新型核苷碱基卤化酶AcmX的原核表达和结晶实验

在链霉菌中发现了一类能对核苷碱基进行特异位点卤化修饰的新型卤化酶,可用于核苷类药物的卤代优化。对新型核苷碱基卤化酶晶体结构的解析,是阐明其生物催化的机理,建立生物催化卤化制备核苷类药物的最直接手段。为了获得一个卤化酶AcmX的晶体结构,首先通过大肠杆菌原核表达制备AcmX蛋白样品,但只能得到没有活性的AcmX的蛋白包涵体。通过构建AcmX、分子伴侣蛋白共表达系统,注意控制合适的分子伴侣蛋白表达水平,并通过高分辨率的分子筛凝胶层析步骤,去除混入AcmX样品的分子伴侣蛋白GroEL,摸索出表达和纯化高质量AcmX蛋白样品的方法,并将蛋白样品成功用于结晶实验。这不仅为解析卤化酶AcmX的晶体结构打下坚实的基础,还可将本研究的方法用于其他表达困难的蛋白的制备。     

冷休克蛋白及冷休克反应

冷休克蛋白是从真细菌中发现的，它不仅作为ＲＮＡ分子的伴侣蛋白，同时还与细菌的多个生理过程有着密切的联系，如：对低温的适应、细胞生长的控制、营养胁迫等．冷休克蛋白引起冷休克反应的分子调控发生在转录和翻译两个水平上．     

金属硫蛋白研究进展

介绍了金属硫蛋白的结构、理化性质和主要功能，金属硫蛋白转录与表达，并对国内外金属硫蛋白与锌、铅、镉、钙的代谢研究，金属硫蛋白与辐射关系，肿瘤的发生、治疗及预后的一些进展进行了介绍，说明了金属硫蛋白有着广阔的应用前景。     

莱茵衣藻腺苷二磷酸葡萄糖焦磷酸化酶的相互作用蛋白组分析

微藻利用光能和CO₂合成淀粉作为藻细胞的储能物质,是以燃料乙醇为代表的液体生物燃料的可持续原料来源.理解微藻淀粉代谢关键酶的催化机制和调控方式是基因工程操作以提高藻细胞淀粉积累能力的前提. 基于此,以莱茵衣藻淀粉合成代谢中的关键酶腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)催化亚基为探针蛋白,获取该酶的GST融合蛋白后使用GST沉降分析实验结合HPLC-MS/MS的蛋白质检测技术及生物信息学分析方法获得一组与AGPase催化亚基存在相互作用蛋白质,主要包含分子伴侣蛋白、光合作用相关蛋白和信号蛋白分子等. 通过综合分析上述蛋白组的生理功能及亚细胞定位,揭示了它们通过与AGPase相互作用在淀粉合成代谢中的生物学意义.      

通州区：聘请专家指导项目进展

近日，通州区科学技术委员会、区生产力促进中心聘请“生物提取猪肝金属硫蛋白技术”专利发明人亲临生物体取猪肝金属硫蛋白技术的产业化基地，进行技术指导。此次专家的到访为“生物体取猪肝金属硫蛋白技术的引进与产业化开发”项目提供了技术支持和具体实施方案。     

金属硫蛋白的功能及应用前景

阐述了金属硫蛋白的结构特点及理化性质，介绍了金属硫蛋白的功能和应用前景．     

大肠杆菌系列融合表达载体的构建

许多异源蛋白在大肠杆菌内的表达是以不可溶、无生物学活性的包涵体形式存在,这为蛋白质的功能研究带来困难.融合表达是提高蛋白可溶性的有效方案之一.为构建通用型融合表达载体,本研究将5种常见的融合标签即突变型麦芽糖结合蛋白(mMBP)、小分子泛素样修饰蛋白(SUMO)、翻译起始因子(IF2-I)、氮源利用物质A(NusA)和谷胱甘肽转移酶(GST)以及3种伴侣蛋白(GroEL、DnaK和TF)分别克隆至pET30a(+),构建了系列融合表达载体.这些载体含有相同的克隆位点以及位于融合标签羧基端的烟草蚀刻病毒蛋白酶(TEV)的酶切位点.N-乙酰-D-葡萄糖胺2-异构酶基因hRnBP克隆到mMBP融合表达载体后,经IPTG诱导,SDS-PAGE检测表明融合蛋白均得到了高效表达且几乎完全可溶,TEV酶切获得了预期的带型.全细胞偶联生成N-乙酰-D-神经氨酸实验发现mMBP-hRnBP的摩尔转化率较无mMBP标签的体系提高了近60%,证明了融合表达载体中融合标签的适用性.新型的原核融合表达载体为蛋白的融合表达、分离纯化及功能研究提供了更多的选择.     

金属硫蛋白的研究进展

本对近年来金属硫蛋白的结构，特性和功能及其分子生物学的国内外研究进展作一综述，讨论了金属硫蛋白与医学的关系，提出了有待研究解决的问题，展望了其应用前景。     

金属硫蛋白的研究进展

 金属硫蛋白是一类广泛存在于生物体内的多功能诱导性蛋白.作者对近年来在金属硫蛋白的结构、分类、功能及测定方法等方面研究取得的进展做了简要的介绍,同时提出了一些金属硫蛋白研究中存在的问题,并对金属硫蛋白将来可能的应用前景进行了展望.     

谁发明了集装箱运输

不知道大家注意过海港码头的货物装卸吗？宽阔的货场上，一个个巨大的金属箱子整齐地排列着，拖车拖着这些巨型箱子，通过码头的吊桥，把箱子运送到货轮上去，或者用起重机把箱子吊起，稳稳地放进船仓。也许你会觉得这很正常，事实上，这种集装箱货物装卸方法经历了漫长的创新历程。     

Protein-folding location can regulate manganese-binding versus copper- or zinc-binding

Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).     

不同蛋白质来源配合饲料对犬饲喂效果及养分消化率研究

用不同蛋白质饲料 (植物蛋白质饲料、动物蛋白质饲料和动植物混合蛋白质饲料 )的配合饲料饲喂实验比格犬。各组的采食积极性和采食量没有明显差异 ;蛋白质来源对生长犬增重没有明显的影响。动植物混合蛋白饲料组与植物蛋白饲料组蛋白质消化率相近 ,动物蛋白饲料组最低 ;动植物混合蛋白饲料组有机质消化率高于植物蛋白饲料组和动物蛋白饲料组 (P <0 0 5 ) ;钙磷消化率以植物蛋白饲料组最低。因此为控制饲料成本并在不能获得高品质动物性蛋白的情况下 ,用动植物混合蛋白饲料饲喂实验犬效果较好     

蛋白质芯片——蛋白质高表达及固定技术研究进展

就近年来国际上蛋白质芯片的高通量蛋白质生产及固定技术的研究进展进行了系统的总结和分析。蛋白质芯片是一种大规模、高通量的蛋白质组学研究技术,目前己广泛运用于基础研究(如蛋白质/蛋白质相互作用)和应用研究(如病理诊断,药物开发),而该技术的关键步骤是实现蛋白质高表达及固定。及时把握蛋白质高表达及固定技术的研究动向是开展蛋白质芯片研究的基础。     

蛋白质亚细胞定位预测研究进展

蛋白质的功能与其在细胞中的定位有着密切的联系,新合成的蛋白质必须处于适当的亚细胞位置才能正确的行使其功能．预测蛋白质的亚细胞定位,在确定一个未知蛋白质的功能,了解蛋白质相互作用等方面有着重要的意义．机器学习方法在蛋白质亚细胞定位研究中扮演着一个重要的角色．笔者从数据集的构建、蛋白质序列特征提取方法、蛋白质亚细胞定位预测算法以及预测算法的性能评估等四方面总结了过去十几年间机器学习方法在蛋白质亚细胞定位研究中的应用情况,系统阐述了蛋白质亚细胞定位预测研究的进展．     

基于遗传算法的蛋白质折叠模拟系统

在模拟蛋白质折叠的遗传算法基础上，采用晶格模型，设计了一个用于蛋白质折叠模拟的软件系统，并举例说明该系统的用法。该蛋白质折叠模拟系统可用于蛋白质折叠预测和蛋白质进化研究，并可为蛋白质分子设计提供重要信息。     

出血病及粘孢子虫病鲫鱼蛋白质变化研究

利用双向电泳方法，以正常鲫鱼为对照，对感染出血病和粘孢子虫病后鲫鱼肌肉、肝脏组织内蛋白质变化作了比较分析．结果表明，正常鱼与病鱼肌肉组织显示出的蛋白质斑点约100个，其中含量较丰富的蛋白质斑点有20余种，以酸性蛋白居多．肝脏组织约有200余种蛋白质斑点存在，其中含量比较丰富蛋白质斑点有30余种，有较为广泛的分子量范围；感染出血病鲫鱼的肌肉组织中诱导出6个新的蛋白质斑点，肝脏中诱导出19个蛋白质斑点．在感染粘孢子虫病的鲫鱼肌肉中，有3个蛋白质斑点消失，诱导出3个蛋白质斑点，肝脏中有16个斑点消失，这些变化可以作为辅助疾病诊断的生化指标，为从生化水平揭示该病的致病机理及开展早期预防研究奠定基础．     

Low pH-induced conformational changes in 33 kD protein of photosystem Ⅱ

33 kD protein, located on the lumen side ofthylakoid membranes, is one of three extrinsic proteins ofphotosystem Ⅱ (PS Ⅱ ). Previous study showed that NBSmodification of W241, the only tryptophan in 33 kD protein,is helpful for understanding the function of W241 in main-taining functional conformation of 33 kD protein. In thispaper, studies of both circular dichroism and fluorescencespectra showed that upon decreasing pH from 6.2 to 2.5, theconformation of soluble 33 kD protein changed significantly,with an increase or a decrease in percentage of random coilor (z-helix and turns. The changes in secondary structures ofthis protein are pH reversible. After NBS modification at pH2.5, the conformational change of 33 kD protein was keptfixed. The CD ellipticity at 200 nm for NBS-modified 33 kDprotein is much lower than that for control, indicating that the unfolding degree of 33 kD protein was enhanced after the NBS modification. Moreover, the conformational flexibility islost in NBS-modified 33 kD protein, and the conformationalchange becomes pH irreversible, indicating that NBS modi-fication blocked the reversibility of conformational change of33 kD protein. The specific binding capability of NBS-modi-fled 33 kD protein is much lower than that of low pH-treatedcontrol. Furthermore, the rebinding of modified protein on PS Ⅱ membranes cannot restore the activity of oxygen evo-lution. We suggest that it is low pH but not NBS modificationof W241 that leads to the conformational change of 33 kDprotein from one functional to another non-functional state.The significant capability of proton transport of 33 kD pro-tein is discussed.     

Applying genetic algorithms to the study of protein mutation theory

Genetic algorithms were applied to the study of simulation of protein mutation carried out on two_dimensional lattice model; to the study of effects of single mutation and double mutation on protein folding and protein structure stability. It is found that in two_dimensional lattice models, replacement of inner core hydrophobic residue by hydrophilic residue will result in reduction of protein stability; at the same time the number of residues of protein surface relatively grows with increase of protein chain length; and most mutations occur in residues of protein surface, these mutations are all neutral and have no effects on protein natural structure. The two mutations of double mutation are interactive and related to each other.     

Grafting of protein-protein binding sites

A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding C_α and C_β atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.