体外蛋白质复性方法及小分子添加剂在复性过程中的作用

变性蛋白质体外复性的方法主要有传统的辅助复性法包括了:稀释法、透析法、超滤法以及蛋白质折叠液相色谱法(PFLC),也有在此基础上模拟体内分子伴侣、折叠酶、人工分子伴侣、反胶束的辅助复性方法。到目前为止,在复性液中添加小分子试剂是提高体外辅助蛋白质复性效率的策略之一。其目的是为了降低蛋白质复性过程中聚集形式的形成,从而促进变性蛋白质向其天然和活性构象的转变。这种方法在一定程度上能够提高蛋白质的复性效率并且得到了长足的发展。为了捕捉变性蛋白质向其活性结构折叠过程的差异性变化规律,明确小分子添加剂对体外辅助蛋白质分子折叠过程的机制,推测一些小分子添加剂辅助蛋白质色谱复性过程中构象的变化规律,为解决包涵体蛋白质难于复性和复性效率低的问题起到指导性的作用。     

苦荞过敏原TBa和TBb基因的共表达及其包涵体复性的研究

[研究目的]利用大肠杆菌共表达苦荞过敏蛋白的两个亚基,并对表达产物进行包涵体复性研究和免疫学活性分析;[方法]用已构建的表达质pET-28a-TBa和pET-32m-TBb,共转化大肠杆菌BL21(DE3)感受态,利用双抗生素筛选法,获得稳定遗传的双质粒转化子,经IPTG诱导,两个基因在同一宿主菌中共表达,在共表达产物复性过程中,两个亚基互为分子伴侣,相互促进了蛋白质的重新正确折叠;[结果]表迭蛋白以包涵体的形式存在,ELISA检测表明:复性后的蛋白免疫学活性得到了提高;[结论]由此获得了有活性的蛋白质,并且建立了不相容双质粒共表达外源基因和包涵体复性的方法.     

Protein Qnality Control

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response,UPR)和内质网相关性蛋白质 降解途径(ER-associated degradation,ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景.指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制.内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白.其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配.如果这些蛋白仍不能正确折叠,则进入ERAD被降解.     

分子伴侣与错误折叠疾病

近年来研究发现,蛋白质错误折叠可以导致一些疾病。蛋白质错误折叠形成非天然构象,并相互聚集。这些聚集体不仅丧失了原有的蛋白质功能,还对细胞有一定毒性。分子伴侣与错误折叠疾病间的关系正逐渐被了解。分子伴侣可识别并阻止蛋白质的错误折叠,其基因突变会引起一些人类疾病。升高的分子伴侣水平可抑制一些变异蛋白的神经毒性,这将有助于疾病的药物治疗。     

蛋白质分子的一生——蛋白质的品质管理(英文)

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response, UPR)和内质网相关性蛋白质降解途径(ER-associated degradation, ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景。指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制。内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白。其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发 UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配。如果这些蛋白仍不能正确折叠,则进入 ERAD 被降解。     

蛋白质的品质管理

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response,UPR)和内质网相关性蛋白质降解途径(ER-associated degradation,ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景。指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制。内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白。其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配。如果这些蛋白仍不能正确折叠,则进入ERAD被降解。     

抗伊维菌素scFv蛋白质的变复性及纯化

基因工程技术的发展扩大了蛋白质研究领域，简化了研究步骤。原核表达系统表达目的蛋白质具有操作简单、培养周期短、高效经济、便于纯化等优点，但大部分表达的蛋白质是以包涵体的形式存在。包涵体是蛋白质在细胞内凝集形成的无活性不溶于水的固体颗粒，含有重组蛋白质及核糖体元件、内毒素、外膜蛋白质、RNA聚合酶和脂体等。在高浓度的变性剂如尿素、盐酸胍等中由于氢键、疏水键被破坏，包涵体蛋白质完全伸展而溶解，在复性剂中可重新折叠形成天然结构而重新获得活性[1]。     

HSP70研究进展及其在粘孢子虫分类学与病害学中的应用

热休克蛋白(HSPs)是生物细胞在受热、生物应激、理化因素等应激原刺激后所产生的一类在生物进化过程中最保守的蛋白,在原核生物和真核生物中普遍存在。其中HSP70是最受关注、研究最为深入的一种,也是含量最丰富的HSPs家族。HSP70具有高度保守的序列,常作为分子伴侣在协助新生多肽链折叠、蛋白质复合体的装配、调节、修复和降解变性方面起着重要作用。对近年来国内外HSP70的研究概况、生物学特性、生物学功能及在粘孢子虫中的应用研究现状进行了综述,并对HSP70在粘孢子虫中的研究前景进行了讨论。     

四种分子伴侣促进牛支原体膜蛋白在大肠杆菌中可溶性表达研究

为研究分子伴侣是否可以促进牛支原体膜蛋白在大肠杆菌中以可溶性形式表达及表达的蛋白是否具有活性,应用原核表达和Western blot方法进行检测,结果表明:1)牛支原体一个膜蛋白M1的截短片段在大肠杆菌表达系统中以包涵体形式表达,改变诱导时间、温度以及诱导剂浓度均未改变其表达形式;2)将4种分子伴侣pGKJE8、pGro7、pG-Tf2和pTf16分别与含有目的蛋白的重组质粒在表达工程菌(BL21)中共表达,当加入终浓度为1.0mmol/L IPTG以及0.5mg/mL L-阿拉伯糖或5.0ng/mL四环素的诱导剂,37℃诱导5h,发现分子伴侣pGTf2和pG-KJE8能显著提高M1截短片段的可溶性表达,其他2种分子伴侣未改变M1的表达形式;3)可溶性表达的M1截短片段与牛支原体阳性血清可以发生特异性反应。因此,研究发现2种分子伴侣可以使牛支原体膜蛋白在大肠杆菌表达系统中以可溶性形式表达,但未改变其生物活性,该研究结果可为建立牛支原体有效的血清学诊断方法及亚单位疫苗的研制奠定基础。     

DnaJ-like蛋白在植物胁迫应答中的作用及机制研究进展

DnaJ-like蛋白是一种重要的分子伴侣,调节分子伴侣HSP70的ATP酶活性,并参与新生肽的折叠、装配和运输过程。大量研究表明,DnaJ-like蛋白具有功能多样性,在植物的形态建成、生长发育以及胁迫应答过程中具有极其重要的作用。综述了DnaJ-like蛋白在植物生物与非生物胁迫响应方面的功能及可能的机制,并对未来的研究方向提出了建议。     

Posttranslational quality control: folding, refolding, and degrading proteins

Polypeptides emerging from the ribosome must fold into stable three-dimensional structures and maintain that structure throughout their functional lifetimes. Maintaining quality control over protein structure and function depends on molecular chaperones and proteases, both of which can recognize hydrophobic regions exposed on unfolded polypeptides. Molecular chaperones promote proper protein folding and prevent aggregation, and energy-dependent proteases eliminate irreversibly damaged proteins. The kinetics of partitioning between chaperones and proteases determines whether a protein will be destroyed before it folds properly. When both quality control options fail, damaged proteins accumulate as aggregates, a process associated with amyloid diseases.     

Molecular chaperones in the cytosol: from nascent chain to folded protein

Efficient folding of many newly synthesized proteins depends on assistance from molecular chaperones, which serve to prevent protein misfolding and aggregation in the crowded environment of the cell. Nascent chain--binding chaperones, including trigger factor, Hsp70, and prefoldin, stabilize elongating chains on ribosomes in a nonaggregated state. Folding in the cytosol is achieved either on controlled chain release from these factors or after transfer of newly synthesized proteins to downstream chaperones, such as the chaperonins. These are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. Understanding how the thousands of different proteins synthesized in a cell use this chaperone machinery has profound implications for biotechnology and medicine.     

Harnessing chaperones to generate small-molecule inhibitors of amyloid beta aggregation

Protein aggregation is involved in the pathogenesis of neurodegenerative diseases and hence is considered an attractive target for therapeutic intervention. However, protein-protein interactions are exceedingly difficult to inhibit. Small molecules lack sufficient steric bulk to prevent interactions between large peptide surfaces. To yield potent inhibitors of beta-amyloid (Abeta) aggregation, we synthesized small molecules that increase their steric bulk by binding to chaperones but also have a moiety available for interaction with Abeta. This strategy yields potent inhibitors of Abeta aggregation and could lead to therapeutics for Alzheimer's disease and other forms of neurodegeneration.     

小热休克蛋白家族的研究进展

小热休克蛋白(sHSP)是热休克家族中的成员,是在进化中高度保守的一系列分子量较小的蛋白质,作为分子伴侣,它们部分结合在变性的蛋白上,防止了由于刺激蛋白不可逆转的集聚,并在细胞内发挥着不同的功能.由于利用热处理使热休克蛋白的表达量增加,从而使水果和蔬菜的抗冻性增强的方法已经得到认可,所以研究小热休克蛋白具有重要的生物学意义,在研究植物抗逆功能基因的表达、农业等方面的应用前景十分广阔.     

Molecular chaperones: the plant connection

Molecular chaperones are a family of unrelated proteins found in all types of cell. They mediate the correct assembly of other polypeptides, but are not components of the mature assembled structures. Chaperones function by binding specifically to interactive protein surfaces that are exposed transiently during many cellular processes and so prevent them from undergoing incorrect interactions that might produce nonfunctional structures. The concept of molecular chaperones originated largely from studies of the chloroplast enzyme rubisco, which fixes carbon dioxide in plant photosynthesis; the function of chaperones forces a rethinking of the principle of protein self-assembly.     

Intramembrane proteolysis: theme and variations

Proteases that reside in cellular membranes apparently wield water to hydrolyze the peptide bonds of substrates despite their water-excluding environment. Although these intramembrane proteases bear little or no sequence resemblance to classical water-soluble proteases, they have ostensibly converged on similar hydrolytic mechanisms. Identification of essential amino acid residues of these proteases suggests that they use residue combinations for catalysis in the same way as their soluble cousins. In contrast to classical proteases, however, the catalytic residues of intramembrane proteases lie within predicted hydrophobic transmembrane domains. Elucidating the biological functions of intramembrane proteases, identifying their substrates, and understanding how they hydrolyze peptide bonds within membranes will shed light on the ways these proteases regulate crucial biological processes and contribute to disease.     

Pb isotopic variability in melt inclusions from oceanic island basalts, polynesia

Previous studies have suggested that melting processes are responsible for the trace element variability observed in olivine-hosted basaltic melt inclusions. Melt inclusions from three individual lava samples (two from Mangaia, Cook Islands, and one from Tahaa, Society Islands) have heterogeneous Pb isotopic compositions, even though the erupted lavas are isotopically homogeneous. The range of Pb isotopic compositions from individual melt inclusions spans 50 percent of the worldwide range observed for ocean island basalts. The melt inclusion data can be explained by two-component mixing for each island. Our data imply that magmas with different isotopic compositions existed in the volcanic plumbing system before or during melt aggregation.     

Games played by rogue proteins in prion disorders and Alzheimer's disease

The incidence of Alzheimer's disease (AD) and that of prion disorders (PrD) could not be more different. One-third of octogenarians succumb to AD, whereas Creutzfeldt-Jakob disease typically affects one individual in a million each year. However, these diseases have many common features impinging on the metabolism of neuronal membrane proteins: the amyloid precursor protein APP in the case of AD, and the cellular prion protein PrPC in PrD. APP begets the Abeta peptide, whereas PrPC begets the malignant prion protein PrPSc. Both Abeta and PrPSc are associated with disease, but we do not know what triggers their accumulation and neurotoxicity. A great deal has been learned, however, about protein folding, misfolding, and aggregation; an entirely new class of intramembrane proteases has been identified; and unsuspected roles for the immune system have been uncovered. There is reason to expect that prion research will profit from advances in the understanding of AD, and vice versa.     

Vascular injury and lysis of basement membrane in vitro by neutral protease of human leukocytes

Frozen and thawed granules of human, peripheral-blood leukocytes rapidly produce hemorrhage when injected into animal tissues. The effect is blocked by inhibitors of proteolysis. The granule extract can digest vascular basement membrane in vitro at neutral pH. In addition, basement membranes of blood vessels damaged in vivo by the leukocyte fraction are found to be attenuated when examined by electron microscopy. The proteases of human leukocyte granules differ in several important respects from known lysosomal cathepsins and trypsin-like esterases. Polymorphonuclear neutrophils are a major source of the neutral proteases present in circulating white cells, and release these enzymes during phagocytosis of immune complexes.