pp 83–97Google Scholar
  21. 21.
    Kigawa T (2010) Analysis of protein functions through a bacterial cell-free protein expression system. Methods Mol Biol 607:53–62PubMedCrossRefGoogle Scholar
  22. 22.
    Kigawa T, Yabuki T, Matsuda N et al (2004) Preparation of Escherichia coli extract for highly productive cell-free protein expression. J Struct Funct Genom 5:63–68CrossRefGoogle Scholar
  23. 23.
    Davanloo P, Rosenberg AH, Dunn JJ et al (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 81:2035–2039PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Grodberg J, Dunn JJ (1988) ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol 170:1245–1253PubMedCentralPubMedGoogle Scholar
  25. 25.
    Zawadski V, Gross HJ (1991) Rapid and simple purification of T7 RNA polymerase. Nucleic Acids Res 19:1948CrossRefGoogle Scholar
  26. 26.
    Ding HT, Ren H, Cheng Q et al (2002) Parallel cloning, expression, purification, and crystallization of human proteins for structural genomics. Acta Crystallogr D Biol Crystallogr 58:2102–2108PubMedCrossRefGoogle Scholar
  27. 27.
    Cheng J, Randall AZ, Sweredoski MJ et al (2005) SCRATCH: a protein structure and structural feature prediction server. Nucleic Acids Res 33(Web Server issue):72–76CrossRefGoogle Scholar
  28. 28.
    Frishman D, Argos P (1997) Seventy-five percent accuracy in protein secondary structure prediction. Proteins 27:329–335PubMedCrossRefGoogle Scholar
  29. 29.
    Yang ZR, Thomson R, McMeil P et al (2005) RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics 21:3369–3376PubMedCrossRefGoogle Scholar
  30. 30.
    Ren J, Wen L, Gao X et al (2008) CSS-Palm 2.0: an updated software for palmitoylation sites prediction. Protein Eng Des Sel 21:639–644PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Cheng J, Saigo H, Baldi P (2006) Large-scale prediction of disulfide bridges using kernel methods, two-dimensional recursive neural networks, and weighed graph matching. Proteins 62:617–629PubMedCrossRefGoogle Scholar
  32. 32.
    Radivojac P, Vacic V, Haynes C et al (2010) Identification, analysis, and prediction of protein ubiquitination sites. Proteins 78:365–380PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Ren J, Gao X, Jin C et al (2009) Systematic study of protein sumoylation: development of a site-specific predictor of SUMPsp 2.0. Proteomics 9:3409–3412PubMedCrossRefGoogle Scholar
  34. 34.
    Gao J, Liao J, Yang GY (2009) CAAX-box protein, prenylation process and carcinogenesis. Am J Transl Res 25:312–325Google Scholar
  35. 35.
    Amaya M, Baranova A, van Hoek ML (2011) Protein prenylation: a new mode of host-pathogen reaction. Biochem Biophys Res Commun 416:1–6PubMedCrossRefGoogle Scholar
  36. 36.
    Xu B, Feng X, Burdine RD (2010) Categorical data analysis in experimental biology. Dev Biol 348:3–11PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Betton JM, Miot M (2008) Cell-free production of membrane proteins in the presence of detergents. In: Spirin AS, Swartz JR (eds) Cell-free protein synthesis: methods and protocols. Wiley, Weinheim, pp 165–178Google Scholar
  38. 38.
    Ishihara G, Goto M, Saeki M et al (2005) Expression of G-protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors. Protein Expr Purif 41:27–37PubMedCrossRefGoogle Scholar
  39. 39.
    Tu BP, Weissman JS (2004) Oxidative protein folding in eukaryotes. J Cell Biol 164:341–346PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Kim DM, Swartz JR (2004) Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnol Bioeng 85:122–129PubMedCrossRefGoogle Scholar
  41. 41.
    Yin G, Swartz JR (2004) Enhancing multiple disulfide bonded protein folding in a cell-free system. Biotechnol Bioeng 86:188–195PubMedCrossRefGoogle Scholar
  42. 42.
    Yang J, Kanter G, Voloshin A et al (2004) Expression of active murine granulocyte-macrophage colony-stimulating factor in an Escherichia coli cell-free system. Biotechnol Prog 20:1689–1696PubMedCrossRefGoogle Scholar
  43. 43.
    Kukimoto-Niino M, Tokmakov A, Terada T et al (2011) Inhibitor-bound structures of human pyruvate dehydrogenase kinase. Acta Crystallogr D Biol Crystallogr 67:763–773PubMedCrossRefGoogle Scholar
  44. 44.
    Suyama M, Ohara O (2003) DomCut: prediction of inter-domain linker regions in amino acid sequences. Bioinformatics 19:673–674PubMedCrossRefGoogle Scholar
  45. 45.
    Norman GR, Streiner DL (2000) Biostatistics: the bare essentials. B.C. Decker, HamiltonGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Alexander A. Tokmakov
    • 1
    • 2
    • 3
  • Atsushi Kurotani
    • 4
    • 5
    • 6
  • Mikako Shirouzu
    • 1
  • Yasuo Fukami
    • 2
    • 3
  • Shigeyuki Yokoyama
    • 4
    • 7
  1. 1.RIKEN Systems and Structural Biology CenterTsurumi, YokohamaJapan
  2. 2.Graduate School of ScienceKobe UniversityNadaJapan
  3. 3.Research Center for Environmental GenomicsKobe UniversityNadaJapan
  4. 4.RIKEN Systems and Structural Biology CenterYokohamaJapan
  5. 5.Plant Science CenterYokohamaJapan
  6. 6.Department of Biotechnology and Life ScienceTokyo University of Agriculture and TechnologyTokyoJapan
  7. 7.Department of Biophysics and Biochemistry, Graduate School of ScienceThe University of TokyoBunkyo-kuJapan

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