CAJ | 학술논문

经两次DE52和Sephacryl S-300柱层析,从棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW45粗提液(37 677 mg蛋白)中纯化得到628 mg的NifB-Av1.经考马斯亮蓝R-250染色的SDS凝胶电泳分析表明,该蛋白基本达到SDS凝胶电泳纯,组成它的亚单位的种类与Av1(α和β亚单位)相似.NifB-Av1不能与NifB-Av2重组成具放氢活性的固氮酶,但可使与其保温重组的FeMoco显出高活性.在合适条件下,NifB-Av1可在结晶溶液中析出棕色短斜四棱柱晶体,目前所得最大晶体的二维边长均为0.1 mm.能否出现晶体以及出晶时间、晶体数目、大小、质量和形状等,与沉淀剂溶液各组分的种类和浓度、结晶方法、实验操作等因素密切相关.初步结果表明,所得晶体为NifB-Av1单晶.
경량차DE52화Sephacryl S-300주층석,종종색고담균(Azotobacter vinelandii Lipmann)돌변충UW45조제액(37 677 mg단백)중순화득도628 mg적NifB-Av1.경고마사량람R-250염색적SDS응효전영분석표명,해단백기본체도SDS응효전영순,조성타적아단위적충류여Av1(α화β아단위)상사.NifB-Av1불능여NifB-Av2중조성구방경활성적고담매,단가사여기보온중조적FeMoco현출고활성.재합괄조건하,NifB-Av1가재결정용액중석출종색단사사릉주정체,목전소득최대정체적이유변장균위0.1 mm.능부출현정체이급출정시간、정체수목、대소、질량화형상등,여침정제용액각조분적충류화농도、결정방법、실험조작등인소밀절상관.초보결과표명,소득정체위NifB-Av1단정.
Six hundred and 28 mg of NifB- Av1 was obtained by a chromatography twice on DE 52columns and Sephacryl S-300 column from the crude extract (37 677 mg) of a nifB mutated strain UW45of Azotobacter vinelandii Lipmann. The protein was almost homogeneous as determined by Coomassiestaining of SDS gels. The analysis by SDS-PAGE showed that NifB-Av1 was similar to Av1 from wild-typestrain of A. vinelandii (OP) in the kinds of subunits ( α and β subunit). When complemented with Av2,NifB Av1 had hardly any H-reducing activity, but could be significantly activated by FeMoco extracted fromAv1. Under a suitable condition for crystallization, short dark-brown rhombohedral crystals could beobtained from NifB-Av1. Both of the longest sides of the biggest crystal were 0.1 mm. The time of theformation of crystals and number, size, quality and shape of crystals obviously depended not only on thekinds and concentrations of the components in the precipitant solution, but also on the methods forcrystallization and technical bias, etc. The preliminary results showed that the crystal seemed to beformed from NifB-Av1.