Cloning and expression of kinesins from the thermophilic fungus Thermomyces lanuginosus

Abstract

The motor domain regions of three novel members of the kinesin superfamily TLKIF1, TLKIFC, and TLBIMC were identified in a thermophilic fungus Thermomyces lanuginosus. Based on sequence similarity, they were classified as members of the known kinesin families Unc104/KIF1, KAR3, and BIMC. TLKIF1 was subsequently expressed in Escherichia coli. The expression level was high, and the protein was mostly soluble, easy to purify, and enzymatically active. TLKIF1 is a monomeric kinesin motor, which in a gliding motility assay displays a robust plus-directed microtubule movement up to 2 microm/s. The discovery of TLKIF1 also demonstrates that a family of kinesin motors not previously found in fungi may in fact be used in this group of organisms.

Temperature dependent properties of a kinesin-3 motor protein from Thermomyces lanuginosus

“…cDNA was generated from total RNA was isolated using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo(dT) primers, and subsequently amplified using sequence specific primers (Forward: 5 0 -ATGTCGGGCGGTGGAAATAT CAA-3 0 ; Reverse: 5 0 -GATATCGAATTCCTGCTTCG CTG-3 0 ) based on the previously published sequence (Sakowicz et al, 1999). The resulting 2358-bp amplified product was cloned into the pET-Blue1 expression system (Novagen, Madison, WI), and a 10· poly-Histidine tag was inserted at the 3 0 end of the amplified TKIN sequence.…”

“…Characterization of TLKIF1 suggested that enzymatic activity was lost following incubation above 45°C (Sakowicz et al, 1999). Thus, to further evaluate the temperature-dependent states of TKIN, samples of purified TKIN (3 mg mL À1 ) were heated for 3 min at 10°C intervals between 30 and 80°C, and allowed to cool to room temperature.…”

“…Several kinesin-3 motor proteins have been isolated from, or predicted from the sequenced genomes of ascomycetous and basidiomycetous fungi (Adio et al, 2006;Fuchs and Westermann, 2005;Sakowicz et al, 1999;Schoch et al, 2003;Wedlich-Soldner et al, 2002). A kinesin-3 motor protein was isolated from the thermophilic, anamorphic fungus Thermomyces lanuginosus Tsiklinsky (Sakowicz et al, 1999).…”

“…A kinesin-3 motor protein was isolated from the thermophilic, anamorphic fungus Thermomyces lanuginosus Tsiklinsky (Sakowicz et al, 1999). Initial characterization of this kinesin demonstrated microtubule transport velocities of $2 lm s À1 at room temperature, and the maintenance of catalytic activity up to 45°C (Sakowicz et al, 1999).…”

“…A kinesin-3 motor protein was isolated from the thermophilic, anamorphic fungus Thermomyces lanuginosus Tsiklinsky (Sakowicz et al, 1999). Initial characterization of this kinesin demonstrated microtubule transport velocities of $2 lm s À1 at room temperature, and the maintenance of catalytic activity up to 45°C (Sakowicz et al, 1999). Similarly, a monomeric kinesin related to the kinesin-3 family was identified from the corn smut fungus Ustilago maydis (DeCandolle), and shown to be responsible, along with dynein, for transporting endosomes in fungal hyphae (Wedlich-Soldner et al, 2002).…”