Three-step chromatographic purification procedure for the production of a his-tag recombinant kinesin overexpressed in E. coli.

A kinesin gene has been cloned by RT-PCR (reverse transcription polymerase chain reaction) from Trypanosoma brucei and the corresponding protein overexpressed as a recombinant His-tag (histidine-tag) kinesin in E. coli in order to study its biochemical properties and to determine its three-dimensional structure by X-ray crystallography. Starting from several liters of culture, an ultrasonic homogenizer was used for cell disruption and an unclarified feedstock was obtained. From this homogenate, a protein was then purified by immobilized metal affinity chromatography (IMAC) using expanded bed adsorption (EBA) technology (Streamline chelating). For this capture step, 100% of the recombinant protein was purified with more than 90% of purity. This step was followed by ion-exchange chromatography (Q Sepharose Fast Flow) for intermediate purification (96% purity, 53% recovery) and by size-exclusion chromatography with Superdex 75 as a polishing step (99% purity, 93% recovery). We then separated two forms of kinesin, a dimer (70%) and a monomer (30%). It was then possible to purify His-tag recombinant protein directly from feedstock in a rapid and efficient way and to isolate two forms of kinesin.