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Current Protocols in Molecular Biology
Volume 53, Issue 1 p. 24.5.1-24.5.34
UNIT


PROTEIN SELECTION USING MRNA DISPLAY


Anthony D. Keefe, 

Anthony D. Keefe

Massachusetts General Hospital, Boston, Massachusetts

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Anthony D. Keefe, 

Anthony D. Keefe

Massachusetts General Hospital, Boston, Massachusetts

Search for more papers by this author
First published: 01 May 2001
https://doi.org/10.1002/0471142727.mb2405s53
Citations: 4
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ABSTRACT

mRNA display is an in vitro technique that may be used to search natural or
synthetic DNA libraries for the functional proteins and peptides they encode.
mRNA-displayed proteins are constructs in which a protein is covalently attached
to the RNA that encodes it. This direct covalent association of phenotype
(protein) and genotype (RNA) renders the protein directly amplifiable. This in
turn allows successive cycles of selection, enrichment, and, optionally,
mutagenesis, to be performed upon libraries of displayed proteins. At the end of
this process, functional sequences will dominate the library; cloning and
sequencing will reveal the identity of the selected functional proteins. mRNA
display allows new functional proteins to be discovered without resorting to
protein design. This unit describes generation of mRNA-displayed proteins by the
in vitro translation of mRNA display templates which are mRNA molecules
3'-terminated in puromycin. Puromycin is a translation inhibitor that is able to
enter the ribosome during translation and form a stable covalent bond with the
nascent protein. This allows a stable covalent linkage to be formed between the
mRNA display template and the protein it encodes, resulting in an mRNA-displayed
protein.


LITERATURE CITED

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 * Cho, G., Keefe, A.D., Liu, R., Wilson, D.S., and Szostak, J.W. 2000.
   Constructing high complexity synthetic libraries of long ORFs using in vitro
   selection. J. Mol. Biol. In press.
   
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   1996. Genetic selection of peptide aptamers that recognize and inhibit
   cyclin-dependent kinase 2. Nature 380: 548-550.
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   protein-protein interactions. Nature 340: 245-246.
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   producing and selecting functional proteins by using cell-free translation.
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   cloning: functional identification of natural product receptors using
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   approximate amino acid probablilities with biased nucleotide usage. Protein
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KEY REFERENCES

 * Roberts and Szostak, 1997. See above.
   
   Google Scholar

   The first demonstration of the formation of mRNA displayed proteins
   (RNA-protein fusions).

 * Liu et al., 2000. See above.
   
   Google Scholar

   Describes the optimization of the synthesis and purification of mRNA
   displayed proteins (RNA-protein fusions).

 * Cho et al., 2000. See above.
   
   Google Scholar

   Describes the use of mRNA display and in vitro selection to construct various
   types of high quality library for use in mRNA display protein selections.

INTERNET RESOURCES

 * http://gaiberg.wi.mit.edu/cgi-bin/CombinatorialCodons
   
   Google Scholar

   Combinatorial Codons is an extremely useful tool for the design of protein
   libraries; it generates a nucleotide distribution that iteratively approaches
   an input amino acid distribution.

 * http://xanadu.mgh.harvard.edu/szostakweb/orf.html
   
   Google Scholar

   This site is a database of exact oligonucleotide sequences that have been
   successfully used in the construction of random, patterned, and
   structure-based mRNA-displayed protein libraries.

 * http://paris.chem.yale.edu/extinct.html
   
   Google Scholar

   The Biopolymer Calculator is a very useful general tool for molecular
   biology.

 * http://sun2.science.wayne.edu/%7Ejslsun2/servers/seqanal/
   
   Google Scholar

   A nucleic acid secondary structure prediction algorithm is given by mfold.

CITING LITERATURE


View the latest in Molecular Biology
This article also appears in:
 * Combinatorial Libraries




 * REFERENCES


 * RELATED


 * INFORMATION


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METRICS

Citations: 4



DETAILS

Copyright © 2001 by John Wiley & Sons, Inc.




PUBLICATION HISTORY

 * Issue Online: 01 May 2001
 * Version of Record online: 01 May 2001




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