Current Protocols in Molecular Biology
Featured Protocol

This Featured Protocol presents a cutting-edge method excerpted from Current Protocols in Molecular Biology UNIT 20.1. From UNIT 20.1 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins Contributed by Erica A. Golemis and Ilya Serebriiskii
Fox Chase Cancer Center
Philadelphia, Pennsylvania

Russell L. Finley. Jr. and Mikhail G. Kolonin
(hunt by interaction mating)
Wayne State University School of Medicine
Detroit, Michigan

Jeno Gyuris
Mitotix, Inc.
Cambridge, Massachusetts

Roger Brent
Massachusetts General Hospital and Harvard Medical School
Boston, Massachusetts

To understand the function of a particular protein, it is often useful to identify other proteins with which it associates. This can be done by a selection or screen in which novel proteins that specifically interact with a target protein of interest are isolated from a library. One particularly useful approach to detect novel interacting proteins--the two-hybrid system or interaction trap (see Figs. 20.1.1 and 20.1.2)--uses yeast as a "test tube" and transcriptional activation of a reporter system to identify associating proteins (see Background Information). This approach can also be used specifically to test complex formation between two proteins for which there is a prior reason to expect an interaction.

In the basic version of this method (see Fig. 20.1.2), the plasmid pEG202 or a related vector (see Fig. 20.1.3 and Table 20.1.1) is used to express the probe or "bait" protein as a fusion to the heterologous DNA-binding protein LexA. Many proteins, including transcription factors, kinases, and phosphatases, have been successfully used as bait proteins. The major requirements for the bait protein are that it should not be actively excluded from the yeast nucleus, and it should not possess an intrinsic ability to strongly activate transcription. The plasmid expressing the LexA-fused bait protein is used to transform yeast possessing a dual reporter system responsive to transcriptional activation through the LexA operator. In one such example, the yeast strain EGY48 contains the reporter plasmid pSH18-34. In this case, binding sites for LexA are located upstream of two reporter genes. In the EGY48 strain, the upstream activating sequences of the chromosomal LEU2 gene--required in the biosynthetic pathway for leucine (Leu)--are replaced with LexA operators (DNA binding sites). pSH18-34 contains a LexA operator-lacZ fusion gene. These two reporters allow selection for transcriptional activation by permitting selection for viability when cells are plated on medium lacking Leu, and discrimination based on color when the yeast is grown on medium containing Xgal (UNIT 13.6).

In Basic Protocol 1, EGY48/pSH18-34 transformed with a bait is characterized for its ability to express protein (Support Protocol 1), growth on medium lacking Leu, and for the level of transcriptional activation of lacZ (see Fig. 20.1.2A). A number of alternative strains, plasmids, and strategies are presented which can be employed if a bait proves to have an unacceptably high level of background transcriptional activation.

In an interactor hunt (Basic Protocol 2), the strain EGY48/pSH18-34 containing the bait expression plasmid is transformed (along with carrier DNA made as described in Support Protocol 2) with a conditionally expressed library made in the vector pJG4-5 (see Fig. 20.1.6). This library uses the inducible yeast GAL1 promoter to express proteins as fusions to an acidic domain ("acid blob") that functions as a portable transcriptional activation motif (act) and to other useful moieties. Expression of library-encoded proteins is induced by plating transformants on medium containing galactose (Gal), so yeast cells containing library proteins that do not interact specifically with the bait protein will fail to grow in the absence of Leu (see Fig. 20.1.2B). Yeast cells containing library proteins that interact with the bait protein will form colonies within 2 to 5 days, and the colonies will turn blue when the cells are streaked on medium containing Xgal (see Fig. 20.1.2C). The DNA from interaction trap positive colonies can be analyzed by polymerase chain reaction (PCR) to streamline screening and detect redundant clones in cases where many positives are obtained in screening (see Alternate Protocol 1). The plasmids are isolated and characterized by a series of tests to confirm specificity of the interaction with the initial bait protein (Support Protocols 3 to 5). Those found to be specific are ready for further analysis (e.g., sequencing). BASIC PROTOCOL 1 CHARACTERIZING A BAIT PROTEIN

The first step in an interactor hunt is to construct a plasmid that expresses LexA fused to the protein of interest. This construct is transformed into reporter yeast strains containing LEU2 and lacZ reporter genes, and a series of control experiments is performed to establish whether the construct is suitable as is or must be modified, and whether alternative yeast reporter conditions should be used. These controls establish that the bait protein is made as a stable protein in yeast, that it is capable of entering the nucleus and binding LexA operator sites, and that it does not appreciably activate transcription of the LexA operator-based reporter genes. This last is the most important constraint on use of this system. The LexA-fused bait protein must not activate transcription of either reporter-- the EGY48 strain (or related strain EGY191) that expresses the LexA fusion protein should not grow on medium lacking Leu, and the colonies should be white on medium containing Xgal. The characterized bait protein plasmid is used for Basic Protocol 2 to screen a library for interacting proteins.

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

Materials

DNA encoding the protein of interest

Plasmids (see Table 20.1.1): pEG202 (see Fig. 20.1.3), pSH18-34 (see Fig. 20.1.4), pSH17-4, pRFHM1, and pJK101 for basic characterization; other plasmids for specific circumstances as described (Clontech, Invitrogen, Origene, or R.Brent)

Yeast strain EGY48 (ura3 trp1 his3 3LexA-operator-LEU2), or EGY191 (ura3 trp1 his3 1LexA-operator-LEU2; Table 20.1.2)

Complete minimal (CM) medium dropout plates (UNIT 13.1), supplemented with 2% (w/v) of the indicated sugars (glucose or galatose), in 100-mm plates:

Glu/CM, -Ura, -His

Gal/CM, -Ura, -His

Gal/CM, -Ura, -His, -Leu

Z buffer (UNIT 13.6) with 1 mg/ml Xgal

Gal/CM dropout liquid medium (UNIT 13.1) supplemented with 2% Gal

Antibody to LexA or fusion domain: monoclonal antibody to LexA (Clontech, Invitrogen) or polyclonal antibody to LexA (available from R. Brent or E. Golemis)

H₂O, sterile
 

30°C incubator

Nylon membrane

Whatman 3MM filter paper

Additional reagents and equipment for subcloning DNA fragments (UNIT 3.16), lithium acetate transformation of yeast (UNIT 13.7), liquid assay b-galactosidase (UNIT 13.6), preparation of protein extracts for immunoblot analysis (see Support Protocol 1), and immunoblotting and immunodetection (UNIT 10.8)

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

1. Using standard subcloning techniques (UNIT 3.16), insert the DNA encoding the protein of interest into the polylinker of pEG202 (see Fig. 20.1.3) or other LexA fusion plasmid to make an in-frame protein fusion.
    

   The LexA fusion protein is expressed from the strong alcohol dehydrogenase (ADH) promoter. pEG202 also contains a HIS3 selectable marker and a 2µm origin for propagation in yeast. pEG202 with the DNA encoding the protein of interest inserted is designated pBait. Uses of alternative LexA fusion plasmids are described in Background Information.

2. Perform three separate lithium acetate transformations (UNIT 13.7) of EGY48 using the following combinations of plasmids:

   pBait + pSH18-34 (test)

   pSH17-4 + pSH18-34 (positive control for activation)

   pRFHM1 + pSH18-34 (negative control for activation).
     

   Use of the two LexA fusions as positive and negative controls allows a rough assessment of the transcriptional activation profile of LexA bait proteins. pEG202 itself is not a good negative control because the peptide encoded by the uninterrupted polylinker sequences is itself capable of very weakly activating transcription.

   pSH18-34 contains a 2µm origin and a URA3 selectable marker for maintenance in yeast, as well as a bacterial origin of replication and ampicillin-resistance gene. It is the most sensitive LacZ reporter available and will detect any potential ability to activate LacZ transcription. pSH17-4 is a HIS3 2µm plasmid encoding LexA fused to the activation domain of the yeast activator protein GAL4. This fusion protein strongly activates transcription. pRFHM1 is a HIS3 2µm plasmid encoding LexA fused to the N-terminus of the Drosophila protein bicoid. This fusion protein has no ability to activate transcription.
    
    

3. Plate each transformation mixture on Glu/CM -Ura, -His dropout plates. Incubate 2 days at 30°C to select for yeast that contain both plasmids.

   Colonies obtained can be used simultaneously in tests for the activation of lacZ (steps 4 to 7) and LEU2 (steps 12 to 13) reporters.

4. Streak a Glu/CM -Ura, -His master dropout plate with at least five or six independent colonies obtained from each of the three transformations in step 3 (test, positive control, and negative control) and incubate overnight at 30°C.
   The filter assay described in Steps 5a to 7a (based on Breeden and Nasmyth, 1985) provides a rapid assay for b-galactosidase transcription. Alternatively, a liquid assay (UNIT 13.6) or a plate assay (described in Steps 5b to 7b) may be used.

Perform filter assay for b-galactosidase activity:

5. a. Lift colonies by gently placing a nylon membrane on the yeast plate and allowing it to become wet through. Remove the membrane and air dry 5 min. Chill the membrane, colony side up, 10 min at -70°C.
   Whatman 3MM filters can be cut to the size of the yeast plate as a more economical alternative to nylon membranes for performing lifts. In addition, two or three 5-min temperature cycles (-70°C to room temperature) can be used instead of a single cycle to promote better lysis; this may be worth doing if there is difficulty visualizing blue color.
    
    

6. a. Cut a piece of Whatman 3MM filter paper slightly larger than the colony membrane and soak it in Z buffer containing 1 mg/ml Xgal. Place colony membrane, colony side up, on Whatman 3MM paper, or float it in the lid of a petri dish containing ~2 ml Z buffer with 1 mg/ml Xgal.
   Acceptable results may be obtained using as little as 300 µg/ml Xgal.
    

7. a. Incubate at 30°C and monitor for color changes.
   It is generally useful to check the membrane after 20 min, and again after 2 to 3 hr. Strong activators will produce a blue color in 5 to 10 min, and a bait protein (LexA fusion protein) that does so is unsuitable for use in an interactor hunt using this lacZ reporter plasmid. Weak activators will produce a blue color in 1 to 6 hr (compare versus negative control pRFHMI which will itself produce a faint blue color with time) and may or may not be suitable. Weak activators should be tested using the repressor assay described in steps 8 to 11.

Perform Xgal plate assay for lacZ activation:

5. b. Prepare Xgal plates as described in UNIT 13.1.
   For activation assays, plates should be prepared with glucose as a sugar source. For repression assays (steps 8 to 11), galactose should be used as a sugar source. In our experience, when patching from a master plate to Xgal plates, sufficient yeast are transferred that plasmid loss is not a major problem even in the absence of selection; this is balanced by the desire to assay sets of constructs on the same plate to eliminate batch variation in Xgal potency. Hence, plates should be made either with complete minimal amino acid mix, or by dropping out only uracil (-Ura), to make the plates universally useful.
    

6. b. Streak yeast from master plate to Xgal plate and incubate at 30°C.
    

7. b. Examine plates for color development at intervals over the next 2 to 3 days.

   
   Strongly activating fusions should be visibly blue on the plate within 12 to 24 hr; moderate activators will be visibly blue after ~2 days.

   When a bait protein appreciably activates transcription under these conditions, there are several recourses. The first and simplest is to switch to a less sensitive lacZ reporter plasmid; use of pJK103 and pRB1840 may be sufficient to reduce background to manageable levels. If this fails to work, it is frequently possible to generate a truncated LexA fusion that does not activate transcription.

For LexA fusions that do not activate transcription, confirm by performing a repression assay (Brent and Ptashne, 1984) that the LexA fusion protein is being synthesized in yeast (some proteins are not) and that it is capable of binding LexA operator sequences (Figure 20.1.5). The following steps can be performed concurrently with the activation assay.

8. Transform EGY48 yeast with the following combinations of plasmids (three transformations):

   pBait + pJK101 (test)

   pRFHM1 + pJK101 (positive control for repression)

   pJK101 alone (negative control for repression).
    

9. Plate each transformation mix on Glu/CM -Ura, -His dropout plates or Glu/CM -Ura dropout plates as appropriate to select yeast cells that contain the indicated plasmids. Incubate 2 to 3 days at 30°C until colonies appear.
    

10. Streak colonies to a Glu/CM -Ura, -His or Glu/CM -Ura dropout master plate and incubate overnight at 30°C.
     

11. Assay b-galactosidase activity of the three transformed strains (test, positive control, and negative control) by liquid assay (using liquid Gal/CM dropout medium), filter assay (steps 5a to 7a, first restreaking to Gal/CM plates to grow overnight), or plate assay (steps 5b to 7b, using Gal/CM -Ura XGal plates).
     

    This assay should not be run for more than 1 to 2 hr for membranes, or 36 hr for Xgal plates, as the high basal lacZ activity will make differential activation of pJK101 impossible to see with longer incubations. Use of Xgal plates, and inspection 12 to 24 hr after streaking, is generally most effective.

    The plasmid pJK101 contains the GAL upstream activating sequence (UAS) followed by lexA operators upstream of the lacZ coding sequence. Thus, yeast containing pJK101 will have significant b-galactosidase activity when grown on medium in which Gal is the sole carbon source because of binding of endogenous yeast GAL4 to the GAL_UAS. LexA-fused proteins that are made, enter the nucleus, and bind the lexA operator sequences block activation from the GAL_UAS, repressing b-galactosidase activity 3- to 20-fold. Note that on Glu/Xgal medium, yeast containing pJK101 should be white, because GAL_UAS transcription is repressed.
     

12. If a bait protein neither activates nor represses transcription, perform immunoblot analysis by probing an immunoblot of a crude lysate with antibodies against LexA or the fusion domain to test for protein synthesis (see Support Protocol 1).
     

    Even if a bait protein represses transcription, it is generally a good idea to assay for the production of full-length LexA fusions, as occasionally some fusion proteins will be proteolytically cleaved by endogenous yeast proteases. If the protein is made but does not repress, it may be necessary to clone the sequence into a LexA fusion vector that contains a nuclear localization motif, e.g., pJK202 (see Table 20.1.1), or to modify or truncate the fusion domain to remove motifs that target it to other cellular compartments (e.g., myristoylation signals).

    Test for Leu requirement

    These steps can be performed concurrently with the lacZ activation and repression assays.

13. Disperse a colony of EGY48 containing pBait and pSH18-34 reporter plasmids into 500 ml sterile water. Dilute 100 ml of suspension into 1 ml sterile water. Make a series of 1/10 dilutions in sterile water to cover a 1000-fold concentration range.
     

14. Plate 100 ml from each tube (undiluted, 1/10, 1/100, and 1/1000) on Gal/CM -Ura, -His dropout plates and on Gal/CM -Ura, -His, -Leu dropout plates. Incubate overnight at 30°C.
     

      
     

    There will be a total of eight plates. Gal/CM -Ura, -His dropout plates should show a concentration range from 10 to 10,000 colonies and Gal/CM -Ura, -His, -Leu dropout plates should have no colonies.

    Actual selection in the interactor hunt is based on the ability of the bait protein and acid-fusion pair, but not the bait protein alone, to activate transcription of the LexA operator-LEU2 gene and allow growth on medium lacking Leu. Thus, the test for the Leu requirement is the most important test of whether the bait protein is likely to have an unworkably high background. The LEU2 reporter in EGY48 is more sensitive than the pSH18-34 reporter for some baits, so it is possible that a bait protein that gives little or no signal in a b-galactosidase assay would nevertheless permit some level of growth on -Leu medium. If this occurs, there are several options for proceeding, the most immediate of which is to substitute EGY191(see Table 20.1.2), a less sensitive screening strain, and repeat the assay.

    As outlined in this protocol, the authors recommend the strategy of performing the initial screening using the most sensitive reporters and then, if activation is detected, screening with increasingly less sensitive reporters (see Critical Parameters for further discussion).

Plasmid	Selection		Comments
name/source	In yeast	In E. coli
LexA fusion plasmids
pEG202c,d,e	HIS3	Apr
pJK202	HIS3	Apr	Like pEG202, but incorporates nuclear localization sequences between LexA and polylinker; used to enhance translocation of bait to nucleus
pNLexA	HIS3	Apr	Contains an ADH promoter that expresses polylinker followed by LexA; used with baits where amino-terminal residues must remain unblocked
pGildad	HIS3	Apr	Contains a GAL1 promoter that expresses same LexA and polylinker cassette as pEG202; for use with baits whose continuous presence is toxic to yeast
pEE202I	HIS3	Apr	An integrating form of pEG202 that can be targeted into HIS3 following digestion with KpnI; used where physiological screen requires lower levels of bait to be expressed
pRFHM1e,f (control)	HIS3	Apr	Contains an ADH promoter that expresses LexA fused to the homeodomain of bicoid to produce nonactivating fusion; used as positive control for repression assay, negative control for activation and interaction assays
pSH17-4e,f (control)	HIS3	Apr	ADH promoter expresses LexA fused to GAL4 activation domain; used as a positive control for transcriptional activation
pMW101f	HIS3	Cmr	Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
pMW103f	HIS3	Kmr	Same as pEG202, but with altered antibiotic resistance markers; basic plasmid used for cloning bait
pHybLex/Zeof,g	Zeor	Zeor	Bait cloning vector compatible with interaction trap and all other two-hybrid systems; minimal ADH promotor expresses LexA followed by extended polylinker
Activation domain fusion plasmids
pJG4-5c,d,e,f	TRP1	Apr	Contains a GAL1 promoter expresses nuclear localization domain, transcriptional activation domain, HA epitope tag, cloning sites; used to express cDNA libraries
pJG4-5I	TRP1	Apr	An integrating form of pJG4-5 that can be targeted into TRP1 by digestion with Bsu36I (New England Biolabs); to be used with pEE202I to study interactions that occur physiologically at low protein concentrations
pYESTrpg	TRP1	Apr	Contains a GAL1 promoter that expresses nuclear localization domain, transcriptional activation domain, V5 epitope tag, multiple cloning sites; contains f1 ori and T7 promoter/flanking site; used to express cDNA libraries (Invitrogen)
pMW102f	TRP1	Kmr	Same as pJG4-5, but with altered antibiotic resistance markers; no libraries yet available
pMW104f	TRP1	Cmr	Same as pJG4-5, but with altered antibiotic resistance markers; no libraries yet available
LacZ reporter plasmids
pSH18-34d,e,f	URA3	Apr	Contains eight  LexA operators that direct transcription of the lacZ gene; one of the most sensitive indicator plasmids for transcriptional activation
pJK103e	URA3	Apr	Contains two LexA operators that direct transcription of the lacZ gene; an intermediate reporter for transcriptional activation
pRB1840e	URA3	Apr	Contains one LexA operator that directs transcription of the lacZ gene; one of the most stringent reporters for transcriptional activation
pMW112f	URA3	Kmr	Same as pSH18-34, but with altered antibiotic resistance marker
pMW109f	URA3	Kmr	Same as pJK103, but with altered antibiotic resistance marker
pMW111f	URA3	Kmr	Same as pRB1840, but with altered antibiotic resistance marker
pMW107f	URA3	Cmr	Same as pSH18-34, but with altered antibiotic resistance marker
pMW108f	URA3	Cmr	Same as pJK103, but with altered antibiotic resistance marker
pMW110f	URA3	Cmr	Same as pRB1840, but with altered antibiotic resistance marker
pJK101e,f (control)	URA3	Apr	Contains a GAL1 upstream activating sequences followed by two LexA operators followed by lacZ gene; used in repression assay to assess bait binding to operator sequences