家蚕30 kD特异性变应原的分析、纯化与质谱鉴定

以Coca's提取液分别提取到不同时期家蚕Bombyx mori的粗浸液,利用SDS-PAGE和Western blotting鉴定其特异性变应原,然后用DEAE-52离子交换层析及切胶纯化出30 kD的特异性变应原,再经MALDI-TOF在线联机分析,所得质谱数据进入网站搜索分析。结果显示：1～5龄家蚕均有20条左右蛋白带,其中 5龄家蚕有23条蛋白带,主带有11条（82、79、60、51、46、38、32、30、28、24和18 kD）。选用家蚕过敏患者阳性血清进行免疫印迹,1～4龄家蚕均显示出82和79 kD的特异性变应原;但只有5龄家蚕的30 kD蛋白为特异性变应原,通过离子交换层析和经切胶纯化出30 kD蛋白,再经MALDI-TOF-MS鉴定该蛋白为外膜蛋白。提示家蚕不同时期抗原成分有所变化,5龄家蚕新出现的30 kD蛋白为特异性变应原。     

意蜂工蜂酸性磷酸酶的纯化及其酶学特性

从意蜂Apis mellifera工蜂体内分离提纯酸性磷酸酶（ACPase, EC3.1.3.2）,并对其性质进行了研究。将工蜂酸性磷酸酶的初提物经分段盐析、DEAE-Sepharose FF离子交换层析及Sephadex G-200 凝胶过滤等纯化步骤,得到经聚丙烯酰胺凝胶电泳为单一蛋白区带的酶液。提纯倍数为77.24,酶液比活力为16.22 U/mg（对硝基苯磷酸二钠作底物）。利用凝胶过滤法测定酶的相对分子质量为135 kD,SDS-PAGE测定酶的亚基相对分子质量为63 .1 kD。酶的等电点为4.46和4.79。非还原/还原（NR/R）单向、双向SDS-PAGE显示酶分子含有链内二硫键。对二级结构圆二色谱分析显示,酶分子中α-螺旋占13.84%,β-折叠占25.68%,无规则卷曲占56.34%。氨基酸组成分析结果表明, 酸性磷酸酶约含有507个氨基酸残基,富含门冬氨酸残基。     

HPV18 E7致癌蛋白的高效表达、纯化和鉴定

目的为进一步研究人乳头状瘤病毒18（Human papillomavirus18,HPV18）E7蛋白的结构与功能。方法构建HPV18 E7的谷胱甘肽S-转移酶融合蛋白质粒pGEX-6P-1-GST-HPV18 E7,重组质粒转入大肠埃希菌BL21进行可溶性融合蛋白的高效表达。结果柱上切除法去除GST标签,表达产物经glutathione Sepharose 4B亲和层析纯化,获得了SDS-PAGE和HPLC-ESI-MS纯度的HPV18 E7均质蛋白,非变性PAGE和凝胶过滤表明HPV18 E7以稳定的单体形式存在于水溶液中。高压液相色谱-电喷雾质谱（HPLC-ESI-MS）分析得到HPV18 E7精确分子量为12865．0 Da,与其理论值吻合。纯化蛋白经HPLC-ESI-MS／MS鉴定为目的产物,鉴定出的9个匹配肽段覆盖率为HPV18 E7整个氨基酸序列的96．5％。结论本文所建立的技术可以有效地大量制备HPV18 E7,为进一步研究其结构与功能和致癌机制奠定了重要的物质基础。     

聚乙二醇伴随式离子交换层析分离重组乙肝病毒表面抗原

对由中国仓鼠卵巢细胞(CHO)表达的多聚亚基蛋白HBsAg在离子交换层析过程中容易因亚基解离而导致蛋白解聚和丧失生物活性的难题,实验中选择聚乙二醇（PEG）作为保护剂伴随式（Polyethylene Glycol-Accompanied）离子交换层析分离纯化HBsAg。实验表明,在流动相中加入1% PEG10000(W/V)作为纯化伴侣, HBsAg的回收率由55% 左右提高到80%以上,纯化倍数基本保持在12左右。对纯化产物进行SDS_PAGE分析表明,1% PEG10000的纯化伴侣伴随式离子交换层析能全部保留HBsAg的糖基化蛋白单体（27kD和30kD）,高效液相色谱联用多角度激光散射(High Performance Size Exclusion Chromatography_Multiangle Laser Light Scattering, HPSEC-MALLS )进一步分析阐明了PEG能促使HBsAg颗粒尺寸分布更均一,结构更接近天然乙肝表面抗原。     

人尿激肽原酶的纯化与鉴定

采用两性离子胶体沉淀和乙醇沉淀相结合的粗提方法,经离子交换、疏水层析、亲和层析及凝胶过滤4个步骤有效地将人尿激肽原酶（hk-1）粗提物纯化,比活提高了1 755倍,总得率为70%.用以慈菇蛋白酶抑制剂为配体的亲和层析纯化hk-1,效果理想,整个工艺路线适合产业化生产.纯化产物在SDS-聚丙烯酰胺凝胶电泳上为单带,高压液相色谱(HPLC)上为单峰,基质辅助激光解析电离飞行时间质谱测得分子质量为33 450 u,等电聚焦测得pI在4.3附近,为含糖蛋白.同时测定了该酶的热稳定性和pH稳定性.纯化过程中同时分离得到另一种药用蛋白——人尿胰蛋白酶抑制剂（HUTI）.     

猪血超氧化物歧化酶分离纯化工艺改进及其抗氧化活性研究

以新鲜猪血为原料,首次利用改进的新工艺提取分离超氧化物歧化酶,经过溶血,热变,丙酮沉淀,超滤浓缩,SephadexG-75凝胶过滤层析和DEAE-sepharose-fast flow离子交换层析纯化,冷冻干燥等步骤,得到高纯度酶,并对酶的相关性能进行研究。试验结果显示产品粗酶活性在3 000 U/mg左右,分别经SephadexG-75和DEAE-sepharose-fast flow层析纯化后,酶活分别达到5 585 U/mg和6 148 U/mg产品得率分别为13.4%和10.32%,SDS-PAGE凝胶电泳显示为单一条带达到电泳纯,其分子量在31 kD附近,其临苯三酚抗氧化活性明显。     

聚球藻PCC 7942氢酶的分离纯化及特性研究

报道了室温、空气环境下聚球藻Synechococcus sp.PCC7942氢酶的分离纯化.经过超声破碎、超速离心、离子交换层析、疏水层析及凝胶层析等步骤,氢酶被纯化了218倍,得率为6.5%,比活为1.46U·mg^-1蛋白.纯化氢酶的SDS-PAGE图显示五条蛋白带,分子量约为83kDa,60kDa,47kDa,30kDa和27kDa.该氢酶为可溶性的双向氢酶,其催化放氢的最佳电子供体为还原态的甲基紫精,最适温度50℃,最适pH8.0.     

异养硝化好氧反硝化菌株Agrobacterium tumefaciens LAD9羟胺氧化酶的分离纯化

羟氨氧化酶(Hydroxylamine oxidase,HAO)的作用方式直接决定了异养硝化好氧反硝化细菌的代谢途径,分离得到纯度较高的HAO也就成为研究这类细菌脱氮机制的重要环节。以异养硝化好氧反硝化细菌Agrobacterium tumefaciens LAD9为代表,建立了该菌株HAO的分离纯化方法:首先采用渗透压休克法提取细胞周质液,然后采用DEAE Sepharose CL-6B离子交换层析和Sephacryl S-100凝胶过滤层析对细胞周质液进行分离纯化。结果表明,经过离子交换层析可得到分子量分别为55.3、35.7和19.2kD的杂蛋白,进一步经过凝胶过滤层析即可得到电泳纯的HAO,纯化倍数为5.79,产率为39.71%。对其酶学性质的初步研究表明,该菌株HAO的分子量为18.8 kD,能够将羟胺氧化为亚硝酸盐氮,且Fe2+的加入可显著增强其酶活。     

银杏种仁中一种抗氧化活性蛋白的纯化及性质

银杏种仁经破碎,提取缓冲液4℃浸取后离心得上清液。上清液经硫酸铵沉淀,DEAE-52离子交换层析,MonoQ离子交换层析,UltroGelACA-54凝胶过滤层析后,分离得到一种具有抗氧化活性的蛋白。该蛋白经UltroGelACA-54凝胶过滤层析测定分子量为60 kD,经PAGE和SDS-PAGE鉴定均为单一蛋白质条带。SDS-PAGE测定其亚基分子量为10 kD。该蛋白具有一定的还原能力和清除超氧阴离子自由基及DPPH自由基能力,并在30～60℃温度下具有良好的稳定性。     

一个未知的子宫雌激素反应蛋白ULF-250的鉴定研究

摘要 鉴定一个未知的子宫雌激素反应蛋白（ULF-250）。去卵巢大鼠补充给予雌二醇造成子宫产生大量宫腔液（ULF）,受雌激素调节的蛋白分泌其中。收集ULF分别进行SDS-PAGE和双向电泳（2-DE）分离;通过Western 和2D-Western确认抗ULF-250抗体识别的蛋白成分并与凝胶上的条带或斑点相对应;对目标蛋白分别用MALDI-TOF-MS和HPLC-ESI-MS/MS两种方法进行质谱分析并获得肽段序列数据;与蛋白数据库比对及文献检索鉴定未知蛋白。2D-Western显示抗ULF-250抗体特异识别的蛋白成分。从2D胶上切下对应的蛋白斑点进行MALDI-TOF-MS。 结果提示：ULF-250是ebnerin/DMBT1。另外,经SDS-PAGE分离的250 kD蛋白条带进行液-质联用分析。结果同样提示：ULF-250是ebnerin/DMBT1。与文献报道比较,ULF-250与ebnerin/DMBT1在子宫的组织定位和调节是相同的。因此,我们认为ULF-250是子宫表达的ebnerin/DMBT1。通过2-DE结合质谱分析以及查阅文献确认未知的雌激素反应蛋白ULF-250是ebnerin/DMBT1。此外,我们的研究提示,该蛋白不仅在子宫上皮细胞表达而且分泌到子宫腔液中,其功能值得进一步研究。     

水稻氨基酰化酶的分离纯化与性质分析

氨基酰化酶（N－acylamino－acidamidohydrolase或acylaseⅠ,EC3．5．1．14）是专一水解N-酰基化L-氨基酸的蛋白酶．从水稻黄化苗得到的抽提液,经过硫酸铵分级沉淀、丙酮分级沉淀和阴离子交换层析三个步骤,纯化得到了该酶,比活达到100U／mg蛋白,在无还原剂存在的SDS-聚丙烯酰胺凝胶电泳上显单一条带,分子量为40kD．而凝胶层析分析表明活性分子的分子量约90kD,因此可推测它的活性分子由两个亚基通过非共价键作用组合而成．进一步研究此酶的性质,在所测的五种乙酰化氨基酸中,最适底物为N-乙酰-L-甲硫氨酸．该酶的最适温度为50℃,最适pH为7．0～8．0．Co2＋和Zn2＋能增强酶活性,但烷基化试剂对酶活性没有影响,表明酶活性中心不含活化的巯基或羟基基因     

火菇素的分离纯化及其分子量的测定*

火菇素是一种从金针菇FlammulinaVelutipes中分离纯化出来的具有抗癌活性的简单蛋白。用SDS-PAGE系统,通过与已知分子量的标准参照蛋白比较,确定火菇素的分子量为24kDa,这一结果与其氨基酸组成分析相比偏大,进一步通过电喷雾离子化质谱法(ESIMS)分析,精确测定火菇素的分子量为19891.13Da。推测由于火菇素蛋白与SDS的非正常结合是造成蛋白质在SDS-PAGE中迁移变慢导致偏差的原因。     

Identification and characterization of major bovine serum tyrosine-O-sulfate-binding protein as a complement factor H

A major tyrosine-O-sulfate (TyrS)-binding protein present in bovine serum was purified to electrophoretic homogeneity using a combination of TyrS-Affi-Gel 10 affinity chromatographyy, DEAE-Bio-Gel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified TyrS-binding protein migrated as doublet protein bands with apparent molecular weights of ca. 160, 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. N-termini of the two forms of purified TyrS-binding protein contain most likely identical sequence for the first fifteen amino acids residues, which displays a high degree of homology to those of human and mouse complement factor H. Furthermore, the purified TyrS-binding protein exhibited immunologic cross-reactivity with anti-human complement factor H. These results indicate the identity of the purified TyrS-binding protein being bovine complement factor H. The two forms of the purified bovine factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of 25 kD, 45 kD and 100 kD, respectively. Limited V8 protease mapping of the two forms yielded similar, yet unidentical, peptide band patterns. Purified bovine factor H appeared to bind agarose-bonded heparin through its anion-binding domain and the binding was inhibited by the presence of free heparin or dextran sulfate.Abbreviations HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - NP-40 Nonidet P-40 - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TyrS tyrosine-O-sulfate     

A highly purified allergen from excretory and secretory products of Dirofilaria immitis

Preceding data revealed that the allergen concentrated mainly in excretory and secretory (ES) products exhausted by adult Dirofilaria immitis. The present paper reported that a highly purified allergen was obtainable from ES products more easily and effectively. An allergen in ES products was purified by a combination of DEAE-Sephadex A-50 chromatography, Sephadex G-200 and Sephadex G-100 gel filtration. The purified preparation was proved to be one protein by sodium dodecyl sulfate (SDS) gel electrophoresis and to be exact the same allergen with the one obtained from the crude extract of adult Dirofilaria worms. The molecular weight of the purified allergen was estimated to be 15,000, and the allergen was inclined to aggregate in the buffered solution.     

Characteristics of human milk glutathione peroxidase

Human milk glutathione peroxidase (GPx) was purified 4500-fold using acetone precipitation and purification by repetitive ion-exchange and gel filtration chromatography with an overall yield of 34%. Homogeneity was established by gel electrophoresis. Using gel filtration, the molecular weight (mol wt) of the enzyme was estimated to be 92 kdalton (kD). The monomeric molecular weight was estimated to b 23 kD from polyacrylamide gel electrophoresis, indicating that the native enzyme consists of four identical subunits. The molecular weight of each subunit was supported by amino acid analysis. Selenium (Se) content of the purified enzyme was 0.31%, in a stoichiometry of 3.7 g-atoms/mol. Data from these studies reveal that GPx provided approximately 22% of total milk Se, but only 0.025% of the total protein.     

巨噬细胞源性神经营养因子的纯化和鉴定

Macrophage-derived neurotrophic factor (M phi DNF) is purified from macrophage conditioned medium by a procedure consisting of column chromatography with Sephacryl S-100-HR, high-performance liquid chromatography (HPLC), and a final step using reverse-phase HPLC. The product shows a single protein band in sodium dodecyl sulfate-polyacrylamide gel. It has a molecular weight of 60.5 kD and an isoelectric point of pI 5.1 and contains more leucine, lysine, glutamine and aspartic acids in its amino acid composition. Purified M phi DNF can promote the survival, activity, and neurite outgrowth of cultured cerebellar cortical neurons and that this effect reaches maximal levels with concentrations of the M phi DNF ranging from 500-1000 ng/ml.     

Characterization of the major allergen of plum as a lipid transfer protein

Background: Allergy to Prunoideae fruit (plum, peach, cherry and apricot) is one of the most frequent food allergies in southern Europe. All these fruits cross-react in vivo and in vitro, as they share their major allergen, a 9 kD lipid transfer protein (LTP). Objective: The aim of the study was the identification and molecular characterization of the major allergen of plum. Methods: The IgE pattern of reactivity to plums was investigated by SDS–PAGE and immunoblotting with the sera of 23 patients. The identified major allergen was purified by HPLC, using a cationic-exchange column followed by gel-filtration. Further characterization was achieved by periodic-Schiff stain, isoelectrofocusing and N-terminal amino acid sequencing. Results and conclusions: The major allergen of plum is a 9 kD lipid transfer protein, not glycosylated and with a basic character (pI>9), highly homologous to the major allergen of peach.     

Isolation,Purification and Some Properties of Invertase from Leaves of Mandarin Orange

Highly active acid invertase was found in the young leaf extract of mandarin orange Citrus reticulata Blanco). The invertase was isolated and purified from the young leaf extract of mandarin orange through the procedures of ammonium sulphate precipitation, DEAE-Sepharose column chromatography and Sephacryl S-200 gel filtration. 6.4% of the invertase activity was recovered. Invertase was 179.2-folds purified. The purified invertase preparation was homogeneous as shown in polyacrylamide gel electrophoresis and Sephacryl S-200 molecular sieve chromatography. The molecular weight of the native invertase determined by gel filtration was 80 kD. The invertase consists of two identical subunits with apparent equal subunit weight of 40 kD as determined on SDS-PAGE. The invertase followed typical Michaelis-Menten Kinetics with apparent Km Of 1. 6 × 10-2 mol/L for sucrose. Vmax of the invertase was 100 mg reducing sugar · mg-1 protein · h-1 The optimum pH was 5.0 (stable from 4.5—5.5). The optimum temperature was 55℃.