凤凰木种子蛋白的分离提纯和生化性质

研究了植物凤凰木种子中的蛋白质成分,试图分离出新的核糖体失活蛋白,凤凰木种子经磷酸盐缓冲液抽提,硫酸铵分级盐析,分子筛和阴离子交换剂等多次柱层析,分离出三种组分ＤｒＩ,ＤｒⅡ和ＤｒⅢ．ＳＤＳ－ＰＡＧＥ和ＩＥＦ实验表明这三种蛋白质均达到单一纯,而ＨＰＬＣ实验则表明它们的纯度不低于９０％,它们的分子量据ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ实验分别约２３５００、２６０００、２８５蛋０．ＩＥＦ实验测得三者的等电点均     

大熊猫子宫钙调素的分离纯化和性质的研究

以大熊猫子宫为材料分离纯化了钙调素,经ＳＤＳ－ＰＡＧＥ,ＰＡＧＥ和等电聚焦电泳鉴定,表现均一。分子量为１８８００道尔顿,等电点为３．６。该蛋白质分子的Ｎ－末端为封闭的。大熊猫子宫钙调素具有其它来源钙调素所特有的一些性质。对环核苷酸磷酸二酯酶有明显的激活作用,还发现对超氧化物歧化酶也有一定激活作用。电泳行为受Ｃａ＾２＋影响而出现特征性电泳改变,在含有Ｃａ＾２＋的ＳＤＳ凝胶电泳中,电泳速度比ＥＧＴＡ存     

铜绿假单胞菌外毒素A的生产,分离纯化和鉴定

通过对培养基组成、种子活化、接种量和培养条件进行优化,使铜绿假单胞菌外毒素Ａ（ＰＥ）产量达到每毫升５－１０μｇ和１９２小鼠ＬＤ５０,不低于国外报道水平。经二步纯化,ＰＥ蛋白回收率为３３．３３％（ＰＥ）和１６．６７％（ＬＤ５０）,提纯系数为４３８．５（ＰＥ）或２１８．５（ＬＤ５０）,ＳＤＳ－ＰＡＧＥ呈现一条带,相对分子质量为６６０００,琼脂糖扩散鉴定与兔抗ＰＥ产生一条沉淀线,小鼠半数致死量为０．１５     

地衣芽孢杆菌β—甘露聚糖酶的纯化及其性质的研究

地衣芽孢杆菌１Ｂａｃｉｕｕｓ Ｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＢＬ－３０６产生的胞外β－甘露聚糖酶经硫酸铵分级盐析,ＤＥＡＥ－纤维素柱层析。Ｓｅｐｈａｄｅｘ－Ｇ１００柱凝胶过滤和ＤＥＡＥ－纤维素柱再层析分离纯化,得到ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）均一样品。用ＳＤＳ－ＰＡＧＥ测得纯化后β－甘露聚糖酶分子量为２６０００道尔顿。用凝胶等电聚焦电泳（ＰＡＧＥＩＥＦ）测得等电点ＰＩ为５．０。该酶     

应用亲和层析法是纯鸡马立克氏病病毒PP38基因重组产物

利用鸡马立氏病病毒（ＭＤＶ）Ｉ型特异单克隆抗体Ｈ１９致敏Ｓｅｐｈａｒｏｓｅ４Ｂ－ＣＮＢｒ,从感染重组病毒ＢＰ３８Ⅱ的昆虫细胞中提纯鸡马立克氏病毒ＰＰ３８基因重组产物,获得良好效果,提纯的蛋白质的ＳＤＳ－ＰＡＧＥ中表现出一条分子量约为３８ｋＤａ的蛋白质条带,在免疫印迹试验中该蛋白质带也能被单克隆抗体Ｈ１９识别。利用该提纯的重组ＰＰ３８免疫小鼠,所制备的小鼠抗血清在免疫荧光染色试验中不仅能与感染重组病     

凤凰木种子蛋白的分离提纯和生化性质

研究了植物凤凰木种子中的蛋白质成分,试图分离出新的核糖体失活蛋白凤凰木种子经磷酸盐缓冲液抽提,硫酸铵分级盐析,分子筛和阴离子交换剂等多次柱层析,分离出三种组分ＤｒⅠ、ＤｒⅡ和ＤｒⅢ．ＳＤＳ－ＰＡＧＥ和ＩＥＦ实验表明这三种蛋白质均达到单一纯,而ＨＰＬＣ实验则表明它们的纯度不低于９０％。它们的分子量据ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ实验分别约２３５００、２６０００、２８５００．ＩＥＦ实验测得三者的等电点均为５．２左右。测定了组分ＤｒⅠ的蛙卵泡裂解活性,其毒性约为天花粉蛋白的１／４０．分析了它们的氨基酸组成。估算了三种蛋白质的二级结构含量,结果表明三者均富含β折叠结构。已有的实验事实表明这三种蛋白质的生化行为明显异于核糖体失活蛋白,因而很可能不属于这一蛋白质家族。     

苦瓜籽核糖体失活蛋白的理化性质及生物活性

采用硫酸铵分级分离,假配基亲和层析和ＳｅｐｈａｃｒｙｌＳ－１００分子筛层析等方法,从苦瓜籽中获得核糖体失活蛋白（ＲＩＰ）．经ＳＤＳ－ＰＡＧＥ、ＰＡＧＥ、ＩＥＦ和ＰＡＳ方法分析均表明为单一蛋白着色带或单一糖蛋白着色带．根据ＳＤＳ－ＰＡＧＥ和Ｓｅｐｈａｄｅｘ Ｇ－１５０分子筛层析结果计算其相对分子质量为３．０×１０４,经ＩＥＦ－ＰＡＧＥ结果计算其ｐＩ为８．９～９．０．对无细胞系统中蛋白质生物合成抑制活性明显,其ＩＣ５０为５．３×１０－ １０ ｍ ｏｌ／Ｌ左右．体外生物活性试验结果表明其对人肝癌细胞、Ｖｅｒｏ、ＳＰ２／０、３Ｔ３、Ｋｂ、Ｎａｖａｎａ 等肿瘤细胞株均表现有不同程度的抑制作用．而对完整细胞人胚肺二倍体细胞却毒性极小．因此,上述实验结果为该ＲＩＰ的进一步深入研究和有可能开发成免疫毒素的高效弹头药物提供了一定的工作基础．     

地衣芽孢杆菌β—甘露聚糖酶的纯化及酶学性质

地衣芽孢杆菌（Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＮＫ－２７菌株发酵产生的β－甘露聚糖酶（β－ｍａｎｎａｎａｓｅ）经硫酸铵盐析沉淀,两次ＤＥＡＥ纤维素和Ｓｅｐｈａｄｅｘ Ｇ－１００离子交换柱层析以及制备ＰＡＧＥ等步骤,获得了凝胶电泳均一的样品。用ＳＤＳ－凝胶电泳测得纯化后的β－甘露聚糖酶分子量为２６ｋＤ,用凝胶聚焦电泳测得等电点Ｐ１为５．０。酶反应的最适ｐＨ为９．０,最适温度为６０℃,稳     

尖吻蝮蛇蛇毒出血毒素的纯化与部分性质

目的　被尖吻蝮蛇 （ Ｄｉｅｎａｇｋｉｓｔｒｏｄｏｎ ａｃｕｔｕｓ） 咬伤会引起严重的出血, 对蛇毒出血毒素的研究有利于治疗蛇伤出血药物筛选。 方法　采用 Ｓｅｐｈａｄｅｘ Ｇ７５, Ｄ Ｅ Ａ Ｅ Ｓｅｐｈａｄｅｘ Ａ５０, Ｓｅｐｈａｄｅｘ Ｇ２００ 和两次 Ｐ Ｂ Ｅ 聚焦层析纯化。 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 电泳和等电聚焦电泳测定纯化样品的纯度和等电点。氨基酸组成用自动氨基酸分析仪测定。以小鼠背部皮下注射部位出血斑的面积来确定最小出血剂量和常规的方法测定酶活性。结果　从尖吻蝮蛇毒中纯化到一个相对分子量为５６ ０００ 的出血毒素 （ Ｄａ Ｈ Ｔ３）, 经氨基酸组成测定计算,它由 ４８７ 个氨基酸残基组成。此成分在 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ上显示出一条均一的蛋白染色带, 其ｐ Ｉ为５５０。该出血成分的最小出血剂量是 ２６μｇ, 具有蛋白水解酶活力, 其活力为 ３６８, 但没有精氨酯酶和磷脂酶 Ａ２ 活力。当加入 Ｅ Ｄ Ｔ Ａ 螯合剂去除金属离子后, 它们的出血活力和蛋白水解酶活力均丧失。 结论　这是从大陆尖吻蝮蛇毒中获得的一个新的出血金属蛋白酶 （ Ｄａ Ｈ Ｔ３）。     

皖南尖吻蝮蛇毒出血毒素IV的纯化与初步鉴定

从皖南尖吻蝮蛇毒液中经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ和Ｓｅｐｈａｃｒｙｌ Ｓ－２００两步凝胶柱层析首次纯化出一种中分子量血毒素（简称ＡａＨⅣ）。经ＳＤＳ－ＰＡＧＥ和等电聚焦凝胶电泳测定其分子量为４４ｋＤ,等电点为ｐＨ５．０。从５００ｍｇ粗毒中可获得２０ｍｇ ＡａＨⅣ纯品。ＡａＨⅣ有较强的出血活性,最小出血剂量（ＭＨＤ）为０．４μｇ。     

MPTP慢性帕金森病小鼠纹状体差异蛋白质的研究

目的分离、鉴定MPTP诱导慢性帕金森病模型小鼠纹状体差异表达的蛋白质,对MPTP慢性PD动物模型的特异性蛋白质组进行初步探讨,为PD的发病机制提供一定的蛋白质组学依据。方法成功建立MPTP诱导慢性帕金森病小鼠模型,提取模型组和对照组小鼠脑纹状体蛋白质,分别以固相pH梯度等电聚焦为第一向,SDS-PAGE垂直电泳为第二向进行2-DE。图像分析软件PDQUEST8.0分析电泳图谱找出差异表达蛋白,运用MALDI-TOF MS质谱鉴定;其肽质量指纹图（PMF）经MS Fit检索。结果比较MPTP诱导慢性PD模型小鼠和正常对照小鼠纹状体二向电泳图,发现12个蛋白表达异常,最终鉴定出其中4个蛋白质：线粒体裂殖调节因子1（mitochondrial fission regulator 1）、类泛素样蛋白3前体（ubiquitin-like protein 3 precursor）表达下调;S100蛋白A10（proteinS100-A10）、Lin-7 homolog B为新出现点。结论初步鉴定出MPTP慢性PD模型小鼠纹状体部分差异表达蛋白,所发现4个表达异常的蛋白质与帕金森病线粒体的损伤和兴奋性神经毒性密切相关,与PD的发病机制相符,为深入研究帕金森病病理机制奠定了基础。     

青海高原春小麦PEG胁迫与复水后叶片差异蛋白质组学研究

春小麦是青海省的主要粮食作物,青海高原干旱频繁且严重,尤以春旱为首,对小麦生长发育造成严重影响．为了从蛋白质组学水平分析小麦对干旱胁迫的应答特征,探讨小麦可能的抗旱机制,本研究对青海主栽春小麦品种青春38幼苗进行聚乙二醇 (polyethylene glycol,PEG) 6000胁迫和复水处理,采用IEF/SDS PAGE双向凝胶电泳技术,对PEG胁迫和复水处理的小麦叶片总蛋白质分别与正常浇水的对照进行差异蛋白质组学研究,经考马斯亮蓝G 250染色获得清晰度和重复性较好的双向电泳图谱．PD Quest软件处理分析各对照和处理图谱,在等电点4.0～7.0线性范围内,均可识别650个以上清晰蛋白质点,获得比较明显的差异表达蛋白点43个,其中8个蛋白点重复.从35个差异蛋白点中选取24个差异点进行MALDI-TOF-TOF-MS肽质量指纹图谱分析,应用Mascot软件在NCBInr数据库中搜索鉴定蛋白质,得到22个阳性结果．对鉴定得到的差异表达蛋白进行功能分析,它们分别参与了光合作用、蛋白质合成、能量代谢途径、细胞防御、氧化还原、运输、信号转导等过程,而且根据差异表达蛋白功能分类所占比例,发现干旱胁迫与光合作用关系最为紧密.     

红莲型水稻细胞质雄性不育花粉总蛋白质初步比较分析

采用固相pH梯度/SDS-PAGE双向电泳对红莲型细胞质雄性不育水稻的不育系(YTA)和保持系(YTB)二核期花粉总蛋白质进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱。用PDQuest2DE软件可识别约1500个蛋白质点,其中差异表达的蛋白质点数为120。将其中15个差异点采用基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption/ionizaton time of flight mass spectrometry,MALDI-TOF-MS)进行了肽质指纹图分析,通过采用Mascot软件对MSDB数据库查询,其中7个蛋白质点得到了鉴定。YTA相对于YTB有部分参与物质和能量代谢的蛋白质缺失或表达量降低,这些蛋白质分别是水稻线粒体H -转运ATPase(H -ATPase)α链、盐诱导型膜联蛋白、线粒体NAD -依赖型苹果酶和磷酸核糖焦磷酸合成酶等。这些蛋白质的表达下调或缺失可能与线粒体提供能量不足而导致的花粉不能正常发育有关。线粒体电压依赖性阴离子通道(VDAC)这一重要蛋白质在YTA中的上调表达有可能与花粉败育过程中细胞的程序性死亡相关。     

Method development for analysis of proteins extracted from the leaves of Orthosiphon aristatus

Orthosiphon aristatus is a traditionally used medicinal plant. In order to study the proteome of the plant, we have developed a simple plant protein extraction method by direct extraction of protein using a modified 2D-gel compatible tris-sucrose buffer followed by a double TCA-acetone precipitation. This method omitted the use of toxic phenol which is widely used in the studies of plants proteins. Moreover, it shortens the lengthy extraction procedure of phenol extraction and back-extraction method and therefore reduced the extraction time (by 2h) while increased in protein yields (by 50%). Comparison of the 2D-gel images of the two extracts revealed that >60 extra protein spots were detected in the extract of our current method. The method was applied on the leaves of O. aristatus collected from six geographical areas in Malaysia. The correlation coefficient of each replicate gels from the six areas ranged from 0.70 to 0.90 indicating good reproducibility of the method.     

Environmental and transgene expression effects on the barley seed proteome

The barley (Hordeum vulgare) cultivar Golden Promise is no longer widely used for malting, but is amenable to transformation and is therefore a valuable experimental cultivar. Its characteristics include high salt tolerance, however it is also susceptible to several fungal pathogens. Proteome analysis was used to describe the water-soluble protein fraction of Golden Promise seeds in comparison with the modern malting cultivar Barke. Using 2D-gel electrophoresis to visualise several hundred proteins in the pH ranges 4-7 and 6-11, 16 protein spots were found to differ between the two cultivars. Eleven of these were identified by mass spectrometric peptide mass mapping, including an abundant chitinase implicated in defence against fungal pathogens and a small heat-shock protein. To enable a comparison with transgenic seed protein patterns, differences in spot patterns between field and greenhouse-grown seeds were analysed. Four spots were observed to be increased in intensity in the proteome of greenhouse-grown seeds, three of which may be related to nitrogen availability during grain filling and total protein content of the seeds, since they also increased in field grown seeds supplied with extra nitrogen. Finally, the fate of transgene products in barley seeds was followed. Spots containing two green fluorescent protein constructs and the herbicide resistance marker phosphinothricin acetyltransferase were observed in 2D-gel patterns of transgenic seeds and identified by mass spectrometry. Phosphinothricin acetyltransferase was observed in three spots differing in pI suggesting that post-translational modification of the transgene product had occurred.     

Identification of plant mitochondrial proteins: A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a FastBlot cycle

Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a 2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing, and evaluates its performance. The paper also describes a “FastBlot” sequencing cycle with the cycle time for protein sequencing from PVDF membranes reduced to less than 29 min with femtomole sensitivity. Other benefits of the cycle include reduced lag, reduced background, reduced loss of labile residues, and increased initial and repetitive yields. The procedure gave excellent results with maize mitochondrial proteins: of six protein spots that we tried to sequence, only one was blocked. The other spots yielded considerable sequence information. One spot was identified from the sequence as superoxide dismutase, while another spot corresponded to an unidentified cDNA from rice. The results of these experiments show that modifications of our previous procedures can provide good N-terminal protein sequencing from individual spots on 2D gels. The technique makes it possible to obtain sequence data, prepare gene probes, and identify many of the polypeptides in the 2D-gel map for plant mitochondria.     

An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins

Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.     

Comparative proteomic analysis of whole saliva from chronic periodontitis patients

Chronic periodontal disease is a chronic inflammatory process affecting tooth supporting tissues in the presence of pathogenic bacterial biofilm. There is some evidence for changes in the protein composition of whole saliva from chronic periodontitis patients, but there have been no studies using a proteomic approach. Hence, the aim of this study was to compare the protein profiles of unstimulated whole saliva from patients with periodontitis and healthy subjects by two complementary approaches (2D-gel electrophoresis and liquid chromatography). Protein spots of interest were analyzed by MALDI-TOF-TOF, and the data was complemented by an ESI-Q-TOF experiment. The analyses revealed that subjects with periodontal disease have increased amounts of blood proteins (serum albumin and hemoglobin) and immunoglobulin, and they have a lower abundance of cystatin compared to the control group. A higher number of protein spots were observed in the periodontitis group, of which most were identified as alpha-amylase. This higher number of alpha-amylase variants seems to be caused by hydrolysis by cysteine proteases under such inflammatory conditions. This approach gives novel insights into alterations of salivary protein in presence of periodontal inflammation and may contribute to the improvement of periodontal diagnosis.     

Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques

In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.