Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Summary

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Abstract

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast¹ to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Introduction

Defining the molecular pathways or networks that mediate a particular biological process is one of the ultimate goals of biomedical research. Fly geneticists have depended heavily on forward genetics, especially modifier genetic screens (both enhancer and suppressor screens), to identify factors that work together, in parallel with, or upstream or downstream of a gene of interest. However, forward genetics screens often times fail to identify essential genes which, when mutated, cause lethality at early developmental stages, or genes with functional redundancy and compensation whose loss of function only cause subtle defects that are hard to score. One way to overcome this difficulty is to screen for direct protein-protein interactions. For more than a decade, a growing list of biochemical methods, including yeast two-hybrid, phage display, chemical cross-linking, Co-IP, Tandem Affinity Purification (TAP), etc. have been used to investigate protein-protein interactions. Each of these approaches has its own set of strengths and weaknesses in regards to sensitivity and specificity.  Among them, the TAP method allows for detection of physical interaction under near-physiological conditions,  preserves specificity and consistency² and includes the ability to extend to high-throughput analyses^3,4.

The TAP method was originally developed in yeast by Rigautand colleagues¹. In this method, a protein of interest is expressed with a TAP tag. The TAP tag harbors two independent affinity-binding domains: a Protein A domain that binds to IgG and a calmodulin-binding domain. The two domains are separated by a TEV (Tobacco Etch Virus) cleavage site. Such a combination allows for two independent rounds of affinity purifications to sufficiently reduce nonspecific bindings and enrich specific bindings¹. For this instance, the TAP method is a very powerful method to identify in vivo interactions of a given protein, although overexpressing the exogenous protein may make it more prone to associate with proteins that normally don't complex with its endogenous counterpart. Since its development, the TAP method has been applied in many other systems, including cell-culture-based systems^5,6 and other in vivo model systems^6-9. Here we describe the adaptation of the TAP method in Drosophila. We first generate pUAST-NTAP and pUAST-CTAP vectors to facilitate cloning and fusion of the TAP tag to either the N- or C-terminal of the gene of interest. The UAS-TAP-tagged transgene is then expressed in the nervous system under the control of a neuronal GAL4 driver¹⁰. Next, a large number of adult fly heads will be collected, which have high content of neural cells and are easy to separate from other body parts after freezing based on size differences. The adult heads are homogenized and cleared by sequential centrifugations, and the supernatant is subject to a TAP procedure described below.

Protocol

1. Generate UAS-TAP-tagged Transgenic Flies

1. Generate pUAST-TAP-tagged DNA constructs.
   1. Decide which side (N- or C-terminus) of the bait protein the TAP tag should be fused to, based on the protein's structure/function. See discussion for more details.
   2. Subclone the cDNA coding region of the gene of interest into the multiple cloning sites (MCS) of the pUAST-NTAP or pUAST-CTAP vectors to generate N- or C-terminal-tagged UAS-TAP transgenes, respectively. See Figure 1 for detailed maps and usable restriction sites and reading frames.
2. Generate UAS-TAP-tagged transgenic flies.
   1. Generate transgenic flies following standard protocols using P-element-mediated insertion¹¹. A number of injection services are commercially available.
   2. Cross neuronal GAL4 driver (i.e. BG380-GAL4) to each individual transgenic line and determine the protein expression levels of each line by western blot (with Peroxidase Anti-Peroxidase antibody) and/or immunostaining (with anti-TAP antibody). In general, a transgenic line with a protein expression level that is close to the endogenous protein is recommended for  TAP procedures. See discussion for more details.
   3. PerformGAL4/UAS-based rescue experiments to confirm the functionality of the TAP-tagged transgenes if loss-of-function mutants of the genes of interest are available. Choose a transgene that can substantially rescue the mutant phenotypes for the following TAP experiments.

2. Prepare Samples for TAP Procedure

1. Generate a fly stock that carries both a neuronal GAL4 driver (e.g. BG380-Gal4) and the chosen TAP-tagged transgene in order to ease expansion of fly samples. Collect the F1 progenies of the GAL4 driver and the UAS-transgene cross in rare cases when the above combination causes survival and growth disadvantage.
2. Collect small scale samples and optimize lysis condition for solubilizing the TAP-tagged protein.
   1. Make a series of lysis buffers using a combination of the nonionic detergents NP-40 (0.1-1%), NaDOC (0.1-1%) and Triton X-100 (0.05-0.5%). See Table 1 and discussion for more information.
   2. On top of a CO2 pad, use #5 forceps to dissect 20 adult heads from the TAP transgene expressing flies and collect them in a 1.5 ml tube to test each lysis buffer condition.
   3. Add 100 ul testing lysis buffer to the tube and homogenize the heads by stroking up and down with a plastic pestle, then add another 100 ul testing buffer.
   4. Spin the head lysate at 21,500 x g for 10 min (4 °C), and separate the supernatant and pellet after centrifugation. Add 25 ul 2x SDS loading buffer to the pellet and 10 ul 4x SDS loading buffer to 30 ul of supernatant respectively.
   5. Boil the two samples for 5 min and analyze them side-by-side using SDS-PAGE and subsequent Western Blot with the PAP antibody. Determine the solubility by the ratio of TAP-protein levels in supernatant vs the pellet.
3. Prepare sample in large scale
   1. Expand and collect adult flies.
      1. Expand the neuronal-GAL4/UAS-TAP-transgene stock in bottles and flip the bottles every 3 days till accumulative 250 bottles are used for collection.
      2. Collect 1-3 days old adult flies into 50 ml conical tubes, put the tube in liquid nitrogen immediately to deep freeze the flies.  Store the flies in a -80 °C freezer. Note that the volume of the flies must not exceed 2/3 of the 50 ml tube.
   2. Collect fly heads (perform this step on top of powdered dry ice).
      1. Take out the prechilled sieves and the mortar and pestle from the -80 °C freezer and put them on dry ice, ideally inside a large ice bucket. Stack two U.S.A. standard test sieves with a No. 25 on the top and No. 40 at the bottom.
      2. Take the frozen flies out and drop them in liquid nitrogen and keep the flies in there for about 10 min.  Vortex or shake the tubes vigorously to break the heads, legs, and wings from the bodies.
      3. Pour the mixture to the top sieve, and then shake the sieves vigorously while holding both sieves together. After sieving, the bodies will stay on the top sieve, fly heads will be retained on the bottom sieve, and legs, wings, and other debris will fall to the dry ice. Separate the two sieves and carefully transfer the fly heads to the cold mortar.
   3. Homogenize the fly heads
      1. On top of dry ice, grind the heads with the mortar and the pestle to powdery particles, then transfer the powder to a 15 ml glass Dounce Tissue Grinder that was prechilled on ice.
      2. Measure the weights of the grinder before and after the head sample was poured into it, and then calculate how much the head sample weighs. A total of 6-15 grams of fly heads will be sufficient for each TAP experiment adjusting accordingly to the expression levels of the protein. Add 15 ml of ice-cold homogenization buffer (lysis buffer optimized in step 2.2) to the powder and then stroke with the large clearance pestle until it is easy for the pestle to go up and down. Keep the glass grinder on ice at all times.
   4. Prepare the supernatant for TAP
      1. Transfer the homogenate to a high-speed centrifuge tube and spin for 20 min at ~50,000 x g (4 °C). Transfer the supernatant to a new high-speed centrifuge tube and repeat the centrifugation one more time.
      2. Transfer the supernatant to an ultracentrifuge tube and perform a 40 min ~250,000 x g spin to further clear the supernatant. The supernatant is ready for the tandem affinity purification procedures after ultracentrifugation.

3. TAP Purification

The following sections were derived from the Séraphin lab TAP protocol¹² (http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/Bertran%20Seraphin%27s%20TAP%20page.pdf )

1. Perform IgG bead affinity purification
   1. Prepare IgG sepharose bead while the samples are being centrifuged. Wash 400 µl IgG bead 3x in a 15 ml Falcon tube with 10 ml cold IgG washing buffer. For each wash, rock the tube gently for 2 min, and then spin down the beads at 1,000 x g for another 2 min. At the end of the third wash, remove the buffer and leave only the beads in the tube.
   2. Carefully transfer the cleared supernatant (~15 ml) into the 15 ml tube containing the IgG beads. Incubate the beads and brain lysate mix at 4 ºC on a nutator for 2 hr.
   3. Set up a clean and empty micro column with about 15 ml total volume in the cold room. Load the IgG bead mixture by steadily pouring the mix into the column; try not to trap any air bubbles inside the column. Allow the beads to settle in the column and the buffer to slowly drain by gravity flow.
   4. Wash the column thoroughly with 10 ml of cold IgG washing buffer after all of the brain lysate has flowed through the settled IgG column. Repeat the wash 2x. Note: never let the bead dry in the air.
2. Perform TEV cleavage
   1. After the third wash, wash the column again with 10 ml TEV cleavage buffer. This step prepares the IgG bead that sequestrates the bait complex for TEV cleavage.
   2. Right before the last drop of TEV buffer is about to drip out, put a cap at the bottom of the column to block the flow, add 1.3 ml TEV buffer containing 130 units of TEV enzyme to the column, and then securely cap the top of the column. Make sure the column is sealed well at both ends.
   3. Rotate the column at 18 ºC for 2 hr to allow the TEV enzyme to cleave the peptide at the TEV site and release the protein complex while leaving behind the protein A domain peptide bound to the IgG sepharose beads.
3. Perform Calmodulin bead affinity purification
   1. Prepare the Calmodulin beads while the IgG beads are incubated with the TEV enzyme. Wash 200 μl Calmodulin beads in a 15 ml Falcon tube 3x, each time with 10 ml of cold Calmodulin binding buffer. For each wash, gently rock the tube for 2 min on a nutator, and then spin down the beads at 1,000 x g for 2 min. At the end of the third wash, take out all the buffer and leave only the beads in the tube.
   2. At the end of the TEV incubation (step 3.2.3), return the IgG column back to the cold room and set it straight up. Let the beads settle for 10 min.
   3. Remove the top cap and then the bottom cap, and then collect the 1.3 ml TEV cleavage product in a 15 ml Falcon tube. Let the buffer drain completely. Add an additional 200 µl TEV buffer to the column to push out the dead volume of the column, collect the flow-out in the same tube.
   4. Add 4.5 ml of Calmodulin binding buffer and 4.5 µl 1 M CaCl₂ to the 1.5 ml TEV cleavage product collected above. The CaCl₂ serves to titrate the EDTA in the TEV buffer. Transfer the 6 ml mixture to the tube containing the Calmodulin beads and rotate the tube at 4 ºC on a nutator for 1 hr.
   5. Set up another clean and empty micro column with about 10 ml total volume in the cold room. Load the Calmodulin bead mixture to the column and allow it to drain by gravity.
   6. When all the solution has flowed through the settled Calmodulin column, wash the column two times, each with 10 ml of cold Calmodulin binding buffer. Note: avoid disturbing the Calmodulin beads and try to keep the surface of the beads as flat as possible during the wash.
4. Elute the bait complex from Calmodulin column.
   Right after washing, elute the Calmodulin column with five fractions of 200 µl cold Calmodulin elution buffer. For each fraction, gently add 200 µl of elution buffer to the column and collect the eluate with a marked 1.5 ml Eppendorf tube. Repeat this 4x.
5. Analyze the protein complex by SDS-PAGE
   1. Take a small aliquot from each of the five fractions (about 30 µl) and add SDS loading buffer. Boil the samples for 5 min and load the samples side-by-side with protein molecular markers in a gradient (4-15%) SDS-PAGE gel.
   2. After the samples have fully resolved in the gel, stop the electrophoresis and stain the gel with any G-250-based sensitive colloidal Coomassie staining procedures such as 'blue silver' staining¹³. Silver staining is optional but not preferable because it is not fully compatible with the subsequent mass spectrometry analysis.  Store the rest of the eluate in a -80 °C freezer for further analysis such as mass spectrometry for uncovering the molecular identities of the purified protein complex. See discussion.

Results

Here we demonstrate our effort in identifying Highwire-interacting proteins in the fly brain. Highwire (Hiw) and its vertebrate and invertebrate homologues are huge ubiquitin ligases that regulate the development and repair of the nervous system¹⁴. They share a number of highly conserved functional domains. However, their molecular actions are not entirely clear. Work done in worm, fly and mouse led to the current working model that Hiw functions as an E3 ligase and as a scaffolding protein to facilitate ...

Discussion

Tandem affinity purification (TAP) method offers a dual purification protocol that allows the isolation and enrichment of protein complexes through two independent affinity purification steps. The design of the TAP tag is not restricted to what is presented in this protocol, other protein binding domains and motifs are also applicable if buffer conditions are adjusted accordingly. A good example of other TAP tags is the GS-TAP tag, a combination of a G protein and a streptavidin-binding motif, designed by Giulio Superti-...

Materials

Name	Company	Catalog Number	Comments
U.S.A. standard test sieve No. 25	Fisher Scientific	04-881-18
U.S.A. standard test sieve No. 40	Fisher Scientific	04-881-21
Kontes Dounce Tissue Grinders 15 ml	Kimble Chase	885300-0015
IgG sepharose beads	Pharmacia	17-0969-01
Econo-column 0.7 cm x 20 cm	Bio-Rad	737-4721
Econo-column 0.5 cm x 15 cm	Bio-Rad	737-4716
Calmodulin beads	Stratagene	214303
Coors Mortar and Pestle	CoorsTek	60311
AcTEV Protease	Invitrogen	12575-015
Protease Inhibitor Cocktail	Roche	11836153001
Protease Inhibitor Mix	Sigma	P8340