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
基金国家重点研发计划(2018YFC1903500)FoSE New Researchers Grant(I01210100011)+4 种基金澳大利亚Edith CowanUniversity Awards Grant(SOE20161124)浙江省钱江人才计划:QJD1803014Faculty Inspiration Grant UNNC(FIG2019)Sum mer Research Grant Support from UNNC 2020宁波市科技局公益项目(2019C10033&2019C10104)。
基金国家重点研发计划(2018YFC1903500)FoSE New Researchers Grant(I01210100011)+4 种基金澳大利亚Edith Cowan University AwardsGrant(SOE20161124)浙江省钱江人才计划:QJD1803014Faculty Inspiration Grant UNNC(FIG2019)Summer Research GrantSupport from UNNC 2020宁波市科技局工艺项目(2019C10033&2019C10104)。
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.