This project was partially funded by an NSF IOS (0849230); Hatch, McIntire-Stennis (AD421 CRIS), USDA Seed Grant (2011-04375), and Sir Frederick McMaster Research Fellowship to ABD.

A graphic depiction of the procedure described below (Figure 1) highlights the two primary components for affinity selection using a phage display library: A) a phage display cDNA library likely to encode proteins with affinity for the bait and; B) a purified recombinant bait protein. Production of bait (recombinant protein) has been extensively examined and literature outlining best practices for securing soluble, active recombinant protein from E. coli12-13, eukaryotic yeast14<...

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Chem. 285, 37281-37292 (2010).</li><li>Jung, S., Honegger, A., Pluckthun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+for+improved+protein+stability+by+phage+display.">Selection for improved protein stability by phage display.</a> J. Mol. Biol. 294, 163-180 (1999).</li><li>Battaglia, M., Olvera-Carrillo, Y., Garciarrubio, A., Campos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enigmatic+LEA+proteins+and+other+hydrophilins.">The enigmatic LEA proteins and other hydrophilins.</a> Plant Physiol. 148, 6-24 (2008).</li><li>Egli, D. B., Bruening, W. 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The authors have nothing to disclose.

By running the experiment in three replicated wells, independently acquired phage of the same protein binding to the bait can be distinguished even if they are the same clone (i.e. no difference in the nucleotide sequence of the CDS region that has been acquired but these have been retrieved from independent wells). Otherwise, the only way to distinguish among phage encoding the same protein binding to the bait is if they are independently reverse transcribed regions that differ in some part of the nucleotide se...

Why use phage display and affinity selection instead of the myriad other techniques available for discovering and investigating protein interactions with other molecules? Phage display can claim some unique advantages over other methods of detecting protein-ligand interactions1-3 including the following:
Very wide repertoire of bait molecules
The foremost reason is in the diversity of molecules capable of acting as bait in affinity selection 4. Phage display is a very powerful means of isolating protein fragments that interact with other proteins, nucleotides, carbohydrates, etc.5&#160;Essentially, if a polymer/molecule can be attached to a recoverable support, it can be screened for affinity with phage displayed proteins. Additionally, the bait molecule can be accessed to determine if it retains biological activity when immobilized6, if conditions used to reduce/eliminate its activity are effective6 or, to introduce post-translational modifications to the bait prior to affinity selection.
Phage resistance to extraneous factors
A second reason for using phage display, is that it is possible that some baits may require environmental stress (heat, high osmolyte concentrations, specific cofactors, etc.) to capture their interacting proteins, or the phage displayed proteins may need to be somehow altered prior to affinity selection. The primary factor being studied is the interaction between bait and protein, not the condition that allows the interaction to occur or if it is lethal to the test organism. The T7 phage is particularly well suited to such studies because it can withstand harsh experimental conditions both intact and viable (e.g. the published thermal maximum for T7 viability is ~60 &#176;C 7). As an example of altering phage displayed proteins, when examining the Arabidopsis thaliana seed proteome for protein substrates of the repair enzyme, Protein Isoaspartyl Methyl Transferease (PIMT), the virus used in these studies had been &#34;aged&#34; for one week, prior to each affinity selection round, to encourage introduction of isoaspartate (isoAsp) residues in susceptible proteins6 which is not possible in organisms that can recognize and repair/metabolize such abnormalities.
Metabolically inert
Furthermore, the phage are usually resistant to metabolic poisons and interfering small molecules that would, at the least, result in pleiotropic effects on metabolically active test organisms. Following the stringency washes, the poison is removed before a large volume of bacteria are introduced for infection so the poison is diluted to a range innocuous to the bacteria or subsequent phage replication. While investigating protein targets of the PIMT repair enzyme, S-Adenosyl Methionine (AdoMet) was used to activate the micro-titer-plate-well immobilized enzyme to permit target protein capture while relying on S-Adenosyl Homocysteine (AdoHcy) to inactivate the enzyme and provide a useful negative control secure in the knowledge that the virus would not be adversely influenced by either AdoMet or AdoHcy6. Additionally, members of certain Late Embryogenesis Abundant&#160;(LEA) protein families, investigated in this lab, are known to alter their shape in the presence of additives such as sucrose8, which can attain concentrations as high as 200 mM in soybean seeds at the point of physiological maturity9. The virus is not anticipated to be influenced by addition of 200 mM sucrose in each affinity selection round, possibly necessary for certain LEA-client protein interactions, which is not the case for autonomously viable test organisms10.
This lab has focused on discovering protein-protein interactions in mature, dehydrated or germinating seeds that underlie the mechanism of stored proteome protection during dehydration11 or repair of components of the stored proteome that are susceptible to isoAsp formation once the seed has imbibed6. Thus, the production and purification of the recombinant proteins required as bait in an active state, and ensuring that they remain so, before and after they are immobilized, although frequently difficult, is a cornerstone to our work. However, as each recombinant protein production scenario is different, optimizing conditions for recombinant protein production will not be addressed here. Users are urged to attempt, wherever possible, to determine if the immobilized protein is still functional (e.g. if it is an enzyme, do an enzyme assay in the microtiter plate wells). This will provide some confidence that the bait is biologically active and therefore, that any discovered interactions are somewhat more likely to have biological relevance.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tryptone</td>
 <td>Becton Dickinson Co.</td>
 <td>211705</td>
 <td></td>
 </tr>
 <tr>
 <td>Yeast Extract</td>
 <td>Becton Dickinson</td>
 <td>212750</td>
 <td></td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Becton Dickinson</td>
 <td>214010</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Fisher Scientific</td>
 <td>BP358-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Research Products International</td>
 <td>A20090</td>
 <td></td>
 </tr>
 <tr>
 <td>Blocking Reagent</td>
 <td>EMD Chemicals Inc.</td>
 <td>69064</td>
 <td></td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Fisher Scientific</td>
 <td>BP337-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Hydroxide</td>
 <td>Fisher Scientific</td>
 <td>BP359-212</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Dodecyl Sulfate</td>
 <td>Fisher Scientific</td>
 <td>BP166-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Fisher Scientific</td>
 <td>BP152-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Fisher Scientific</td>
 <td>BP1145-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Escherichia coli BLT5403</td>
 <td>EMD Chemicals Inc.</td>
 <td>BLT5403</td>
 <td>Genotype: F- ompT hsdSB (rB - mB -) gal dcm pAR5403 (AmpR)</td>
 </tr>
 <tr>
 <td>ampicillin, Sodium Salt</td>
 <td>Research Products International</td>
 <td>A40040</td>
 <td></td>
 </tr>
 <tr>
 <td>ethidium bromide</td>
 <td>Fisher Scientific</td>
 <td>BP102-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin (BSA)</td>
 <td>Sigma-Aldrich Corp.</td>
 <td>A-9647</td>
 <td></td>
 </tr>
 <tr>
 <td>penta-HIS primary antibody</td>
 <td>Qiagen Inc.</td>
 <td>34660</td>
 <td></td>
 </tr>
 <tr>
 <td>Goat anti mouse alkaline phosphatise conjugate</td>
 <td>Sigma-Aldrich Corp.</td>
 <td>A-5153</td>
 <td></td>
 </tr>
 <tr>
 <td>para-nitrophenylphosphate</td>
 <td>Sigma-Aldrich Corp.</td>
 <td>N-7653</td>
 <td></td>
 </tr>
 <tr>
 <td>T7-UP primer</td>
 <td>EMD Chemicals Inc.</td>
 <td></td>
 <td>5'-GGAGCTGTCGTATTCCAGTC-3'</td>
 </tr>
 <tr>
 <td>T7-DOWN primer</td>
 <td>EMD Chemicals Inc.</td>
 <td></td>
 <td>5'-AACCCCTCAAGACCCGTTTA-3'</td>
 </tr>
 <tr>
 <td>Qiaquick PCR Purification kit</td>
 <td>Qiagen Inc.</td>
 <td>28104</td>
 <td></td>
 </tr>
 <tr>
 <td>Qiaquick Gel Extraction kit </td>
 <td>Qiagen Inc.</td>
 <td>28706</td>
 <td></td>
 </tr>
 <tr>
 <td>Big Dye Terminator v3.1 Cycle Sequencing Kit</td>
 <td>Invitrogen Life Technologies</td>
 <td>4336917</td>
 <td></td>
 </tr>
 <tr>
 <td>Heating Block</td>
 <td>Pierce Chemical Co. (Thermo Fisher Scientific Co.)</td>
 <td>18780</td>
 <td></td>
 </tr>
 <tr>
 <td>96 Well Cell Culture Plates</td>
 <td>Corning</td>
 <td>Costar 3590</td>
 <td></td>
 </tr>
 <tr>
 <td>Isotemp Incubator Oven</td>
 <td>Fisher Scientific</td>
 <td>516D</td>
 <td></td>
 </tr>
 <tr>
 <td>Pasteur Pipets, 5 &frac34;"</td>
 <td>Fisher Scientific</td>
 <td>13-678-20A</td>
 <td></td>
 </tr>
 <tr>
 <td>ChromaView Transilluminator</td>
 <td>UVP Inc.</td>
 <td>TS-15</td>
 <td></td>
 </tr>
 <tr>
 <td>UV Shield</td>
 <td>Oberon</td>
 <td>071AF</td>
 <td></td>
 </tr>
 <tr>
 <td>Gloves, Nitrile</td>
 <td>Fisher Scientific</td>
 <td>19-170-010C</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile 1.5 ml tubes</td>
 <td>USA Scientific Inc</td>
 <td>1615-5500</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml borosilicate Pipets</td>
 <td>Fisher Scientific</td>
 <td>13-678-25E</td>
 <td></td>
 </tr>
 <tr>
 <td>Borosilicate culture tubes</td>
 <td>Fisher Scientific</td>
 <td>14-961-27</td>
 <td></td>
 </tr>
 <tr>
 <td>Uniskan I, ELISA plate reader</td>
 <td>Labsystem and Flow Laboratories, Helsinki, Finland</td>
 <td>Type 362</td>
 <td></td>
 </tr>
 </tbody>
</table>

a protocol for phage display and affinity selection using recombinant protein baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Being able to impair the capacity of the bait to interact with phage-displayed proteins (metabolically poisoning the bait) provides a potent negative control for this technique. It is also advisable to determine if the bait, when bound to the microtiter plate well, retains its function. Both of these checks will increase the confidence that interacting, phage-displayed proteins recovered by the nonpoisoned bait are legitimate.
Sampling three triplicates from each well adds considerably to the ...

Watch this Scientific Journal Video about A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits at JoVE.com

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

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Biology

33.6K visualizações.  University of Kentucky.  A exibição em fagos é uma técnica poderosa para captura de proteínas ou fracções de proteínas que interagem com uma molécula de interesse imobilizada. Uma vez tomada uma decisão sobre o tipo de biblioteca de cDNA de fagos para criar e tela, o protocolo descrito aqui permite a seleção de afinidade eficiente levando à identificação de interagentes. 