小远志多糖一级结构的初步研究

从药用植物小远志中提取、精制得多糖PP-1,PP-2,采用纸层析、薄层层析和气相色谱等方法确定了多糖中的单糖组成.通过高碘酸氧化、Smith降解和甲基化分析等方法确定了相邻单糖基连接方式,初步推测了两个小远志多糖可能的一级结构,为进一步合成和研究其生物活性提供了数据支持.     

冬小麦麸皮抗冻蛋白一级结构的研究

冬小麦麸皮抗冻蛋白(TaAFP)的一级结构是研究其高级结构和功能的基础.本研究结合N-末端测序技术和肽指纹图谱技术,测定TaAFP的一级结构,并对其同源性进行分析.MALDI-TOF-MS质谱分析显示TaAFP的分子量为13637.711 Da.使用Trypsin(胰蛋白酶)、Hydroxylamine(羟胺)和Chymotrypsin(胰凝乳蛋白酶)分别对TaAFP酶解,并对各个酶解片段的肽指纹图谱进行解析和连接,最终确定TaAFP的一级结构为MARKVIALAFLLLLTISLSKSNAARVKYNGGESGGGGGGGGGGGGGGNGSG-SGSGYGYNYGKGGGQSGGGQGGGGGGGGGGGSNGSGSGSGYGYGYGQGNGGAQGQGSGGGGGGGGGGGGGGSGQGSGSGYGYGYGKGGGGGGGGGGDGGGGGGGGSAYVGRHE,测定覆盖度达到了100%.序列比对分析以及同源性分析结果显示,TaAFP是一种与植物细胞壁和抵抗寒冷相关的蛋白质.     

蛋白质的结构预测和分子设计

蛋白质的结构预测及分子设计是适应基因工程的需要发展起来的对蛋白质的空间结构进行研究并在此基础上提供蛋白质改造方案的方法。通过对已知的蛋白质结构数据进行总结、分析,结合分子力学、分子动力学等计算方法可以进行某些种类的结构预测。在对蛋白质的结构与功能研究的基础上可以提出改造方案,从而进行有目的的分子设计。     

天花粉蛋白一级结构的修正及不同产地天花粉蛋白的研究

胰蛋白酶酶解天花粉蛋白, 用高效液相色谱分离酶解肽段, 用顺序仪测定其有关肽段的顺序。用羧肽酶A, B, Y测定了天花粉蛋白C-端和天花粉蛋白溴化氰降解肽CB1的C-端顺序, 修正了我们1985年测定的天花粉蛋白一级结构, 证明天花粉蛋白由246(7)氨基酸残基所组成, 除C-端微观不均一外, 与Collins结果一致。同时比较了芜湖产天花粉蛋白一级结构与平湖产的天花粉蛋白一级结构, 没有发现两者的一级结构有差别。     

从猪血中分离纯化高纯度的猪血红蛋白

为了从猪血中分离纯化高纯度的猪血红蛋白,建立了通过超滤、DEAE-Sepharose Fast Flow离子交换色谱和Sephadex G-75凝胶 排阻色谱三步法制备高纯度猪血红蛋白的方法,并通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、高效凝胶排阻色谱和反相高 效液相色谱方法,对纯化后的猪血红蛋白进行了鉴定。经三步分离纯化后,猪血红蛋白的纯度大于99%,含量为1.328 g/L。     

牛胰腺中活性蛋白质的液相色谱分离纯化方法进展

牛胰腺中含有多种活性蛋白,其中许多蛋白已被开发为有利于人类健康的药物。从牛胰腺中分离纯化得到的蛋白药物是一种有高附加值的高技术产品。现代生物技术中所用的大多数有价值的活性蛋白产品的制备仍然依赖于不同的液相色谱法。本文综述了牛胰腺中活性蛋白质的提取方法以及以色谱分离为主的分离与纯化技术,为开展从天然产品中提取并应用蛋白质提供一定的参考。     

豆浆凝固过程中大豆蛋白质二级结构的研究

用变温ATR-FT-IR测定了豆浆加热、豆浆凝固过程中蛋白质二级结构的变化,结果显示:随着温度的升高,熟豆浆中蛋白质分子间强相互作用氢键(1β)和总相互作用氢键(1β 2β)明显下降,豆浆从20℃升温到80℃时,1β由29.2%下降到16.1%,表明蛋白质分子间的相互作用减弱;豆腐的核心结构(β α)约38.0%变化不大。随豆腐凝胶强度增加,1β由17.4%上升到43.3%,而维持蛋白质紧密结构的氢键(β α)由38.8%下降到17.4%。豆浆凝固过程中1β的变化与其凝胶特性G*变化完全一致,说明蛋白质分子间强相互作用氢键与豆腐的硬度有关。     

用疏水色谱复性并同时纯化蛋白质的机理及其应用

变性蛋白表面的疏水氨基酸残基有与疏水色谱固定相(STHIC)颗粒相互作用的倾向, 两者之间的疏水相互作用能够抑制变性蛋白分子间的相互聚集. 同时疏水色谱固定相还能在分子水平上给变性蛋白分子提供足够高的能量, 使其瞬时脱水并折叠成其天然构象或不同的折叠中间体. 变性蛋白在疏水界面上的折叠不仅取决于其氨基酸之间的特异性相互作用及疏水色谱固定相的结构, 而且还取决于固定相和流动相之间的协同作用. 同时, 还提出了高效疏水相互色谱(HPHIC)进行蛋白折叠的机理及其进行蛋白折叠时能实现质量控制的原理. 在适当的色谱条件下, HPHIC 可使几种变性蛋白一步实现复性及同时纯化. 此外, 还设计制造出了直径比柱长大得多的实验室型和制备型“变性蛋白复性及同时纯化装置, USRPP”, 该“装置”具有完全除去变性剂、使蛋白质复性, 与杂蛋白分离及易于回收变性剂的“一石四鸟”功能. 该“装置”对变性蛋白的复性和纯化效率与通常使用的长柱相当. 在制备规模情况下, 该“装置”可以在低压梯度条件下简便、快速、而经济地应用于重组蛋白药物的制备. 文中以重组人干扰素-γ为例, 说明了制备型“装置”在其复性及同时纯化生产工艺中的应用.     

蛋白质二级结构预测的人工神经网络方法研究

本文比较了五种神经网络方法预测蛋白质二级结构的准确率,并做出初步评价。五种神经网络分别是:误差反传前向网络(BP),径向基函数网络(RBF),广义回归神经网络(GRNN),串并联叠层网络(CF),Elman网络(ELM)。结果显示:GRNN的预测准确率达85.7%,优于其它网络。本文还讨论了训练集样本数及参数的优化对GRNN预测准确率的影响。     

乳腺癌组织中蛋白质二级结构的Fourier变换红外光谱研究

根据乳腺大汗腺癌、小管癌、黏液癌、浸润导管癌、单纯癌和髓样癌组织Ｆｏｕｒｉｅｒ变换红外光谱酰胺Ⅰ带的去卷积和拟合分析,获得组织中蛋白质二级结构的数目及其组成。结果表明：这些组织中蛋白质二级结构的数目及其组成存在着显著的差异,并与组织的类型、分化、坏死等密切相关其中,髓样癌的差异性最大;高分化癌中螺旋结构的含量及非典型螺旋和α－螺旋含量的比值均比低分化癌的相应值高;肿瘤边缘坏死的乳腺癌组织也表现出明     

Purification and Characterization of Cyclic AMP-Binding Protein from Ganoderma lucidum

Cyclic AMP-binding protein was purified 30 fold from the mycelia of Ganoderma lucidum by the methods of ammonium sulfate precipitation, DEAE-cellulose, phospho-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration. The molecular mass of the purified protein is 34.5 kDa and 17 kDa by Sephacryl S-100 gel filtration and SDS-ployacrylamide gel electrophoresis, respectively. From these results it is suggested that the protein has a homometric dimmer structure. The pI of the purified protein is pH 8.2 by native isoelectric focusing gel. The half-life of the protein activity in 10% glycerol at 4 ℃ is 7 d in crude extract, but its half-life is only 3 d under purifying conditions. The optimal conditions of the protein activity are at 1 ℃ and pH 7.5. Its activity is increased 6 times by 1 mmol/L Zn^2 and is slightly inhibited by cGMP,Cu^2 and Mn^2 .     

乙型肝炎病毒包膜M蛋白的高效表达和分离纯化

从病人血清中经过聚合酶链反应扩增得到了编码HBV-M蛋白的基因片段(PreS2+S),将其转入原核表达载体pET-His,构建了原核表达质粒pET-His-M。重组质粒转化大肠杆菌BL21,IPTG诱导表达,收获菌体超声波破碎后,蔗糖梯度溶液洗脱杂质。HBV-M在原核系统中高效表达,分子量为30kD,纯化后得到了纯度为98%的HBV-M蛋白,ELISA检测结构表明M蛋白的抗原性比S蛋白的抗原性强。该研究为进一步研究HBV包膜M蛋白的结构、功能及新型乙肝疫苗的研制奠定了基础。     

Purification of a PHA-Like Chitin-binding Protein from Acacia farnesiana Seeds: A Time-dependent Oligomerization Protein

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 +/- 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.     

拟多维高效液相色谱法分离纯化双峰驼精清中的活性蛋白

利用一种新的高效液相色谱洗脱技术，建立了 ｐＨ缓冲液／离子对二元洗脱模式，对双峰驼精清的两种提取物的反相柱上进行了分离，得到了较纯的活性蛋白组分。该法操作简单、快速、比常规ＨＰＬＣ有更高的选择性及峰容量，并可减少活性蛋白的失活。     

Purification and Characterization of PRL Protein Tyrosine Phosphatases

PRLs constitute a subfamily of protein tyrosine phosphatases(PTPs). In the present paper are reported the molecular cloning, expression, purification, and characterization of all the three members of the PRL enzymes in human and the only PRL in C. elegans. These enzymes were expressed as glutathione S-transferase (GST) fusion proteins in DE3pLysS E. coil cells, and the recombinant fusion proteins were purified on glutathione-Sepharose affinity columns. Having been cleaved with thrombin, GST-free enzymes were further purified on an S-100 Sepharose gel filtration column. The purified proteins show single polypeptide bands on SDS-polyacrylamide gel electrophoresis. With para-nitrophenyl phosphate(p-NPP) as a substrate, PRLs exhibit classical Michaelis-Menten kinetics with Vmax values two orders of magnitude smaller than those of classic PTPs. The responses of PRLs to ionic strength, metal ions and phosphatase inhibitors are similar to those of other characterized PTPs, but their optimal pH values are different. These data thus reveal distinct common biochemical properties of PRL subfamily PTPs as well.     

Purification and Primary Structure Determination of a Novel Polypeptide Isolated from Mistletoe Viscum coloratum

A novel polypeptide was isolated from mistletoe Viscum coloratum. The primarystructure of the polypeptide ‘named viscotoxin B2‘ was determined to be KSCCKNTTGRNIYNT CRFAGGSRERCAKLSGCKIISASTCPSDYPK by Edman degradation. Viscotoxin B2 shared highsequence homology with viscotoxins isolated from Viscum album. Pharmacological experimentsshowed that viscotoxin B2 had distinct cytotoxic activity on tumor cells. Viscotoxin B2 could beused as a leading compound in cancer therapy.     

乙型肝炎病毒表面抗原(HBsAg)的高效表达和分离纯化

从病人血清中采用PCR扩增得到了编码HBV-HBsAg蛋白的S基因片段,将其转入PET-His表达载体,构建了原核表达质粒pET-His-HBsAg,转化大肠杆菌BL21,IPTG诱导表达。收获菌体超声波破碎后,梯度蔗糖溶液洗脱杂质。HBsAg在原核系统中高效表达分子量为25 kDa,并得到了纯度为97%的HBsAg蛋白。为进一步研究HBsAg奠定了基础。     

Purification of Biliary Protein and Its Effect on Calcium Carbonate Mineralization

The biliary protein (BP) was isolated from pig bile by gel filtration. The interaction between Ca²⁺ and protein was measured by fluorescence spectra. The result showed that there was a strong coordination between biliary protein and Ca²⁺. The CaCO₃ crystals obtained in systems with and without BP were characterized by scanning electron microscopy, Fourier transform infrared spectrography and powder X‐ray diffractometry. The possible formation mechanism of CaCO₃ in biliary protein solution was discussed.     

富硒灵芝中一种新含硒蛋白的纯化、性质及其自由基清除活性研究

通过硫酸铵沉淀、离子交换层析和分子排阻层析等方法, 从富硒灵芝中获得了一种新的含硒蛋白, 命名为Se-GL-P, 并研究了此蛋白的性质、抗氧化活性与其硒含量间的关系. 结果表明, 此蛋白的分子量为36600, 分子中约含有19.8%的糖链, N端的氨基酸残基序列为DINGGGATLPQKLYLTPDVL, 属于DING蛋白家族. 硒含量为4.87 mg/g, 具有较高的羟自由基和超氧自由基清除活性. 研究发现, Se-GL-P的抗氧化活性的提高与其中硒含量的提高相关.