350字英语求翻译

SBP1和STM1的编代码序列被PCR得到使用S. cerevisiae基因组的脱氧核糖核酸阿斯样扳.PCR产品在59和39末端分别有NdeI和XhoI尖刻的地点.碎片是限制级和变为pET- 15b附着为His6-给Sbp1p和Stm1p加上标签的表达产生 pET-SBP1和pET-STM1质粒.HMT1是在实验室中可用的(Chern等等2002).它被把插入pBS-MP(Hwang等等1999),不改变 E.***同里蛋氨酸氨基肽酶((MAP)基因((pBS-MAPp-hmt1)的促进者序列取代编代码地区.有地图促进者was那时的 HMT1基因从pBS-MAPp-hmt1扩大和把一向STM1的停止密码子是11核苷酸39的pGEX-STM1质粒的地点插入NotI.在纵列二马拉的双轮马车中STM1和HMT1基因是那时,随着分别带着NdeI和BamHI地点59和39初步知识扩大.PCR产品在59末端以开始开始密码子的STM1和变为pET-15b矢附着产生pET-STM1-((MAPp-hmt1).有关将His6-标签编码pET-15b矢序列是在NcoI和NdeI地点之间.因此,STM1作为一受到一个T7促进者的控制N-终端His6-标签熔化蛋白质被表达.最后, 在pET-STM1-(MAPp-hmt1)was上STM1用SBP1为代替Sbp1p/hmt1生产((pET-SBP1-[MAPphmt1],Fig. 1).此外,在在所有当中表达质粒的凝血酶分裂地点上精氨酸残渣在上方提到(pET-SBP1,pET-STM1,pET-STM1-[[MAPp-hmt1]和 pET-SBP1-[[MAPp-hmt1)]被用一赖氨酸残渣代替.凝血酶分裂地点和产生Stm1p R236K,R237K,R240K和R243K突变体的地点-导演突变形成被地点-导演有的QuickChange执行根据manufacturer’s指令突变形成配套元件 ((Stratagene).E.***同里BL21((DE3)细胞被使用作为主人为蛋白质表达.直到文化在600 nm达到一吸光率的0.6,用质粒改变细胞被在Lauia原汁清汤肉汤阿特30°C中长出.异丙基b-D-1-thiogalactopyranosid e was then added (IPTG, 1 mM final concentration) and cells were cultured for 4 h before harvesting. After cell lysis, the recombinant proteins were affinity purified with Ni2 chelating beads (Novagen) according to the manufacturer’s instructions.