The macro domain as fusion tag for carrier-driven crystallization.

Rebekka Wild,Michael Hothorn

doi:10.1002/pro.3073

Rebekka Wild, Michael Hothorn

Open Access

https://doi.org/10.1002/pro.3073

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Abstract

Obtaining well-ordered crystals remains a significant challenge in protein X-ray crystallography. Carrier-driven crystallization can facilitate crystal formation and structure solution of difficult target proteins. We obtained crystals of the small and highly flexible SPX domain from the yeast vacuolar transporter chaperone 4 (Vtc4) when fused to a C-terminal, non-cleavable macro tag derived from human histone macroH2A1.1. Initial crystals diffracted to 3.3 Å resolution. Reductive protein methylation of the fusion protein yielded a new crystal form diffracting to 2.1 Å. The structures were solved by molecular replacement, using isolated macro domain structures as search models. Our findings suggest that macro domain tags can be employed in recombinant protein expression in E. coli, and in carrier-driven crystallization.

Highlights

Carrier-driven crystallization,[1,2] or chaperoneassisted crystallization,[3] describes the crystallization of a target protein by fusing it to a well-behaving protein tag, which may contribute to the formationAbbreviations: GST, glutathione-S-transferase; MBP, maltose binding protein; sizeexclusion chromatography (SEC), size-exclusion chromatography; TTM, triphosphate tunnel metalloenzyme; VTC, vacuolar transporter chaperone
Our findings suggest that macro domain tags can be employed in recombinant protein expression in E. coli, and in carrier-driven crystallization
SPX domains can be crystallized as macro domain fusion proteins The VTC SPX domains contain highly conserved Ntermini and are connected to the catalytic TTM domain by a short linker[22] (Fig. 1)

Summary

Introduction

Carrier-driven crystallization,[1,2] or chaperoneassisted crystallization,[3] describes the crystallization of a target protein by fusing it to a well-behaving protein tag, which may contribute to the formation

Methods

Results

Conclusion