Molecular Properties of Purified Human Uncoupling Protein 2 Refolded from Bacterial Inclusion Bodies

One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (K _d) of 0.3–0.5 and 23–92 M. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a K _d of greater than 100 M, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound [8-¹⁴C] ATP and [8-¹⁴C] ADP with K _ds of 4–5 and 16–18 M, respectively. Mg²⁺ (2 mM) reduced the apparent ATP affinity to 53 M, an effect entirely explained by chelation of ATP; with Mg²⁺, K _d using calculated free ATP was 3 M. A combination of gel filtration, Cu²⁺-phenanthroline cross-linking, and ultracentrifugation indicated that 75–80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg²⁺-free nucleotides: the K _d for ATP is about 3–5 M and for Mant-ATP it is about 10 times lower; and (c) in C₁₂E₉ detergent, the monodisperse protein may be in dimeric form.     

功能性包涵体的研究进展

利用原核系统表达外源重组蛋白的一个特点是表达蛋白多以包涵体形式存在。在过去人们一直认为包涵体是错误折叠、无生物活性的蛋白,需要经过变复性的过程重新获得有生物活性、可溶的蛋白,因而变复性条件的摸索迄今仍然是该领域的难点。但近几年的研究表明包涵体并非都是无生物活性的,功能性包涵体(或者称为非传统意义包涵体) 概念的提出是该领域的一个重大研究进展。由于功能性包涵体本身具有生物活性,可在非变性条件下提取,目前已经在生命科学的基础研究、生物制药、生物材料、生物催化等领域展现出良好的应用前景。重点从功能性包涵体的定义、形成机理、提取条件等近期研究进展进行综述,以期为原核细胞表达和工业生产重组蛋白药物提供新的思路。     

重组蛋白包涵体的复性研究

重组蛋白在大肠杆菌中的高表达往往形成不可溶、无生物活性的包涵体,需经过变性溶解后,在适当条件下复性形成天然的构象,才可恢复其生物活性．变复性实验是建立在对蛋白质体外折叠机制的了解的基础上．根据近年来对蛋白质折叠机制的认识和重组蛋白包涵体在复性方面的主要进展,论述以下3个方面的内容：1)蛋白质在细胞内的折叠机制;2)蛋白质体外折叠机制;3)蛋白质复性的策略和方法．     

P小体的研究进展

P小体(processing bodies)即mRNA处理小体,它是一种富含了多种功能相关蛋白以及RNA的胞浆集合体(cytoplasmic foci)。研究表明这种胞浆结构与mRNA的降解过程以及RNA干扰介导的转录后基因沉默效应有关,另外,它还参与了细胞增殖和细胞周期以及宿主的抗病毒感染能力的调控。     

Interconnected Cavernous Structure of Bacterial Fruiting Bodies

The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicellular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to limitations of different imaging methods. A new technique using Infrared Optical Coherence Tomography (OCT) revealed previously unknown details of the internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative high and low spore density regions. To make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high-density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The integration of novel OCT experimental techniques with computational simulations can provide new insight into the mechanisms that can give rise to the pattern formation seen in other biological systems such as dictyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions.     

Mass Purification of Nucleopolyhedrosis Virus Inclusion Bodies in the K-Series Centrifuge

Nucleopolyhedrosis virus inclusion bodies specific for Hemerocampa pseudotsugata, Neodiprion sertifer, Porthetria dispar, and Heliothis zea have been purified by using a continuous-sample-flow-with-isopycnic-banding centrifuge in quantities up to 6 x 10(13) polyhedral inclusion bodies per day. Continuous-flow methods for S-rho type purification have been evolved to deal with mass isolation of bioparticles.     

功率超声方法对蛋白质包涵体的解聚

用低频功率超声波辐照方法,实现了在低浓度变性剂(4mol/L尿素)中对大肠杆菌体系表达产物缩短的人巨噬细胞集落刺激因子(hM-CSF)包涵体的解聚,以期改进普遍采用的强烈变性剂(8mol/L尿素)溶解包涵体方法,超声处理后的SDS-PAGE在分子量17000上显示了与8mol/L尿素溶解结果相同的条带.表现出功率超声方法在基因工程包涵体解聚中的应用前景.     

Titration of Adenovirus by Counting Cells Containing Virus-Induced Inclusion Bodies

A new method for the titration of adenovirus types 2 and 12 based on the enumeration of viral inclusions in infected cells was devised and evaluated. The technique gave virus titers comparable to those obtained by the plaque assay procedure.     

切胶纯化表达蛋白包涵体的可行性分析

目的:高效率地纯化以包涵体形式表达的蛋白。方法:将SDS-PAGE电泳后的目的蛋白用4mol/L的乙酸钠显现出来,切割下来的目的蛋白可直接用于免疫,将切割下来的目的蛋白装在透析袋中,在电场中将游离的目的蛋白洗脱下来可用于ELISA检测。结果:用该法得到的蛋白经ELISA和Western blot检测均能保持其原有的抗原性,并用该法纯化的蛋白成功制备了相应的多抗和单抗。结论:证实了回收的蛋白仍能保留原有的抗原性,与传统的方法比较,方法简单且比较经济实用特别是包涵体,为切胶纯化蛋白提供了一个新的方法。     

包含体内重组蛋白质的复性

具有临床、工业生产、药用功能的真核生物蛋白质的供给常常受到其天然来源的限制。可喜的是基因工程技术的发展使许多真核生物蛋白质能在细菌细胞中进行表达[1] 。大肠杆菌由于培养和基因操作容易而成为最受欢迎的表达系统 ,但是重组蛋白质在大肠杆菌中的高水平表达常常导致以包含体形式存在的胞內聚集的变性蛋白质的形成。这种变性蛋白质的量可高达总的重组蛋白质量的95%。由于以包含体形式存在的聚集蛋白质分子不具有正确的三维结构 (天然结构 ) ,它们在水溶液中通常不溶解且没有活性 ,因此大肠杆菌中包含体的形成就意味着可溶性重组蛋白…