1Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 2Department of Cell Biology, New York University School of Medicine
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Tian, Q. Y., Zhao, Y. P., Liu, C. j. Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin. J. Vis. Exp. (59), e3562, doi:10.3791/3562 (2012).
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.
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 protein that consists of separable domains responsible for DNA-binding and transcriptional activation. The bait protein is expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD), while the prey proteins are expressed as fusions to the GAL4 activation domain (AD). Interaction between bait and prey fusion proteins leads to the transcriptional activations of GAL4-binding sites containing reporter genes that are integrated into the yeast genome. The principle of Y2H is illustrated in Fig. 1 and the experimental procedure is summarized in Fig. 2.
2. Required materials and solutions
3. Bait generation (pDBLeu-PGRN)
A cDNA fragment encoding PGRN lacking signal peptide (a.a.21-588) was directionally cloned into the Sal I-Not I sites of the pDBLeu vector (the ProQuest two-hybrid system, Invitrogen), keeping the same translation reading frame as the GAL4 DNA Binding Domain to generate pDBLeu-PGRN.
4. Small scale transformation of bait plasmid
5. Bait validation
Before performing yeast two-hybrid screen, test pDBLeu-PGRN for self-activation and determine the basal expression levels of the HIS3 reporter gene. This test determines whether baits activate transcription and whether the self-activation can be neutralized by inhibitors. 3-Amino-1, 2, 4-triazole (3-AT) is a competitive inhibitor of the HIS3-gene product and can be used to titrate the minimum level of HIS3 expression required for growth on histidine-deficient media.
6. cDNA library screening
The pDBLeu-PGRN plasmid is introduced into MaV203 using a small-scale transformation as described above. To introduce pPC86-library (Invitrogen) into MaV203 (pDBLeu-PGRN), the procedure described below typically gives ~4 x 104 colonies with 0.5 μg of plasmid library DNA. Hence, 2.5 x 106 yeast transformants will require ~30.0 μg pPC86-cDNA library plasmid DNA, 25 transformations, and fifty 10-cm plates (SD-Leu-Trp-His-Ura+3AT).
7. X-gal assay
8. Retransformation assay
Prey fusion proteins (AD-Y) isolated from library screening should retain the interaction with bait fusion protein (DB-X) to induce the report genes, and the retransformation of prey clones and bait construct into yeast can further eliminate false positives and facilitate additional analysis.
9. Sequencing and bioinformatics analysis
Sequence the plasmid DNA that was isolated from likely true positive clones, compare these sequences to those in the GenBank using the BLAST program, and identify those two-hybrid clones that correspond to known genes. Sequencing data showed that two of the 12 positives obtained above were cell surface TNFR2 (TNFRSF1B/CD120b; Accession #NM_130426). In addition, the interaction between PGRN and TNFR was verified using various protein-protein interaction assays, including in vitro Solid-phase binding assay, Co-immunoprecipitation, Surface plasmon resonance analysis, and Flow cytometry assay 14.
10. Representative Results
The flow chart of the screening is outlined in Fig. 3. Typically 50-100 positive clone candidates will be obtained at this step. We initially isolated 54 positive clone candidates among 2.5 million transformants screened with the PGRN bait. Positive clone candidates were then verified by performing X-gal assay. Typically, approximately 50% false positive clones are removed via X-gal assay. We obtained 23 positive clone candidates at this step for the PGRN bait (Fig. 4). Retransformation of the prey clones and bait construct into yeast further eliminate false positives and clones that still activate the reporter genes likely represent the true positives. Typically, approximately 50% positive clone candidates will be removed. We finally isolated 12 positive clones that interact with PGRN in yeast.
Figure 1. Principle of yeast two-hybrid system
Figure 2. Pipeline of identifying protein binding partners using yeast two-hybrid system
Figure 3. Flow chart of screening yeast two-hybrid library. Click here to view a full-sized version of this image.
Figure 4. Beta-Galactosidase assay of positive clone candidates. A, Positive clones obtained from library screen were transferred to YPD plate and incubated at 30°C overnight; B, All colonies on YPD plate were transferred to nitrocellulose membranes and beta-galactosidase assay performed.
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-hybrid system has limitations, for example, the absence of the required post-translational modifications, the insufficient folding and/or stability of a fusion protein, and the altered subcellular location (proteins are brought to the nucleus and this may not be the real physiological state). Furthermore, the proteins that exhibit spurious activation of reporter genes, e.g. self activators, cannot be directly used as baits for screening the prey cDNA libraries. In order to overcome this drawback, the individual functional domains instead of intact proteins, are usually used as baits followed by the validations of the interactions using various approaches with intact proteins. For example, we have successfully isolated ADAMTS-7 metalloproteinase and progranulin growth factor as the binding partners of COMP using the EGF-like domain of COMP as a bait 10, 13.
The conventional screening procedure is time consuming, since the yeast transformants are plated on the first minus-three plates (SD-Leu-Trp-His +3AT ) to select His+ clones, which are then transferred to the second minus-three plates (SD-Leu-Trp-Ura +3AT) for Ura selection. In addition, the replication of thousands of positive clones needs special tools, and this screen process often results in a large number of false positives. We directly plated the yeast transformants on minus-four plates (SD-Leu-Trp-His-Ura +3AT) rather than two minus-three plates, because true interaction clones should be able to simultaneously activate all of the reporter constructs (i.e. producing histidine, and uracil) integrated into the yeast genome and should survive on minus-four plates (SD-Leu-Trp-His-Ura +3AT). This modification markedly shortens the process (e.g. the conventional screening procedure usually takes 10-20 days, whereas the modified screen procedure only needs 5-10 days), reduces the workload, and, most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable, and we have successfully employed this modified system in isolating the binding proteins of various baits, including sodium channel 12 and extracellular matrix protein 10, 13. Recently, we identified PGRN as a novel ligand of TNF receptors using this approach14, 15, providing a solid foundation for future discoveries relating to this growth factor.
No conflicts of interest declared.
This work was funded by NIH research grants K01AR053210, R01AR061484 and a grant from National Psoriasis Foundation.
|ProQuest two-hybrid system||Invitrogen||10835|
|YPD Growth Medium||Clontech Laboratories||630409|
|YPD Agar Medium||Clontech Laboratories||630410|
|Minimal SD agar Base||Clontech Laboratories||630412|
|-Leu DO Supplement||Clontech Laboratories||630414|
|-Leu/-Trp DO Supplement||Clontech Laboratories||630417|
|-His/-Leu/-Trp/-Ura DO Supplement||Clontech Laboratories||630425|
|Luria broth (LB)||Sigma-Aldrich||L3022|
|Sonicated Salmon Sperm DNA||Stratagene, Agilent Technologies||201190|
|Subcloning Efficiency‚™ DH5őĪ‚™ Competent Cells||Invitrogen||18265-017|
|10-cm petri dish||ITI Scientific||CT-903|
|Incubator (30 °C)||ATR (Ecotron)|