Overexpression and crystallization of a novel nucleoside base halogenase AcmX

Abstract:

Most recently a new group of regioselective adenine halogenases AcmX and AcmY have been found in Streptomyces, which can be employed to develop the halide substitution of nucleoside-like drugs. The crystal structures of this new family of halogenase are the basis to elucidate the catalytic mechanism and biosynthesize nucleoside-like drugs with halogen modifications. In order to solve the structure of AcmX of this new halogenase family, AcmX was overexpressed in Escherichia coli but was initially as the inactive inclusion body. Thus we constructed a dual expression system for AcmX and the chaperone plasmid pGro7, which promotes AcmX to fold properly by controlling the expression level of the chaperone plasmid. The chaperone protein is inclined to co-purify with the target protein but can be removed by a step of gel filtration chromatography, so that the protocol can be established to obtain AcmX in high quality, leading to the successful crystallization. This study will facilitate X-ray diffraction data collection and structure determination for AcmX and provide a useful protocol for the other difficult cases in expression and purification.