Name: modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin
Uploaded: 2012-01-17T13:00:00
Description: Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

This work was funded by NIH research grants K01AR053210, R01AR061484 and a grant from National Psoriasis Foundation.

1. Background information
The yeast two-hybrid system is a powerful genetic technique used to discover protein-protein interactions 16, 17. Several kinds of 2-hybrid systems, such as lexA-based systems, the Sos Recruitment System, and bacteria- or mammalian cell-based 2-hybrids, are commercial available, this paper specifically focuses on the modifications of the most commonly used GAL4 based yeast 2-hybrid system. Briefly, the method is based on the properties of the yeast GAL4</...

<ol>
	<li>He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progranulin+is+a+mediator+of+the+wound+response.">Progranulin is a mediator of the wound response.</a> Nat. Med. 9, 225-229 (2003).</li><li>Kessenbrock, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteinase+3+and+neutrophil+elastase+enhance+inflammation+in+mice+by+inactivating+antiinflammatory+progranulin.">Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin.</a> J. Clin. Invest. 118, 2438-2447 (2008).</li><li>Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+proepithelin+to+epithelins:+roles+of+SLPI+and+elastase+in+host+defense+and+wound+repair.">Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair.</a> Cell. 111, 867-878 (2002).</li><li>Yin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exaggerated+inflammation,+impaired+host+defense,+and+neuropathology+in+progranulin-deficient+mice.">Exaggerated inflammation, impaired host defense, and neuropathology in progranulin-deficient mice.</a> J. Exp. Med. 207, 117-128 (2010).</li><li>Van Damme, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progranulin+functions+as+a+neurotrophic+factor+to+regulate+neurite+outgrowth+and+enhance+neuronal+survival.">Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival.</a> J. Cell. Biol. 181, 37-41 (2008).</li><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+progranulin+cause+tau-negative+frontotemporal+dementia+linked+to+chromosome+17.">Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17.</a> Nature. 442, 916-919 (2006).</li><li>Cruts, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Null+mutations+in+progranulin+cause+ubiquitin-positive+frontotemporal+dementia+linked+to+chromosome+17q21.">Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.</a> Nature. 442, 920-924 (2006).</li><li>Guo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulin-epithelin+precursor+binds+directly+to+ADAMTS-7+and+ADAMTS-12+and+inhibits+their+degradation+of+cartilage+oligomeric+matrix+protein.">Granulin-epithelin precursor binds directly to ADAMTS-7 and ADAMTS-12 and inhibits their degradation of cartilage oligomeric matrix protein.</a> Arthritis Rheum. 62, 2023-2036 (2010).</li><li>Feng, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulin+epithelin+precursor:+a+bone+morphogenic+protein+2-inducible+growth+factor+that+activates+Erk1/2+signaling+and+JunB+transcription+factor+in+chondrogenesis.">Granulin epithelin precursor: a bone morphogenic protein 2-inducible growth factor that activates Erk1/2 signaling and JunB transcription factor in chondrogenesis.</a> FASEB. J. 24, 1879-1892 (2010).</li><li>Xu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+oligomeric+matrix+protein+associates+with+granulin-epithelin+precursor+(GEP)+and+potentiates+GEP-stimulated+chondrocyte+proliferation.">Cartilage oligomeric matrix protein associates with granulin-epithelin precursor (GEP) and potentiates GEP-stimulated chondrocyte proliferation.</a> J. Biol. Chem. 282, 11347-11355 (2007).</li><li>Liu, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ADAMTS-7+and+ADAMTS-12+in+the+pathogenesis+of+arthritis.">The role of ADAMTS-7 and ADAMTS-12 in the pathogenesis of arthritis.</a> Nat. Clin. Pract. Rheumatol. 5, 38-45 (2009).</li><li>Liu, C., Dib-Hajj, S. D., Waxman, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waxman,+S.G.+Fibroblast+growth+factor+homologous+factor+1B+binds+to+the+C+terminus+of+the+tetrodotoxin-resistant+sodium+channel+rNav1.9a+(NaN).">Waxman, S.G. Fibroblast growth factor homologous factor 1B binds to the C terminus of the tetrodotoxin-resistant sodium channel rNav1.9a (NaN).</a> J. Biol. Chem. 276, 18925-18933 (2001).</li><li>Liu, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAMTS-7:+a+metalloproteinase+that+directly+binds+to+and+degrades+cartilage+oligomeric+matrix+protein.">ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein.</a> FASEB. J. 20, 988-990 (2006).</li><li>Tang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Growth+Factor+Progranulin+Binds+to+TNF+Receptors+and+Is+Therapeutic+Against+Inflammatory+Arthritis+in+Mice.">The Growth Factor Progranulin Binds to TNF Receptors and Is Therapeutic Against Inflammatory Arthritis in Mice.</a> Science. 332, 478-484 (2011).</li><li>Wu, H., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progranulin+Resolves+Inflammation.">Progranulin Resolves Inflammation.</a> Science. 332, 427-428 (2011).</li><li>Hollenberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+family+of+tissue-specific+basic+helix-loop-helix+proteins+with+a+two-hybrid+system.">Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.</a> Mol. Cell. Biol. 15, 3813-3822 (1995).</li><li>Vojtek, A. B., Hollenberg, S. M., Cooper, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Ras+interacts+directly+with+the+serine/threonine+kinase+Raf.">Mammalian Ras interacts directly with the serine/threonine kinase Raf.</a> Cell. 74, 205-214 (1993).</li></ol>

Yeast two-hybrid screening has proven to be an effective tool in identifying protein interaction 16, 17. Compared with other approaches for identifying protein-binding partners, such as biochemical co-purification and protein chips, yeast two-hybrid system is a sensitive genetic approach that can be used for screening very high numbers of coding sequences in a relatively simple experiment; in addition, it detects the in vivo interaction and does not need complicated protein purification. Of course yeast two-hy...

<table border="1">
<tbody>
 <tr>
	<td>Name of the reagent</td>
	<td>Company</td>
	<td>Catalogue number</td>
 </tr>
 <tr>
	<td>ProQuest two-hybrid system</td>
	<td>Invitrogen</td>
	<td>10835</td>
 </tr>
 <tr>
	<td>YPD Growth Medium</td>
	<td>Clontech</td>
	<td>630409</td>
 </tr>
 <tr>
	<td>YPD Agar Medium</td>
	<td>Clontech</td>
	<td>630410</td>
 </tr>
 <tr>
	<td>Minimal SD agar Base</td>
	<td>Clontech</td>
	<td>630412</td>
 </tr>
 <tr>
	<td>-Leu DO Supplement</td>
	<td>Clontech</td>
	<td>630414</td>
 </tr>
 <tr>
	<td>-Leu/-Trp DO Supplement</td>
	<td>Clontech</td>
	<td>630417</td>
 </tr>
 <tr>
	<td>-His/-Leu/-Trp/-Ura DO Supplement</td>
	<td>Clontech</td>
	<td>630425</td>
 </tr>
 <tr>
	<td>3-Amino-1,2,4-Triazole (3AT)</td>
	<td>Sigma-Aldrich</td>
	<td>A8056</td>
 </tr>
 <tr>
	<td>Luria broth (LB)</td>
	<td>Sigma-Aldrich</td>
	<td>L3022</td>
 </tr>
 <tr>
	<td>X-Gal</td>
	<td>Invitrogen</td>
	<td>15520-034</td>
 </tr>
 <tr>
	<td>Sonicated Salmon Sperm DNA</td>
	<td>Stratagene</td>
	<td>201190</td>
 </tr>
 <tr>
	<td>Ampicillin</td>
	<td>AMRESCO</td>
	<td>0339</td>
 </tr>
 <tr>
	<td>Kanamycin Sulfate</td>
	<td>Invitrogen</td>
	<td>11815-024</td>
 </tr>
 <tr>
	<td>Subcloning Efficiency&trade; DH5&alpha;&trade; Competent Cells</td>
	<td>Invitrogen</td>
	<td>18265-017</td>
 </tr>
 <tr>
	<td>Trizma&reg; base</td>
	<td>Sigma-Aldrich</td>
	<td>T6066</td>
 </tr>
 <tr>
	<td>Lithium acetate</td>
	<td>Sigma-Aldrich</td>
	<td>L4158</td>
 </tr>
 <tr>
	<td>Ethylenediaminetetraacetic acid</td>
	<td>Sigma-Aldrich</td>
	<td>ED</td>
 </tr>
 <tr>
	<td>Nitrocellulose Membrane</td>
	<td>Bio-Rad</td>
	<td>162-0115</td>
 </tr>
 <tr>
	<td>10-cm petri dish</td>
	<td>ITI Scientific</td>
	<td>CT-903</td>
 </tr>
 <tr>
	<td>Incubator (30 &deg;C)</td>
	<td>ATR (Ecotron)</td>
	<td>&nbsp;</td>
 </tr>
</table>

modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Research

JoVE Journal

Biology

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...