Protein Selection Using mRNA Display

Anthony D. Keefe1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 24.5
DOI:  10.1002/0471142727.mb2405s53
Online Posting Date:  May, 2001
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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.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Preparation and Purification of mRNA‐Displayed Proteins
  • Basic Protocol 2: Purification and Reverse Transcription of the mRNA‐Displayed Proteins
  • Basic Protocol 3: Selection and Amplification of the mRNA‐Displayed Proteins
  • Support Protocol 1: FLAG Tag Purification
  • Support Protocol 2: Mutagenic PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation and Purification of mRNA‐Displayed Proteins

  Materials
  • DNA library
  • 1 M MgCl 2
  • 100 mM nucleotide triphosphate solutions
  • recipe10× transcription buffer (see recipe)
  • Deionized, ultrafiltered water
  • 10 U/µl T7 RNA polymerase
  • Solid EDTA
  • Urea
  • 0.5× TBE buffer ( appendix 22)
  • 3 M NaCl
  • 100% and 70% ethanol
  • 100 mM EDTA
  • Puromycin‐terminated DNA linker
  • 100 mM ATP
  • T4 polynucleotide kinase buffer
  • T4 polynucleotide kinase
  • 10× T4 DNA ligase buffer
  • 10 U/µl T4 DNA ligase
  • 3 M potassium acetate solution, pH 5.3
  • Rabbit reticulocyte lysate translation kit (e.g., Red Nova Lysate kit, Novagen)
    •  Control RNA
    •  12.5× methionine‐free translation mix
    •  2.5 M potassium chloride
    •  25 mM magnesium acetate
    •  Nuclease‐free water
    •  Rabbit reticulocyte lysate
  • 35S‐methionine
  • Electroeluter (VWR or Schleicher & Schuell)
  • Denaturing PAGE gel (unit 2.12)
  • Gel filtration columns (Pharmacia)
  • Additional reagents and equipment for preparative denaturing PAGE purification (unit 2.12), determining nucleic acid concentration by spectrometry ( appendix 3D), synthesis of oligonucleotides (unit 2.11), and SDS‐PAGE in Tris‐tricine buffer systems (unit 10.2)

Basic Protocol 2: Purification and Reverse Transcription of the mRNA‐Displayed Proteins

  Materials
  • Oligo(dT) cellulose (Amersham Pharmacia Biotech)
  • recipeOligo(dT) binding buffer (see recipe)
  • 1.3‐ml translation reaction mRNA displayed proteins (see protocol 1)
  • recipeOligo(dT) wash buffer (see recipe)
  • Ni‐NTA agarose (Qiagen)
  • recipeNi‐NTA binding buffer (see recipe)
  • 2‐Mercaptoethanol
  • recipeNi‐NTA wash buffer 1 (see recipe)
  • recipeNi‐NTA wash buffer 2 (see recipe)
  • recipeNi‐NTA elution buffer (see recipe)
  • 10 mg/ml salmon sperm DNA (Life Technologies)
  • 1 mg/ml BSA
  • 200 µM DNA splint
  • 5× Superscript II reverse transcriptase buffer (NEB)
  • 0.1 M DTT
  • 30 µl (each) 25 mM deoxynucleotide triphosphates (final 0.5 mM)
  • 200 U/ml Superscript II reverse transcriptase (NEB)
  • 25 mM deoxynucleotide triphosphate solutions
  • recipeATP‐aptamer selection binding buffer (see recipe)
  • recipeATP‐aptamer selection elution buffer (see recipe)
  • Chromatography columns (Bio‐Rad)
  • Gel filtration columns (e.g., NAP‐5, Amersham Pharmacia Biotech)
  • For additional reagents and equipment for preparative denaturing PAGE purification (unit 2.12) and SDS‐PAGE in Tris‐tricine buffer systems (unit 10.2)

Basic Protocol 3: Selection and Amplification of the mRNA‐Displayed Proteins

  Materials
  • ATP agarose (Sigma)
  • recipeATP‐aptamer selection binding buffer (see recipe)
  • Purified mRNA‐displayed proteins (see protocol 2)
  • recipeATP‐aptamer selection elution buffer (see recipe)
  • 100 mM EDTA ( appendix 22)
  • 1 M NaOH ( appendix 22)
  • 1 M HCl
  • 10 mg/ml salmon sperm DNA
  • 1 mg/ml BSA
  • 100 µM 3′ primer (specific for cDNA library)
  • 100 µM 5′ primer (specific for cDNA library)
  • 25 mM (each) deoxynucleotide triphosphates
  • 10× PCR buffer containing 15 mM MgCl 2 (Boehringer Mannheim)
  • 5 U/µl Taq DNA polymerase (Boehringer Mannheim)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • Chloroform
  • 1‐Butanol
  • 3 M NaCl
  • 100% ethanol
  • Gel filtration columns (e.g., NAP‐25, Amersham Pharmacia Biotech)
  • Additional reagents and equipment for butanol extraction (unit 2.1)

Support Protocol 1: FLAG Tag Purification

  • Anti‐FLAG M2 agarose (Sigma)
  • recipeFLAG clean buffer (see recipe)
  • recipeFLAG binding buffer (see recipe)
  • FLAG peptide (Sigma)

Support Protocol 2: Mutagenic PCR

  • 2.5 M KCl
  • 100 mM MnCl 2 solution
  • 100 mM Tris⋅Cl, pH 8.3 ( appendix 22)
  • 100 µl PCR tubes (Sarstedt)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 15.1)
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Figures

Videos

Literature Cited

   Cadwell, R.C. and Joyce, G.F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28‐33.
   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.
   Colas, P., Cohen, B., Jessen, T., Grishina, I., McCoy, J., and Brent, R. 1996. Genetic selection of peptide aptamers that recognize and inhibit cyclin‐dependent kinase 2. Nature 380:548‐550.
   Fields, S. and Song, O. 1989. A novel genetic system to detect protein‐protein interactions. Nature 340:245‐246.
   Gold, L., Allen, P., Binkley, J., Brown, D., Schneider, D., Eddy, S.R., Tuerk, C., Green, L., MacDougal, S., and Tasset, D. 1993. The shape of things to come. In The RNA World. (R.F. Gesteland and J.F. Atkins eds.) pp. 497‐ 509. Cold Spring Harbor, New York.
   Jermutus, L., Ryabova, L., and Plückthun, A. 1998. Recent advances in producing and selecting functional proteins by using cell‐free translation. Curr. Opin. Biotechnol. 9:391‐410.
   Joyce, G.F. 1993. Evolution of catalytic function. Pure & Appl. Chem. 65:1205‐1212.
   LaBean, T.H. and Kauffman, S.A. 1993. Design of synthetic gene libraries encoding random sequence proteins with desired ensemble characteristics. Protein Sci. 2:1249‐1254.
   Liu, R., Barrick, J., Szostak, J.W., and Roberts, R.W. 2000. Optimized synthesis of RNA‐protein fusions for in vitro protein selection. Methods Enzymol. 317:268‐293.
   Roberts, R.W. 1999. Totally in vitro protein selection using mRNA‐protein fusions and ribosome display. Curr. Opin. Chem. Biol. 3:268‐273.
   Roberts, R.W. and Ja, W.W. 1999. In vitro selection of nucleic acids and proteins: What are we learning? Curr. Opin. Struct. Biol. 9:521‐529.
   Roberts, R.W. and Szostak, J.W. 1997. RNA‐peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. U.S.A. 94:12297‐12302.
   Sche, P.P., McKenzie, K.M., White, J.D., and Austin, D.J. 1999. Display cloning: functional identification of natural product receptors using cDNA‐phage cloning. Chem. Biol. 6:707‐716.
   Smith, G.P. and Petrenko, V.A. 1997. Phage display. Chem Rev. 97:391‐410.
   Stemmer, W.P.C. 1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370:389‐391.
   Szostak, J.W. and Ellington, A.D. 1993. In vitro selection of functional RNA sequences. In The RNA World. (R.F. Gesteland and J.F. Atkins eds.) pp. 551‐533. Cold Spring Harbor, New York.
   Wilson, D.S. and Szostak, J.W. 1999. In vitro selection of functional nucleic acids. Annu. Rev. Biochem. 68:611‐647.
   Wolf, E. and Kim, P.S. 1999. Combinatorial Codons: A computer program to approximate amino acid probablilities with biased nucleotide usage. Protein Sci. 8:680‐688.
Key References
   Roberts and Szostak, 1997. See above.
  The first demonstration of the formation of mRNA displayed proteins (RNA‐protein fusions).
   Liu et al., 2000. See above.
  Describes the optimization of the synthesis and purification of mRNA displayed proteins (RNA‐protein fusions).
   Cho et al., 2000. See above.
  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
  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
  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
  The Biopolymer Calculator is a very useful general tool for molecular biology.
   http://sun2.science.wayne.edu/%7Ejslsun2/servers/seqanal/
  A nucleic acid secondary structure prediction algorithm is given by mfold.
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