In order to obtain phase information for the X-ray diffraction of tabtoxin resistance protein (TTR) crystal using the MAD phasing method, a selenomethionine (Se-Met) derivative of TTR was overexpressed in E. coli stra...In order to obtain phase information for the X-ray diffraction of tabtoxin resistance protein (TTR) crystal using the MAD phasing method, a selenomethionine (Se-Met) derivative of TTR was overexpressed in E. coli strain M15, with pQE-30 plasmid, through IPTG induction in M9 medium containing Se-Met. The product was purified to an estimated homogeneity of greater than 95% according to SDS-PAGE, by a Ni-NTA metal affinity followed by a Mono Q anion exchange column chromatography. The successful substitution of Se-Met for methionine (Met) was confirmed by MALDI-TOF and ESI-Quadrupole Mass Spectrometry analysis. The derivative crystal was obtained using similar conditions as those for the native.展开更多
基金This work wassupported by the National Natural Science Foundation of China (Grant Nos. 39870174 and 39970155) the National "863" Project (Grant No. 103130306) and the National "973" Project (Grant Nos. G1999075602, G19990II902 and 1998051105).
文摘In order to obtain phase information for the X-ray diffraction of tabtoxin resistance protein (TTR) crystal using the MAD phasing method, a selenomethionine (Se-Met) derivative of TTR was overexpressed in E. coli strain M15, with pQE-30 plasmid, through IPTG induction in M9 medium containing Se-Met. The product was purified to an estimated homogeneity of greater than 95% according to SDS-PAGE, by a Ni-NTA metal affinity followed by a Mono Q anion exchange column chromatography. The successful substitution of Se-Met for methionine (Met) was confirmed by MALDI-TOF and ESI-Quadrupole Mass Spectrometry analysis. The derivative crystal was obtained using similar conditions as those for the native.
