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</...

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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 ...

modified-yeast-two-hybrid-system-to-identify-proteins-interacting

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28.5K visualizações.  NYU Hospital for Joint Diseases. Nós modificamos o fermento convencional de dois híbridos de rastreamento, uma ferramenta eficaz na identificação genética de interação de proteínas. Esta modificação marcadamente encurta o processo, reduz a carga de trabalho, eo mais importante, reduz o número de falsos positivos. Além disso, esta abordagem é reproduzível e confiável.