Purification, crystallization and preliminary crystallographic analysis of the Hermes transposase

Abstract

DNA transposition is the movement of a defined segment of DNA from one location to another. Although the enzymes that catalyze transposition in bacterial systems have been well characterized, much less is known about the families of transposase enzymes that function in higher organisms. Active transposons have been identified in many insect species, providing tools for gene identification and offering the possibility of altering the genotypes of natural insect populations. One of these active transposons is Hermes, a 2749-base-pair element from Musca domestica that encodes its own transposase. An N-terminally deleted version of the Hermes transposase (residues 79-612) has been overexpressed and purified, and crystals that diffract to 2.1 A resolution have been obtained at 277 K by the hanging-drop method.

Figure 1

Schematic diagram of the Hermes transposase. Six conserved primary sequence blocks (designated A–F) are found in most hAT transposases (Rubin et al., 2001 ▶). The three blocks D–F that cluster at the C-terminus are found within the hAT domain, which consists of residues 527–604. The three major proteolytic cleavage sites after residues 78, 162 and 482 are marked by arrows. Three important catalytic residues are Asp180, Asp248 and Glu572.

Figure 2

Elution profile of Hermes 79–612 S163G on a TSK-Gel G3000SW size-exclusion column. (a) Hermes 79–612 S163G elutes as three distinct species corresponding to a large-molecular-weight aggregate (peak 1), a hexamer (peak 2) and a smaller approximately dimeric species (peak 3). (b) SDS–PAGE analysis of the protein content of the three peaks. The arrow indicates the fragment in peak 3 subjected to MALDI–TOF (see text).

Figure 3

(a) Crystals of Hermes 79–612 S163G. (b) SDS–PAGE analysis of dissolved crystals. Lane 1, molecular-weight markers (kDa); lane 2, dissolved crystals.