固定化枯草杆菌色氨酰tRNA合成酶(英文)

为研究ｔＲＮＡＴｒｐ 与色氨酰ｔＲＮＡ合成酶（ＴｒｐＲＳ） 的相互识别及其结构、功能关系, 纯化了枯草杆菌ＴｒｐＲＳ并用溴化氰活化的Ｓｅｐｈａｒｏｓｅ ４Ｂ 将ＴｒｐＲＳ固定化, 固定化ＴｒｐＲＳ的蛋白质回收率为９５ ．５ ％ , 活力回收率为３１．３％ 。研究了固定化ＴｒｐＲＳ的酶学性质, 其热稳定性和贮存稳定性方面均比液相ＴｒｐＲＳ有了较大的提高, 最适温度、最适ｐＨ 均有一定程度的增大, 工作稳定性良好。以固定化ＴｒｐＲＳ为亲和层析介质, 对含有２０ 个核苷酸随机序列、长度为５６ 个核苷酸的单链ＲＮＡ 随机库进行了３ 轮筛选,ＲＮＡ 群体亲和固定化ＴｒｐＲＳ的比例从４ ．３ ％ 上升至１４ ．７ ％ 。筛选得到了与ｔＲＮＡＴｒｐ 氨基酸接受茎类似的ＲＮＡ二级结构。实验结果表明固定化ＴｒｐＲＳ可以作为ＳＥＬＥＸ 亲和层析介质, 进行模拟ｔＲＮＡＴｒｐ 分子的ＲＮＡ 随机库的ＳＥＬＥＸ 筛选。     

固定化村草杆菌色氨酰—tRNA合成酶

为研究ｔＲＮＡ＾Ｔｒｐ与色氨酰－ｔＲＮＡ合成酶（ＴｒｐＲＳ）的相互识别及其结构、功能关系,纯化了枯草杆菌ＴｒｐＲＳ并用溴化氰活化的Ｓｅｐｈａｒｏｓｅ４Ｂ将ＴｒｐＲＳ固定化,固定化ＴｒｐＲＳ的蛋白质回收率为为９５．５％,活力回收率为３１．３％。研究了固定化ＴｒｐＲＳ的酶学性质。其热稳定性和贮存稳定性方面均比液相ＴｒｐＲＳ有了较大的提高,最适温度、最适ｐＨ均有一定程度的增大,工作稳定性良好。以固定化Ｔ     

树脂载体固定S-腺苷甲硫氨酸合成酶的研究

目的:筛选一种适合S-腺苷甲硫氨酸合成酶固定化的树脂载体,进行固定化工艺优化及固定化酶性质研究。方法:以固定化率和表观酶活回收率为指标,筛选固定化效果最佳的一种树脂,采用单因素实验对固定化条件进行优化。结果:阴离子交换树脂载体ESR-2表现出最优的固定化率(94.03%)和酶活回收率(47.45%);最佳固定化条件为加酶量4U/g、pH 8.0、15℃吸附10h,最佳条件下固定化酶表观酶活为2.1U/g,表观酶活回收率达51.6%。固定化酶的最适pH为8.5,最适温度为35℃,连续反应10批次后酶活剩余77.92%。结论:树脂载体ESR-2固定化S-腺苷甲硫氨酸合成酶酶活及稳定性较好,能够用于S-腺苷甲硫氨酸的工业化大规模生产。     

用SELEX技术筛选环孢霉素A适体

目的：运用指数富集的配体系统进化（SELEX）技术筛选并鉴定环孢霉素A（CsA）特异性适体。方法：合成全长78个核苷酸中间含35个随机序列的随机单链DNA（ssDNA）文库,通过SELEX方法,以CsA为靶标、磁珠为筛选介质,利用生物素-链亲和素-辣根过氧化物酶系统检测每轮ssDNA文库与CsA的亲和力,筛选针对CsA的适体,将最后一轮筛选产物克隆测序,并运用相关软件进行一级结构和二级结构分析。结果：经过10轮筛选,ssDNA文库与CsA的亲和力呈上升趋势,随机挑选的19个克隆适体根据一级结构的同源性可分为5个家族,二级结构预测以茎环（发夹）为主。结论：通过改良SELEX方法筛选得到了CsA特异性的适体。     

SELEX技术筛选纤维蛋白适配子的研究

目的:用纤维蛋白作为靶物质对ss DNA随机序列文库进行筛选,旨在获得高亲和力的纤维蛋白适配子。方法:在体外人工合成长度为99个核苷酸的ss DNA随机序列文库,文库中间区域为63个核苷酸的随机序列,两端为18个核苷酸的固定的引物序列;然后以羧基磁珠为介质包被纤维蛋白,利用指数级富集的配体系统进化技术(SELEX)对ss DNA随机序列文库进行反复筛选,当结合率不再提高时对筛选出的适配子进行连接、转化及测序分析。结果:羧基磁珠成功地包被了纤维蛋白,包被效率为87.65%,经15轮逐步递增压力的筛选,获得了纤维蛋白适配子群,经测序分析比对发现适配子有很好的多样性。结论:应用SELEX技术初步筛选出了亲和力较高的纤维蛋白适配子群,为下一步的鉴定及功能研究奠定了良好基础。     

人源色氨酰tRNA合成酶H130R突变体的酶活和初步晶体学分析（英文）

色氨酰t RNA合成酶(tryptophanyl-t RNA synthetase,Trp RS)催化色氨酸的活化及其特异性t RNA的氨酰化,为蛋白质合成提供原料。在IFN-γ刺激下,人源细胞中的Trp RS通过结合血红素增强其氨酰化活性,以调节吲哚胺2,3-双加氧酶表达水平增高所引起的色氨酸缺失。体外研究发现,锌离子和血红素竞争性结合人源Trp RS以增强其氨酰化活性。然而,由于一个氨基酸位点H130R的突变,牛和鼠的Trp RS以及人的H130R突变体都不再受锌离子和血红素的影响,并具有高氨酰化活性。H130R Trp RS模拟了一种结合着锌离子或血红素的高活性构象,但目前还没有对这种构象的结构描述。为了阐明H130R决定Trp RS氨酰化活性种属特异性的原因,以及锌离子和血红素的结合位点,作者对人H130R Trp RS进行了活性分析和初步晶体学研究,此工作将为锌离子和血红素调节Trp RS氨酰化活性的机制研究奠定基础。     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

SELEX筛选中不同筛选介质的富集效果比较

目的:比较SELEX筛选中不同筛选介质的富集效果,为高通量筛选奠定基础.方法:以乙肝表面抗原(HBsAg)为靶蛋白,采用两种不同的筛选介质:硝酸纤维素膜和环氧树脂,分别将HBsAg包被其上,利用SELEX技术从随机单链DNA文库中筛选得到富集的亲和配基库,最后通过聚丙烯酰胺凝胶电泳和实时荧光定量PCR检测各自的富集效果.结果:经过16轮筛选,发现实时荧光定量PCR时,硝酸纤维素膜空白管与阳性管的循环阈值均在14循环,无明显区别;而环氧树脂空白管与阳性管的循环阈值区别明显,前者是25循环,后者是18循环.结论:在SELEX筛选中,以环氧树脂为筛选介质更易富集到与靶蛋白特异性结合的核酸适配体.     

蒜氨酸酶的固定化及其酶学性质研究

为了提高蒜氨酸酶的稳定性并实现酶的反复利用,研究了影响蒜氨酸酶固定化的因素及固定化蒜氨酸酶的酶学性质。蒜氨酸酶的固定化以壳聚糖微球为载体,戊二醛为交联剂,固定化的最适条件为：戊二醛浓度4%,给酶量20.2U,交联时间2h。固定化蒜氨酸酶的最适pH值7.0,最适温度35℃,米氏常数Km 7.9 mmol/L,操作稳定性比较好,连续使用10次后酶活力损失低于10%。     

海因酶产生菌SHNU01的包埋固定化研究

对从土壤中筛选得到的高选择性产D-海因酶菌株SHNU01的性质进行了研究并探讨了用PVA-硼酸法进行包埋固定的效果。研究结果显示:固定化后的细胞与游离细胞相比,最适反应温度由游离细胞的37℃上升为47℃,在此温度下固定化细胞的酶活可达到游离细胞的3倍;连续反应7批后,固定化细胞活力为初始活力的80%。固定化细胞在操作稳定性、贮存稳定性等方面较游离细胞也有较大提高。     

3种水稻线粒体tRNA~(Trp)突变体的克隆和活力鉴定

 设计并完成了 3种水稻线粒体tRNATrp的突变 ,体外转录并用枯草杆菌和人色氨酰tRNA合成酶 (TrpRS)对tRNATrp及其突变体进行了活力测定 .3种突变体的氨酰化活力比野生型水稻线粒体tRNATrp分别上升了 1 8、1 5和 5倍 .说明A1 U72和G5 C68对于提高线粒体tRNATrp被细胞质TrpRS氨酰化能力的作用并不大 ,细胞质tRNATrp与细胞质TrpRS的识别方式并不适用于线粒体tRNATrp与细胞质TrpRS的相互识别 .研究结果对于了解线粒体tRNATrp和细胞质TrpRS的相互识别及药物设计有重要意义     

Recognition by tryptophanyl-tRNA synthetases of discriminator base on tRNATrp from three biological domains

To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNA(Trp) discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNA(Trp), representing the three biological domains. When B. subtilis, A. fulgidus, and human TrpRS were used to acylate these tRNA(Trp), two distinct preference profiles regarding the discriminator base of different tRNA(Trp) substrates were found: G>A>U>C for B. subtilis TrpRS, and A>C>U>G for A. fulgidus and human TrpRS. The preference for G73 in tRNA(Trp) by bacterial TrpRS is much stronger than the modest preferences for A73 by the archaeal and eukaryotic TrpRS. Cross-species reactivities between TrpRS and tRNA(Trp) from the three domains were in accordance with the view that the evolutionary position of archaea is intermediate between those of eukarya and bacteria. NMR spectroscopy revealed that mutation of A73 to G73 in bovine tRNA(Trp) elicited a conformational alteration in the G1-C72 base pair. Mutation of G1-C72 to A1-U72 or disruption of the G1-C72 base pair also caused reduction of Trp-tRNA(Trp) formation. These observations identify a tRNA(Trp) structural region near the end of acceptor stem comprising A73 and G1-C72 as a crucial domain required for effective recognition by human TrpRS.     

Residues Lys-149 and Glu-153 switch the aminoacylation of tRNA(Trp) in Bacillus subtilis

Tryptophanyl-tRNA synthetase (TrpRS) consists of two identical subunits that induce the cross-subunit binding mode of tRNA(Trp). It has been shown that eubacterial and eukaryotic TrpRSs cannot efficiently cross-aminoacylate the corresponding tRNA(Trp). Although the identity elements in tRNA(Trp) that confer the species-specific recognition have been identified, the corresponding elements in TrpRS have not yet been reported. In this study two residues, Lys-149 and Glu-153, were identified as being crucial for the accurate recognition of tRNA(Trp). These residues reside adjacent to the binding pocket for Trp-AMP and show phylogenic diversities in the charge on their side chains between eubacteria and eukaryotes. Single mutagenesis at Lys-149 or Glu-153 reduced the activity of TrpRS in the activation of Trp. The reduction was less than that caused by the double mutant WBHA (K149D/E153R). It is unusual that E153G had no detectable activity in the activation of Trp unless tRNA(Trp) was added to the reaction. In addition, we successfully switched the species specificity of Bacillus subtilis TrpRS recognition of tRNA(Trp). The affinity of WBHA, K149E and E153K to human tRNA(Trp) was 31-, 13.5-, and 12.9-fold greater than that of wild type B. subtilis TrpRS, respectively. Indeed WBHA and E153K were found to prefer genuine human tRNA(Trp) to their cognate eubacteria tRNA(Trp).     

Structure and activity of an aminoacyl-tRNA synthetase that charges tRNA with nitro-tryptophan

The most divergent of two tryptophanyl tRNA synthetases (TrpRS II) found in Deinococcus radiodurans interacts with a nitric oxide synthase protein that produces 4-nitro-tryptophan (4-NRP). TrpRS II efficiently charges transfer RNA(Trp) with 4-NRP and 5-hydroxy-tryptophan (5-HRP). The crystal structures of TrpRS II bound to tryptophan and 5-HRP reveal residue substitutions that accommodate modified indoles. A class of auxiliary bacterial TrpRSs conserve this capacity to charge tRNA with nonstandard amino acids.     

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.     

Structures of tryptophanyl-tRNA synthetase II from Deinococcus radiodurans bound to ATP and tryptophan. Insight into subunit cooperativity and domain motions linked to catalysis

An auxiliary tryptophanyl tRNA synthetase (drTrpRS II) that interacts with nitric-oxide synthase in the radiation-resistant bacterium Deinococcus radiodurans charges tRNA with tryptophan and 4-nitrotryptophan, a specific nitration product of nitric-oxide synthase. Crystal structures of drTrpRS II, empty of ligands or bound to either Trp or ATP, reveal that drTrpRS II has an overall structure similar to standard bacterial TrpRSs but undergoes smaller amplitude motions of the helical tRNA anti-codon binding (TAB) domain on binding substrates. TAB domain loop conformations that more closely resemble those of human TrpRS than those of Bacillus stearothermophilus TrpRS (bsTrpRS) indicate different modes of tRNA recognition by subclasses of bacterial TrpRSs. A compact state of drTrpRS II binds ATP, from which only minimal TAB domain movement is necessary to bring nucleotide in contact with Trp. However, the signature KMSKS loop of class I synthetases does not completely engage the ATP phosphates, and the adenine ring is not well ordered in the absence of Trp. Thus, progression of the KMSKS loop to a high energy conformation that stages acyl-adenylation requires binding of both substrates. In an asymmetric drTrpRS II dimer, the closed subunit binds ATP, whereas the open subunit binds Trp. A crystallographically symmetric dimer binds no ligands. Half-site reactivity for Trp binding is confirmed by thermodynamic measurements and explained by an asymmetric shift of the dimer interface toward the occupied active site. Upon Trp binding, Asp68 propagates structural changes between subunits by switching its hydrogen bonding partner from dimer interface residue Tyr139 to active site residue Arg30. Since TrpRS IIs are resistant to inhibitors of standard TrpRSs, and pathogens contain drTrpRS II homologs, the structure of drTrpRS II provides a framework for the design of potentially useful antibiotics.