Developing genetic tools to exploit Chaetomium thermophilum for biochemical analyses of eukaryotic macromolecular assemblies.

Nikola Kellner,Javier Fernandez-Martinez,Brian T Chait,Miriam Sturm,Johannes Schwarz,Ulrich Kück,Sabine Griesel,Michael P Rout,Wenzhu Zhang,Ed Hurt

doi:10.1038/srep20937

Nikola Kellner, Javier Fernandez-Martinez + Show 8 more

Open Access

https://doi.org/10.1038/srep20937

Copy DOI

Abstract

We describe a method to genetically manipulate Chaetomium thermophilum, a eukaryotic thermophile, along with various biochemical applications. The transformation method depends on a thermostable endogenous selection marker operating at high temperatures combined with chromosomal integration of target genes. Our technique allows exploiting eukaryotic thermophiles as source for purifying thermostable native macromolecular complexes with an emphasis on the nuclear pore complex, holding great potential for applications in basic science and biotechnology.

Highlights

IntroductionThe use of thermostable proteins from prokaryotic thermophiles (bacteria, archaea) has been extensively exploited in the past for various biotechnological applications and in structural biology (reviewed in[1,2])
The use of thermostable proteins from prokaryotic thermophiles has been extensively exploited in the past for various biotechnological applications and in structural biology
The genetic method developed in this study is based on polyethylene glycol (PEG) induced protoplast transformation that was successfully applied in the past for members of the Pezizomycotina clade (e.g. Sordaria macrospora15) to which C. thermophilum belongs

Summary

Introduction

The use of thermostable proteins from prokaryotic thermophiles (bacteria, archaea) has been extensively exploited in the past for various biotechnological applications and in structural biology (reviewed in[1,2]). Several proof-of-principle studies exploiting the C. thermophilum proteome for biotechnological applications[6,7,8,9] or high-resolution 3D characterization of single proteins or reconstituted protein complexes[10,11,12,13,14] have been reported. These investigations required expression of C. thermophilum genes in heterologous mesophilic expression systems, as C. thermophilum has not yet been amenable for genetic manipulation. In an effort to fully exploit the potential of this thermophilic eukaryote as a source for biochemical and structural analyses of difficult proteins and derived complexes, we developed a transformation system for C. thermophilum, allowing the stable integration of constructs into the genome, which renders this thermophile accessible for affinity-purification of native thermostable proteins and protein complexes assembled under physiological conditions

Objectives

Methods

Results