Expression Analysis In Vitro

Abstract

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 (http://books.google.com/ngrams; see also Michel et al., ). Notice the 20‐fold difference in the vertical axis between the upper and lower panels.

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