Abstract
Expression of proteins from cDNA clones by transcription and translation in vitro can be an attractive option for analytical applications that do not require significant quantities of product. Our laboratory has been interested in this technique because the translation of nucleic acid into protein sequence allows use of the unsurpassed resolving power of 2D protein electrophoresis to analyze complex mixtures of gene products. Thus, typically we might compare two cell types that differ in some phenotypic trait, initially by translation of mRNA purified from both sources and 2D electrophoresis of the translation products. If a 2D gel spot varies in an interesting manner between the cell types, we convert the population of mRNA molecules expressed in the positive cell type into a population of cDNA clones, then search among the clones for one that produces the spot of interest after in vitro transcription and translation. The 2D gel spot is used only as the diagnostic signature of an mRNA species, and does not need to be physically handled itself to permit the recovery of cognate cDNA clones; in this way, this approach differs radically from strategies based on microscale sequencing. However, other investigators may wish to use cell-free expression to monitor mutagenesis, epitope mapping or the biological activity of already isolated clones encoding known proteins. The methods described here can be applied equally well to single clones and to complex mixtures of clones.
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References
Coleclough, C. (1993) Cell-free expression of large collections of cDNA clones using positive-selection λ phage vectors. Methods Enzymol. 217, 152–170.
Lefkovits, I., Kettman, J., and Coleclough, C. (1990) A strategy for forming a global lymphocyte proteinpaedia and gene catalogue. Immunol. Today 11, 157–162.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Parks, G. D., Duke, G. M., and Palmenberg, A. C. (1986) Encephalomyocarditis virus 3C protease: efficient cell—free expression from clones which link viral 5′ noncoding sequences to the P3 region. J. Virol. 60, 376–384.
Patterson, T. A. and Dean, M. (1987) Preparation of high titer lambda phage lysates. Nucleic Acids Res. 15, 62–98.
Gurevich, V. V., Pokrovskaya, I. D., Obukhova, T. A., and Zozulya, A. (1991) Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. Anal. Biochem. 195, 207–213.
Clemens, M. J. (1984) Translation of eukaryotic messenger RNA in cell-free extracts, in Transcription and Translation. A Practical Approach (Hames, B. D. and Higgins, S. J., eds.), IRL, Oxford, pp. 232–270.
Jackson, R. J. (1991) Potassium salts infuence the fidelity of mRNA translation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation. Biochim. Biophys. Acta 1088, 345–358.
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© 1997 Humana Press Inc.
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Coleclough, C. (1997). Expression of cDNA-Encoded Proteins by Cell-Free Transcription and Translation. In: Tuan, R.S. (eds) Recombinant Gene Expression Protocols. Methods in Molecular Biology, vol 62. Humana Press. https://doi.org/10.1385/0-89603-480-1:99
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DOI: https://doi.org/10.1385/0-89603-480-1:99
Publisher Name: Humana Press
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