RESUMEN
Objective To construct a prokaryotic expression vector pET-22b+with Middle East respiratory syndrome ( MERS) coronavirus nuclocapsid protein( NP) gene and to express and purify N protein.Methods N gene amplified by PCR was inserted into the prokaryotic expression vector pET-22b+.Recombinant plasmid was confirmed using DNA elec-trophoresis and sequencing.NP was expressed in E.coli BL21(DE3) by IPTG induced and purified by cation exchange chromatography using the AKTA purification system.Results The NP gene sequence was proved to be correct by sequen-cing and the protein was expressed in both soluble and insoluble forms in E.coli BL21 ( DE3 ) after IPTG induction.The purity and concentration of recombinant protein was improved obviously by cation exchange chromatography and enrich-ment.Conclusion Recombiant NP of high purity and concentration is purified and will facilitate NP functional research.
RESUMEN
OsRacD belonging to rice Rho family of the small GTP binding proteins,is a pivotal gene involved in rice photoperiod fertility conversion of photoperiod sensitive genic male sterile rice,which influences rice fertility via controlling the pollen tube growth.T20N site-directed mutation was introduced into its highly conserved G1 motif by PCR-mediated method to mimic its GDP-binding state based on the sequences alignment and conserved domains analysis.The prokaryotic expression vector of OsRacD and T20N-OsRacD were constructed,and the His6 tag fused proteins were expressed and purified from E.coli.After identified by Western blot,the GTP hydrolysis activities were detected.The results showed that the GTPase activities of T20N-OsRacD were significantly reduced comparing with that of OsRacD,suggested that OsRacD and T20N-OsRacD have different biochemical characteristics.