碱提酸沉法制备燕麦浓缩蛋白的工艺研究

燕麦蛋白具有高营养价值,可采用碱提酸沉工艺以脱脂燕麦粉为原料提取.以燕麦浓缩蛋白的提取率为目标函数,在通过单因素试验对提取的工艺条件进行初步探索的基础上,运用正交试验设计对碱提酸沉法的工艺条件予以优化,确定合适的提取条件为:浸提pH值9.5、液料比10:1、温度45～50℃、时间90min.在此条件下,燕麦浓缩蛋白的提...     

The effect of a secretion-enhanced heavy chain on improving intein-based dual-vector co-delivery of a full-length factor VIII gene

Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene, which is difficult to package in the promising adeno-associated virus (AAV) vectors. Inhibited secretion of FVIII is caused mainly by inefficient secretion of its heavy chain. Previously, we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene, suggesting that the light chain, covalently ligated to a co-expressed heavy chain can improve the secretion of spliced BDD-FVIII. However, its level of secretion was affected by inefficient secretion the heavy chain. Here, we studied the effect of a mutant heavy chain with L303E/F309S substitutions, which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII gene. Eukaryotic vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells. A spliced FVIII protein was seen in co-transfected cells by Western blot analysis. The heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene, which was greater than the secretion of wild-type heavy chain. The amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL, with an activity of (0.61±0.08) IU/mL, which was greater than that of wild-type FVIII co-transfected cells. Spliced FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain transfections. This suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a dual-vector. These results encourage the transfer of this technology to an animal model using a dual-AAV vector.     

A case study of 3D protein structure prediction with genetic algorithm and Tabu search

This paper describes a case study of 3D protein structure prediction of six sequences from protein data bank (PDB) by genetic algorithm and tabu search (GATS), where off-lattice AB model is considered as a simplified model of protein structure. The lowest-energy values required for forming the native conformation of proteins are searched by GATS, and then the coarse structures (i.e., simplified structure) of the proteins are obtained according to the multiple angle parameters corresponding to the lowest energies. All the coarse structures form single hydrophobic cores surrounded by hydrophilic residues, which stay on the right side of the actual characteristic of protein structure. It demonstrates that this approach can predict the 3D protein structure effectively.     

锌指蛋白Zbed3的表达与分离纯化

将编码小鼠锌指蛋白Zbed3的基因克隆到大肠杆菌表达载体pET28c,构建了含组氨酸标签的表达质粒pET28c-Zbed3,并将其转入大肠杆菌BL21-CodonPlus(DE3)-RP;在20℃条件下,采用100μmol/L的异丙基硫代--βD半乳糖苷(IPTG)诱导、培养20 h而获得含Zbed3的菌体;菌体裂解液经超滤浓缩后采用镍(螯合)柱分离纯化得到锌指蛋白Zbed3(浓度达60 mg/L);同时,采用30%醋酸溶解包涵体,用0.5 mol/L的Tris-HCl(pH=8.8)缓冲液中和而使Zbed3在生理pH环境中复性.结果表明,Zbed3复性后在离心上清液中的回收率达到80%,其复性蛋白质与表达产物为可溶性(天然态)Zbed3的圆二色性光谱特征相一致,以此验证了复性后Zbed3空间构象的正确性.     

我国西北地区12个不同硬质度优质蛋白玉米自交系的品质及配合力

以中国西北地区常用的12个优质蛋白玉米(QPM)自交系为试材,采用胚乳硬质度5级分类法,分别对籽粒的物理性状和营养品质进行测定分析;应用NCII设计,以硬质度1,2级3个QPM自交系为测验种与硬质度3,4,5级的9个QPM自交系配制27个测交组合,分析这些测交组合的产量配合力.结果表明:胚乳硬质度越高,籽粒的物理性状越...     

从高分子构象与动力学到蛋白质折叠

针对作用在聚合物刷上的键拉力研究表明作用在接枝基面上的力随着聚合物刷接枝密度的增大反而减小,然而尾端单体上的拉伸张力并没有消失.高分子的构象和动力学转变决定了其物性和多种多样的应用,而生物大分子蛋白质作为由二十种不同属性的氨基酸构成的序列,更是具有由其序列所决定的特别的三维自然结构.本文就聚合物刷、聚合物纳米复合材料、聚合物网络等几种高分子体系的构象与动力学过程,及蛋白质构象和其折叠与去折叠的动力学过程做了介绍.特别是蛋白质的折叠与去折叠速率在单分子操纵实验中受到拉力的调控,通过测量这种拉力依赖的动力学过程、蛋白质的自由能曲面和折叠去折叠路径可以得到系统全面的研究.本文以肌肉蛋白titin的免疫球蛋白结构域I27为例对蛋白质折叠研究进行了阐述.     

基于网络拓扑和多种生物信息融合的关键蛋白质识别算法

关键蛋白质的识别有助于了解细胞存活的基本需求,并为疾病治疗找到新方法,但是蛋白质自身携带着复杂的生物特性,仅依赖网络拓扑特性不能精准地判断其关键性.因此,提出一种新方法来提高识别关键蛋白质的准确率.首先,考虑网络拓扑特性以及蛋白质在不同亚细胞中的重要程度,定义了SNC方法;其次,利用蛋白质在亚细胞与复合物信息中的特性定义了SIDC方法;最后,通过融合网络拓扑结构和多源生物信息,提出了关键蛋白质识别算法CTB.在YDIP、YMIPS和Krogan数据集上利用精准率-查全率等多种评估方法进行实验,结果表明CTB算法提高了识别关键蛋白质的性能.     

基于热处理的鲈鱼肌原纤维蛋白结构和功能关联性研究

以鲈鱼肌原纤维蛋白为原料,以巯基质量摩尔浓度、紫外吸收强度、内源性荧光、浊度、二级结构等为指标,研究热处理对肌原纤维蛋白结构及功能特性的影响.结果显示,30～90℃下,肌原纤维蛋白的巯基质量摩尔浓度减小、紫外吸光度增大、荧光强度下降、 Ca²⁺-ATPase活性下降.随着加热温度升高,肌原纤维蛋白的表面疏水性先增大后减小.热处理使肌原纤维蛋白的α-螺旋含量减少,β-折叠和无规卷曲的含量增加.热处理后的肌原纤维蛋白浊度增加、乳化性和起泡性先增加后减小.主成分分析表明,α-螺旋、β-折叠、无规卷曲和乳化性被主成分介绍的程度高.     

基于蛋白质相互作用网络的蛋白质复合物和功能模块预测算法研究进展

蛋白质相互作用网络中的模块化结构通常对应于蛋白质复合物或者蛋白质功能模块。基于蛋白质相互作用网络预测蛋白质复合物和功能模块不仅有助于理解生命有机体的细胞生物过程,而且可为探讨疾病的发生、发展和治疗以及合理的药物开发提供重要的基础。本文通过回顾近二十年来基于蛋白质相互作用网络的蛋白质复合物和功能模块预测算法研究的发展历程,按照静态蛋白质相互作用网络(SPIN)和动态蛋白质相互作用网络(DPIN)两个方向分别梳理预测算法所涉及的方法和技术,同时归纳常用的数据集并分析所面临的问题,为进一步研究提供有价值的参考。     

基于rbcL蛋白质序列的石斛属植物系统发育研究

以生物分类的数学模型和生物演化的数学模型为基础,探讨25种石斛属植物rbcL蛋白质序列的系统发育关系．用MATLAB7．1构建NJ系统树和UPGMA系统树,用MEGA4．1构建MP系统树,并进行系统发育分析,为研究石斛属植物的系统发育提供蛋白质序列方面的证据．