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 overe xpressed as a recombinant His-tag (histidine-tag) kinesin in E. coli in ord er to study its biochemical properties and to determine its three-dimension al structure by X-ray crystallography. Starting from several liters of cult ure, an ultrasonic homogenizer was used for cell disruption and an unclarif ied feedstock was obtained. From this homogenate, a protein was then purifi ed by immobilized metal affinity chromatography (IMAC) using expanded bed a dsorption (EBA) technology (Streamline chelating). For this capture step, 1 00% of the recombinant protein was purified with more than 90% of purity. T his step was followed by ion-exchange chromatography (Q Sepharose Fast Flow ) for intermediate purification (96% purity, 53% recovery) and by size-excl usion chromatography with Superdex 75 as a polishing step (99% purity, 93% recovery). We then separated two forms of kinesin, a dimer (70%) and a mono mer (30%). It was then possible to purify His-tag recombinant protein direc tly from feedstock in a rapid and efficient way and to isolate two forms of kinesin. (C) 2000 Elsevier Science B.V. All rights reserved.