星海链霉菌卤化酶重组蛋白在大肠杆菌中可溶表达

大肠杆菌(Escherichia coli,E.coli)是表达异源蛋白的良好宿主,但常遇到蛋白表达产物不可溶的困难.在对来自星海链霉菌的卤化酶基因sinH进行大肠杆菌异源表达时,为克服蛋白表达产物可溶性低的问题,探讨了不同载体对蛋白可溶表达的影响.利用pET28a进行SinH表达时不能得到可溶的目标蛋白;使用含有泛素标签及内含肽的载体pHUIE实现了卤化酶蛋白SinH的可溶表达,但纯化后纯度不高;利用带有链霉菌分子伴侣蛋白基因的载体pET28a-SinH与pETcoco-pL1SL2在E.coli BL21(DE3)中共表达,在30℃,0.1mmol/L IPTG诱导条件下表达4h,成功获得大量可溶蛋白,使用亲和层析(Ni-NTA)纯化,获得了较纯的目标蛋白SinH,上述研究结果为在大肠杆菌中进行其他链霉菌蛋白的可溶表达提供了参考.     

棉铃虫泛素基因的克隆及序列分析

泛素介导的泛素-蛋白酶体通路(Ubiquitin-proteasome pathway,UPP)是真核细胞内依赖ATP的非溶酶体蛋白质降解途径,该途径对细胞内蛋白的选择性降解起着重要作用.设计一对简并引物,从棉铃虫Helicoverpa armigera卵巢细胞Ha831中克隆了泛素基因的编码区,GenBank登录号AY456195.序列分析表明,该编码区的长度为228bp,编码76个氨基酸、其编码蛋白的相对分子质量为8 560,等电点为6.56.同源性比较发现,棉铃虫泛素基因与其他真核生物泛素基因在氨基酸水平上具有96%以上的相似性,而与棉铃虫核多角体病毒泛素的相似性为76%,所有已知的泛素关键功能位点在该泛素蛋白中均保守存在.     

BICP0 and its RING finger domain act as ubiquitin E3 ligases <Emphasis Type="Italic">in vitro</Emphasis>

Bovine infected-cell protein 0 (BICP0) encoded by bovine herpes virus 1 (BHV-1) immediate early gene is necessary for efficient productive infection, in a large part,because it activates all 3 classes of BHV-1 genes. It also has the ability to efficiently transactivate promoters that are not derived from BHV-1. To investigate the mechanism by which BICP0 achieves these effects, we expressed and purified BICP0 and its different mutants in E. coil In vitro assays showed that both full-length BICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in BICP0 activity in other assays. Based on these, we conclude that BICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and its RING finger domain is necessary for this function. These strongly support the hypothesis that BICPO might influence virus infection through its ability to interact with the ubiquitin-proteasome pathway.     

Ubiquitin-proteasome pathway as a primary defender against TRAIL-mediated cell death

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death as well as expression of proinflammatory genes such as CXCL8 in malignant human astrocytoma cells. However, the molecular mechanisms that determine the fate of cells are not yet understood. The ubiquitin (Ub)-proteasome pathway regulates a wide range of cellular functions through degradation of various regulatory proteins; given this, we hypothesized that this pathway may play a central role in TRAIL-mediated signaling. We demonstrate here that inhibition of the Ub-proteasome pathway enhanced TRAIL-mediated cell death of human astrocytoma CRT-MG cells within hours by blocking degradation of active caspase-8 and -3. Proteasome inhibitors suppressed TRAIL-mediated activation of NF-B; however, inhibition of the NF-B pathway alone was not sufficient to enhance TRAIL-mediated cell death. Collectively, these results suggest that the Ub-proteasome pathway may play an important role as an antiapoptotic surveillance system by eliminating activated caspases as well as mediating NF-B-dependent signals.Received 30 December 2003; received after revision 9 February 2004; accepted 13 February 2004     

泛素调节的蛋白质降解--2004年诺贝尔化学奖成果简介

以色列科学家阿龙·切哈诺沃(Aaron Ciechanover)、阿夫拉姆·赫什科(Avram Hershko)和美国科学家欧文·罗斯(Irwin Rose)因发现泛素调节的蛋白质降解被授予2004年诺贝尔化学奖.泛素是一种含76个氨基酸的多肽,存在于除细菌外的许多不同组织和器官中,具有标记待降解蛋白质的作用.被泛素标记的蛋白质在蛋白酶体中被降解.泛素控制的蛋白质降解具有重要的生理意义,它不仅能够清除错误蛋白质,对细胞生长周期、DNA复制以及染色体结构都有重要的调控作用.     

Protein oxidation and 20S proteasome-dependent proteolysis in mammalian cells

The generation of reactive oxygen species is an inevitable aspect of aerobic life. In addition to being exposed to free radicals in the environment, aerobic organisms must also deal with oxygen radicals generated as byproducts of a number of physiological mechanisms - for example, by the mitochondrial and endoplasmic reticulum electron transport chains, and by cells of the immune system. Although most organisms are equipped with several lines of defense against oxidative stress, these defensive mechanisms are not 100% effective, and oxidatively modified forms of proteins accumulate during aging, and in many pathological conditions.?Oxidatively modified proteins can form large aggregates due to covalent cross-linking or increased surface hydrophobicity. Unless repaired or removed from cells, these oxidized proteins are often toxic and can threaten cell viability. Mammalian cells exhibit only limited direct repair mechanisms, and oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the major cytosolic proteinase, the proteasome. Interestingly, it appears that the 20S 'core' proteasome conducts the recognition and elimination of oxidized proteins in an ATP-independent and ubiquitin-independent pathway. Received 31 May 2001; accepted 26 June 2001     

泛素-蛋白水解酶复合体通路在小鼠精子变态中的作用

应用重力沉降法分离睾丸各层细胞,通过蛋白聚丙烯酰胺凝胶电泳和蛋白免疫迹观察各种生精细胞中泛素结合蛋白的表达规律,并通过免疫组织化学方法对其进行组织和细胞水平的定位,表明,在圆形精子和长形精子细胞中泛素结合蛋白增加,在成熟精子中量较低,在精原和精母细胞中量微弱。总蛋白电泳的结果表明,在精子变态过程中蛋白 变化以长形精子细胞转变为成熟精子阶段最为剧烈,其间许多蛋白消失同时半随一些新蛋白的出现,此阶段消     

Tbfmoc-蛋氨酸在泛素固相合成中的应用

将合成的Tbfmoc-蛋氨酸替代Fmoc-蛋氨酸作为泛素的固相多肽化学合成原料。利用Tbfmoc-泛素衍生物与各种杂质在凝胶过滤层析及紫外吸收的显著差异,通过凝胶过滤层析将Tbfmoc-泛素从杂质中分离。在pH=8.5的温和条件下脱除Tbfmoc基团后,经氨基酸分析、质谱测定及高效液相色谱检测得到高纯度泛素产物。所用策略可有效克服采用Tbfmoc-Cl时导致反应时间长、效率低、增加副产物及产物分离纯化难度大等弊病,为用途广泛的泛素化学合成及分离纯化提供了高效简便的新方法。     

拟南芥Rad23原核重组蛋白构建和活性检测

在拟南芥中,Rad23基因通过参与泛素/26s蛋白酶体途径调节植物生长发育和激素应答.为进一步研究该基因编码蛋白的分子作用机制及其生物学功能,构建了pCold-Rad23融合蛋白重组表达载体,将该载体转化到大肠杆菌BL21中诱导表达目的蛋白.SDS聚丙烯酰胺凝胶电泳检测结果表明,分子质量为94 ku左右的融合重组蛋白在...     

拟南芥 BAH1 与大肠杆菌热胁迫耐受的关系分析

拟南芥BAH1含有保守的C3H4型RING结构域,与DnaJ锌指结构类似.利用原核表达纯化的BAH1进行体外泛素化实验证明了BAH1具有E3连接酶活性.然后通过表型回复实验发现BAH1融合J-domain结构域后(JdBAH1)和DnaJ一样能明显弥补danJ突变株MF634的热敏表型,在43℃存活;而转入突变锌指结构的JdBAH1C231S,C234S,C276S,C279S(JdBAH1△Zn1/2)菌株在43℃高温条件下不能存活,说明BAH1在大肠杆菌内具有类似DnaJ锌指结构的功能.因此,BAH1在E.coli中的功能有可能与DnaJ相似,通过锌指结构参与了DnaK/DnaJ伴侣系统发挥功能.