Expression Analysis In Vitro


Expression analysis in vitro is a constantly evolving field, consolidated in the fourth quarter of the last century and essentially based on the use of a template of ribonucleic acid (RNA), for a translation reaction, or of deoxyribonucleic acid (DNA), in a coupled transcription–translation system. Traditional applications of expression analysis in vitro cover a wide range of structural and functional studies on proteins and nucleotides using methodologies like yeast one‐, two‐ and three‐hybrid systems, reporter genes, phage display, DNase footprinting, methylation interference assays and gel‐shift assays. Moreover, in the last decades in‐vitro expression analyses benefitted from substantial advancements, mostly associated with the use of a number of refined cell‐free protein synthesis methods and of microarrays and nanodevices. The frequency trends of related keywords in a huge database of English books published all over the world and covering a wide, recent time window provide an indirect – although highly suggestive – estimate of their relative importance in the next years.

Key Concepts:

  • In‐vitro expression systems can: (1) be used for the expression of toxic, proteolytically sensitive or unstable proteins; (2) incorporate unnatural amino acids and (3) allow the addition of exogenous factors to study enzymatic activity, and of microsomal membranes to study post‐translational modifications.

  • Application of in‐vitro expression systems include: (1) site‐specific methods that utilise tRNA charged with any number of unnatural amino acids; (2) the use of putative DNA‐binding proteins such as transcription factors and (3) improving particular features of preexisting molecules like ultraspecificity, affinity and reaction rate.

  • The intrinsic appealing of in‐vitro expression analysis has been reinforced in the last decades thanks to refined cell‐free protein synthesis (CFPS) methods, microarrays (MA) and nanodevices (ND), whose evolution occurred at a remarkably fast pace. The data flow streaming out of the above‐mentioned techniques demands, in any case, massive statistical analyses and systematic cross‐checking of results by independent strategies.

Keywords: reporter gene studies; DNase footprinting; methylation interference assays; gel‐shift assay; yeast one‐, two‐ and three‐hybrid system; phage display; microarrays

Figure 1.

Schematic overview of yeast one‐, two‐ and three‐hybrid systems. DNA‐BP and ‐AD are, respectively, the DNA‐binding domain and the activation domain, identified in many eukaryotic transcriptional activators as functionally and physically independent units. (a) In one‐hybrid systems, the two domains must be present in the same chimaeric protein to allow generation of the transcriptional signal by the reporter gene, generally consisting of growth or colour selection; (b) in two‐hybrid systems, they are coupled to proteins P1 and P2, whose physical interaction is a necessary prerequisite for a successful transcription of the reporter gene and (c) in three‐hybrid systems, a third hybrid molecule acts to bring together the DNA‐BP fused to the receptor for one ligand with the DNA‐AD fused to the receptor for the second ligand, thus reconstituting a functional transcriptional activator.

Figure 2.

Occurrence of selected keywords in a corpus of English books published all over the world in the last 50 years. The results are depicted in the form of fractional n‐grams, namely short and ordered phrases of n words, reckoned on a yearly basis over all the n‐grams of the same length in a corpus of English books containing 361 billion words (; see also Michel et al., ). Notice the 20‐fold difference in the vertical axis between the upper and lower panels.



Arduengo M, Schenborn E and Hurst R (2007) The role of cell‐free rabbit reticulocyte expression systems in functional proteomics. In: Kudlicki W, Katzen F and Bennett R (eds) Cell‐Free Expression. Austin, Texas: Landes Bioscience.

Bai C and Elledge SJM (1996) Gene identification using the yeast two‐hybrid system. Methods in Enzymology 273: 331–347.

Bundy BC, Franciszkowicz MJ and Swartz JR (2008) Escherichia coli‐based cell‐free synthesis of virus‐like particles. Biotechnology and Bioengineering 100: 28–37.

Carey M and Smale ST (2007) Methylation interference assay. Cold Spring Harbor Protocols. doi: 10.1101/pdb.prot4812.

Carlson ED, Gan R, Hodgman CE and Jewett MC (2011) Cell‐free protein synthesis: applications come of age. Biotechnology Advances 30(5): 1185–1194.

Chalmeau J, Monina N, Shin J, Vieu C and Noireaux V (2011) α‐Hemolysin pore formation into a supported phospholipid bilayer using cell‐free expression. Biochimica et Biophysica Acta – Biomembranes 1808: 271–278.

Dudoit S, Yang YH, Callow MJ and Speed TP (2002) Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Statistica Sinica 12: 111–139.

Frydman J and Hartl FU (1996) Principle of chaperon‐assisted protein folding: differences between in vitro and in vivo mechanisms. Science 272: 1497–1502.

Goshima N, Kawamura Y, Fukumoto A et al. (2008) Human protein factory for converting the transcriptome into an in vitro‐expressed proteome. Nature Methods 5: 1011–1017.

Hampshire A, Rusling D, Broughton‐Head V and Fox K (2007) Footprinting: a method for determining the sequence selectivity, affinity and kinetics of DNA‐binding ligands. Methods 42: 128–140.

Huang J, Ru B, Zhu P et al. (2012) MimoDB 2.0: a mimotope database and beyond. Nucleic Acids Research 40: 271–277.

Jewett MC, Calhoun KA, Voloshin A, Wuu JJ and Swartz JR (2008) An integrated cell‐free metabolic platform for protein production and synthetic biology. Molecular Systems Biology 4: 220.

Jungmann R, Renner S and Simmel FC (2008) From DNA nanotechnology to synthetic biology. HFSP Journal 2(2): 99–109.

Kim HC, Kim TW and Kim DM (2011) Prolonged production of proteins in a cell‐free protein synthesis system using polymeric carbohydrates as an energy source. Process Biochemistry 46: 1366–1369.

Lehming N, Thanos D, Brickman JM et al. (1994) An HMG‐like protein that can switch a transcriptional activator to a repressor. Nature 371: 175–179.

Licitra EJ and Liu JO (1996) A three‐hybrid system for detecting small ligand‐protein receptor interactions. Proceedings of the National Academy of Sciences of the USA 93: 12817–12821.

Marioni JC, Mason CE, Mane SM, Stephens M and Gilad Y (2008) RNA‐seq: an assessment of technical reproducibility and comparison with gene expression arrays, Genome Research 18: 1509–1517.

Michel JB, Shen YK, Aiden AP et al. (2011) Quantitative analysis of culture using millions of digitized books. Science 331: 176–182.

Nair S, Arathy DS, Issac A and Sreekumar E (2011) Differential gene expression analysis of in vitro duck hepatitis B virus infected primary duck hepatocyte cultures. Virology Journal 8: 363.

Naujok O, Francini F, Picton S et al. (2009) Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin producing cells in vitro and in vivo. Diabetes/Metabolism: Research and Reviews 25: 464–476.

O'Brien T.P., Bult CJ, Cremer C et al. (2003) Genome function and nuclear architecture: from gene expression to nanoscience. Genome Research 13: 1029–1041.

Pan Q, Shai O, Lee LJ, Frey BJ and Blencowe BJ (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high‐throughput sequencing. Nature Genetics 40: 1413–1415.

Ritchie ME, Dunning MJ, Smith ML, Wei S and Lynch AG (2011) BeadArray expression analysis using bioconductor. PLoS Computational Biology 7: 1–6.

Roberts BE and Paterson BM (1973) Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell‐free system from commercial wheat germ. Proceedings of the National Academy of Sciences of the USA 70: 2230–2334.

Robinson MK, McCarthy DJ and Smyth GK (2010) edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26: 139–140.

Sakalian M, Parker SD, Weldon RA Jr and Hunter A (1996) Synthesis and assembly of retrovirus Gag precursor into immature capsids in vitro. Journal of Virology 70: 3706–3715.

Schmidt M (2012) Synthetic Biology: Industrial and Environmental Applications (3rd ed.). Weinheim, Germany: Wiley‐Blackwell. pp 1–67. ISBN 3‐527‐33183‐2.

Schwarz D, Dötsch V and Bernhard F (2008) Production of membrane proteins using cell‐free expression systems. Proteomics 8: 3933–3946.

Slonim DK and Yanai I (2009) Getting started in gene expression microarray analysis, PLoS Computational Biology 10: 1–7.

Smith AJ and Humphries SE (2009) Characterization of DNA‐binding proteins using multiplexed competitor EMSA. Journal of Molecular Biology 385(3): 714–717.

Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigen on the virion surface. Science 228: 1315–1316.

Stapleton JA and Swartz JR (2010) Development of an in vitro compartmentalization screen for high‐throughput directed evolution of [FeFe] hydrogenases. PLoS One 5(12): e15275.

Suzuki T, Ito M, Ezure T et al. (2007) Protein prenylation in an insect cell‐free protein synthesis system and identification of products by mass spectrometry. Proteomics 7: 1942–1950.

Takai K, Sawasaki T and Endo Y (2010) Practical cell‐free protein synthesis system using purified wheat embryos. Nature Protocols 5: 227–238.

Valk PJ, Verhaak RG, Beijen MA et al. (2004) Prognostically useful gene‐expression profiles in acute myeloid leukemia. New England Journal of Medicine 350: 1617–1628.

Warner DR, Basi NS and Rebois RV (1995) Cell‐free synthesis of functional type IV adenylyl cyclase. Analytical Biochemistry 232: 31–36.

Welsh JP, Bonomo J and Swartz JR (2011) Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell‐free system. Biotechnology and Bioengineering 108: 1739–1748.

Wei LQ, Xu WY, Deng ZY et al. (2010) Genome-scale analysis and comparison of gene expression profiles in developing and germinated pollen in Oryza sativa. BMC Genomics 11: 338.

Williams C, Helguero L, Edvardsson K, Haldosé LA2 and Gustafsson JA (2009) Gene expression in murine mammary epithelial stem cell‐like cells shows similarities to human breast cancer gene expression. Breast Cancer Research 11: 1–17.

Wrighton NC, Farrelle FX, Chang R et al. (1996) Small peptides as potent mimetics of the protein hormone erythropoietin. Science 273: 458–464.

Yokota T, Mishra M, Akatsu H et al. (2006) Brain site‐specific gene expression analysis in Alzheimer's disease patients.European Journal of Clinical Investigation 36: 820–830.

Zawada JF, Yin G, Steiner AR et al. (2011) Microscale to manufacturing scale‐up of cell‐free cytokine production – a new approach for shortening protein production development timelines. Biotechnology and Bioengineering 108: 1570–1578.

Zhao L, Helms JB, Brunner J and Wieland FT (1999) ATP‐dependent binding of ADP‐ribosylation factor to octamer in close proximity to the binding site for dilysine retrieval motifs and p23. Journal of Biological Chemistry 274: 14198–14203.

Further Reading

Allen JB, Walberg MV, Edwards MC and Elledge SJ (1995) Finding prospective partners in the library: the two‐hybrid system and phage display find a match. Trends in Biochemistry 20: 511–516.

Canalesi RD, Luo Y, Willey JC et al. (2006) Evaluation of DNA microarray results with quantitative gene expression platforms. Nature Biotechnology 24: 1115–1122.

Carey J (1991) Gel retardation. Methods in Enzymology 208: 103–117.

Chien CT, Bartel PL, Sternglanz R and Fields S (1991) The two‐hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proceedings of the National Academy of Sciences of the USA 88: 9578–9582.

Felici F, Castagnoli L, Musacchio A, Jappelli R and Cesareni G (1991) Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. Journal of Molecular Biology 222: 301–310.

Goerke AR and Swartz JR (2008) Development of cell‐free protein synthesis platforms for disulfide bonded proteins. Biotechnology and Bioengineering 99: 351–367.

Grange T, Bertrand E, Espinàs ML et al. (1997) In vivo footprinting of the interaction of proteins with DNA and RNA. Methods 11: 151–163.

Griffith EC, Licitra EJ and Liu JO (2000) Yeast three‐hybrid systems for detecting ligand‐receptor interactions. Methods in Enzymology 328: 89–102.

Gstaiger M, Knoepfel L, Georgiev O, Schaffner W and Hovens CM (1995) A B‐cell coactivator of octamer‐binding transcription factors. Nature 373: 360–362.

Ikeuchi A, Nakano H, ad Kamiya T, Yamane T and Kawarasaki Y (2010) A method for reverse interactome analysis: High‐resolution mapping of interdomain interaction network in Dam1 complex and its specific disorganization based on the interaction domain expression. Biotechnology Progress 26: 945–953.

Li JJ and Herskowitz I (1993) Isolation of ORC6, a component of the yeast origin recognition complex by a one‐hybrid system. Science 262: 1870–1874.

Rothschild KJ and Gite S (1999) tRNA‐mediated protein engineering. Current Opinion in Biotechnology 10: 64–70.

Sachdev SS, Lowman HB, Cunningham BC and Wells JA (2000) Phage display for selection of novel binding peptides. Methods in Enzymology 328: 333–363.

Sinclair AM, Todd MD, Forsythe K et al. (2007) Expression and function of erythropoietin receptors in tumors. Cancer 110: 477–488.

Singh R, Saxena A and Mozumdar S (2008) Calcium phosphate‐DNA nanocomposites: morphological studies and their bile duct infusion for liver‐directed gene therapy. International Journal of Applied Ceramic Technology 5: 1–10.

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Colosimo, Alfredo(Sep 2013) Expression Analysis In Vitro. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0005678.pub2]