英语翻译如题 350字英文求翻译 跪谢.The coding sequence of SBP1 and STM1 wer

英语翻译
如题 350字英文求翻译 跪谢.
The coding sequence of SBP1 and STM1 were obtained by PCR using the S.cerevisiae genomic DNA as template.The PCR products have NdeI and XhoI cutting sites at the 59 and 39 ends,respectively.The fragments were restricted and inserted into pET- 15b to generate the pET-SBP1 and pET-STM1 plasmids for the expression of His6-tagged Sbp1p and Stm1p.
HMT1 is available in the laboratory (Chern et al.2002).It was inserted into pBS-MP (Hwang et al.1999) to replace the coding region without changing the promoter sequence of the E.coli methionine aminopeptidase (MAP) gene (pBS-MAPp-hmt1).The HMT1 gene with the MAP promoter was then amplified from pBS-MAPp-hmt1 and inserted into the NotI site of a pGEX-STM1 plasmid,which is 11 nucleotides 39 to the stop codon of STM1.The STM1 and HMT1 genes in tandem were then amplified with 59 and 39 primers carrying NdeI and BamHI sites,respectively.The PCR product starts with the initiating codon of STM1 at the 59 end and inserted into the pET-15b vector to generate pET-STM1-(MAPp-hmt1).The sequence on the pET-15b vector encoding the His6-tag is between the NcoI and NdeI sites.Hence,STM1 is expressed as an N-terminal His6-tag fusion protein under the control of a T7 promoter.Finally,the STM1 on pET-STM1-(MAPp-hmt1) was replaced with SBP1 for Sbp1p/hmt1 production (pET-SBP1-[MAPphmt1],Fig.1).In addition,the arginine residue on the thrombin cleavage site of all of the expression plasmids mentioned above (pET-SBP1,pET-STM1,pET-STM1-[MAPp-hmt1] and pET-SBP1-[MAPp-hmt1]) was replaced with a lysine residue.Site-directed mutagenesis of the thrombin cleavage site and generation of the Stm1p R236K,R237K,R240K,and R243K mutants were carried out with QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.
E.coli BL21(DE3) cells were used as host for protein expression.Cells transformed with plasmids were grown in Lauia broth at 30°C until the culture reached an absorbance of 0.6 at 600 nm.Isopropyl b-D-1-thiogalactopyranoside 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.

在SBP1编码序列及STM1 PCR技术,获得了使用酿酒酵母基因组DNA为模板.PCR产物已在59和39结束,分别NdeI和切酶切割的网站.碎片受到限制,在出口的宠物插入- 15B条产生的PET - SBP1和pET - STM1的His6表达质粒标记Sbp1p和Stm1p.
HMT1可以在实验室（陈省身等.2002年）.这是插入到公共广播,手机（黄等.1999年）,以取代不改变大肠杆菌的蛋氨酸氨肽酶启动子序列（MAP）的基因的编码区（公共广播,马普- hmt1）.与地图基因启动子HMT1然后从公共广播扩增马普- hmt1成一个网站中插入pGEX诺蒂- STM1质粒,这是11个核苷酸39条STM1终止密码子.在串联的STM1和HMT1基因,然后扩增引物59和39载NdeI和BamHI位点,分别为.开始的PCR产物与STM1开始在59个密码子,并到年底的PET - 15B条载体产生聚脂STM1（马普- hmt1插入）.关于在PET - 15B条编码His6矢量标记序列之间的NcoI和NdeI网站.因此,STM1表示为N末端的His6 -标签下的T7启动子控制的融合蛋白.最后,在PET - STM1 -（马普STM1 - hmt1）是与Sbp1p/hmt1生产SBP1取代（聚脂SBP1 - [MAPphmt1],图.1）.此外,在上述的质粒（聚脂SBP1,宠物,STM1,宠物,STM1 - [马普- hmt1]和pET - SBP1 - [马普- hmt1]）中提到的表达凝血酶裂解位点的精氨酸残改为与赖氨酸残基.定点的凝血酶裂解位点及Stm1p R236K,R237K,R240K,并R243K代诱变突变体进行了有快速转换定点突变试剂盒（Stratagene）根据制造商的说明.
大肠杆菌BL21（DE3）中细胞蛋白表达作为主机使用.与质粒转化细胞生长在30℃至文化达到600纳米的0.6吸光度Lauia肉汤.异丙基的BD - 1 -半乳糖苷当时说（经IPTG,终浓度为1毫米）和细胞培养为4小时收获前.细胞裂解后,重组蛋白的亲和力与镍（Novagen）螯合珠纯化根据制造商的指示.