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....

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. Generat....

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 forma.......

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

List of materials used in this article

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