Protein stability for single substitution mutants and the extent of local compactness in the denatured state

The stability changes caused by single amino acid substitutionsare studied by a simple, empirical method which takes accountof the free energy change in the compact denatured state aswell as in the native state. The conformational free energyis estimated from effective inter-residue contact energies,as evaluated in our previous study. When this method is applied,with a simple assumption about the compactness of the denaturedstate, for single amino acid replacements at Glu49 of the tryptophansynthase subunit and at Ile3 of bacteriophage T4 lysozyme,the estimates of the unfolding Gibbs free energy changes correlatewell with observed values, especially for hydrophobic aminoacids, and it also yields the same magnitudes of energy as theobserved values for both proteins. When it is also applied foramino acid replacements at various positions to estimate theaverage number of contacts at each position in the denaturedstate from the observed value of unfolding free energy change,those values for replacements with Gly and Ala at the same residueposition in staphylococcal nuclease correlate well with eachother. The estimated numbers of contacts indicate that the proteinis not fully expanded in the denatured state and also that thecompact denatured state may have a substantially native-liketopology, like the molten globule state, in that there is aweak correlation between the estimated average number of contactsat each residue position in the denatured state and the numberof contacts in the native structure. These results provide somefurther evidence that the inter-residue contact energies asapplied here (i) properly reflect actual inter-residue interactionsand (ii) can be considered to be a pairwise hydrophobicity scale.Also, the results indicate that characterization of the denaturedstate is critical to understanding the folding process.     

过敏原肌质钙结合蛋白在毕赤酵母中的表达、优化及免疫反应性研究

目的 探究天然肌质钙结合蛋白(sarcoplasmic calcium binding protein, SCP)的可替代物,为蟹类过敏原的检测提供基础材料,本研究首次利用毕赤酵母(Pichia pastoris, P. pastoris)高效表达表达三疣梭子蟹(Portunus trituberculatus)重要过敏原SCP,并检验其免疫反应性。方法 根据毕赤酵母的密码子偏好性优化SCP基因并构建重组质粒。将其热激转化至P. pastoris GS115菌株后经遗传霉素(Geneticin, G418)筛选获得阳性高拷贝子。最后通过甲醇诱导表达重组SCP并结合免疫印记(Western blotting, WB)和间接酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)验证其免疫反应性。结果 SCP在P. pastoris GS115中实现了可溶性高效表达,其表观分子量约为28 kDa。在摇瓶水平下,最佳诱导条件为pH为6.0、每24 h添加1.0%(v/v)甲醇,于28℃发酵144 h,在此条件下,纯度为91.6%的SCP产量可达15 mg/L。WB和间接ELISA结果表明,重组SCP具有IgG结合能力。结论 毕赤酵母表达系统可以得到纯度较高且免疫反应性良好的重组SCP。本研究为SCP的理化研究及产业化应用奠定了基础,并有望促进特异性甲壳类过敏原检测的发展。     

BP神经网络结合变量选择方法在牛奶蛋白质含量检测中的应用北大核心CSCD

牛奶中的蛋白质含量会影响牛奶的品质,利用高光谱图像的光谱特征信息研究对牛奶蛋白质含量预测的可行性。本文提出一种基于竞争性自适应重加权算法(competitive adaptive reweighted sampling, CARS)和连续投影算法(successive projections algorithm, SPA)结合多层前馈神经网络(back propagation, BP)的预测建模方法,实验以含有不同浓度蛋白质的牛奶为对象,利用可见光/近红外高光谱成像系统共采集到5种牛奶共计250组高光谱数据,通过实验对比选择采用标准化方法对获取到的吸收光谱预处理,然后采用CARS结合SPA筛选特征波长,得到18个特征波长,建立CARS-SPA-BP模型,经过试验,CARS-SPA-BP模型的训练集决定系数和测试集决定系数R;和R;分别达到0.971和0.968,训练集均方根误差(root mean square error of calibration,RMSEC)和测试集均方根误差(root mean square error of prediction,RMSEP)达到了0.033和0.034。研究发现,采用CARS结合SPA筛选的牛奶特征波长建立的多层前馈神经网络模型,其模型预测结果与全波长建模相比并没有明显降低,因此将CARS结合SPA用于波长筛选并且结合BP神经网络基本可以完成对牛奶蛋白质含量的预测。为验证CARS-SPA-BP模型的预测能力,在相同数据环境下,使用较为传统的偏最小二乘回归(partial least squares regression, PLSR)进行建模,实验结果表明,CARS-SPA-BP相较于PLSR,R;和RMSEP均有明显提升。研究表明,CARS-SPA-BP可充分利用牛奶光谱特征信息实现较高精度的牛奶蛋白质含量检测。     

Development of a water-in-oil-in-water multiple emulsion system integrating biomimetic aqueous-core lipid nanodroplets for protein entity stabilization. Part II: process and product characterization

The aqueous-core enclosed in lipid nanoballoons integrating multiple emulsions of the type water-in-oil-in-water mimic, at least in theory, the environment within viable cells, thus being suitable for housing hydrophilic protein entities such as bioactive proteins, peptides and bacteriophage particles. This study reports a complete physicochemical characterization of optimized biomimetic aqueous-core lipid nanoballoons housing hydrophilic (BSA) protein entities, evolved from a statistical 2³×3¹ factorial design study (three variables at two levels and one variable at three levels) that was the subject of the first paper of a series of three, aiming at complete stabilization of the three-dimensional structure of protein entities attempted via housing the said molecular entities within biomimetic aqueous-core lipid nanoballoons integrating a multiple (W/O/W) emulsion. The statistical factorial design followed led to the production of an optimum W/O/W multiple emulsion possessing quite homogeneous particles with an average hydrodynamic size of (186.2?±?2.6) nm and average Zeta potential of (?36.5?±?0.9) mV, and exhibiting a polydispersity index of 0.206?±?0.014. Additionally, the results obtained for the diffusion coefficient of the lipid nanoballoons integrating the optimized W/O/W multiple emulsion were comparable and of the same order of magnitude (10^?12 m² s^?1) as those published by other authors since, typically, diffusion coefficients for molecules range from 10^?10 to 10^?7 m² s^?1, but diffusion coefficients for nanoparticles are typically of the order of magnitude of 10^?12 m² s^?1.     

以序列特征值预测酶和非酶蛋白及内含肽

利用生物信息学快速准确鉴别酶、非酶蛋白及内含肽能极大提高实验效率,而测序数量的指数型增长使酶、非酶蛋白及内含肽的自动分类尤显重要。本文获取了同一性小于25%的序列共计3853条,采用Z标度的伪氨基酸组成和氨基酸组成分布提取序列特征值识别酶、非酶蛋白及内含肽。结果表明,该特征值提取方法经参数优化后,即当λ=5,w=0.15时,以支持向量机为分类器,其10倍交叉验证的精度可达81.3%,ROC曲线下面积为0.83;其精度高于其它方法0.5%到12.9%不等;独立样本测试的预测精度可达71.2%,ROC曲线下面积为0.782,其精度高于其它方法0.4%到6.4%不等,效果均优于其它常见的序列特征值方法。本文结果说明从序列出发判断其归属是可行的,3种不同功能的分子在序列特征上存在一定的差异,所建立的Z标度的伪氨基酸组成和氨基酸组成分布法可用于其它类似的生物信息学问题。建立了从序列出发预测酶、非酶蛋白及内含肽的新方法。     

An integrated microfluidic system for fast, automatic detection of C-reactive protein

C-reactive protein (CRP) is a well-known inflammation marker in human beings. This study reports a new microfluidic system for fast, automatic detection of CRP. It contains pneumatic micropumps, a vortex-type micromixer, a pneumatic micro-injector and several microvalves to automatically perform the entire protocol for CRP detection. This includes sample/reagent transportation, incubation between the target CRP and a CRP-specific aptamer, washing processes, and the chemiluminescence development process. In addition, the chemiluminescence signal is measured by using a custom-made optical system which consists of a photomultiplier tube, a portable air compressor and eight electronic magnet valves to quantify the concentration of CRP. When compared to previous works, not only can this new microfluidic system automatically perform the entire process via a new integrated micro-injector and new micropumps, but a new CRP-specific DNA aptamer with a higher affinity and specificity is also used for CRP measurement. Experimental data show that the developed system can automatically complete the entire protocol within 30 min with a detection limit of 0.0125 mg/L, which is superior to previous published results. Moreover, this study also measures CRP concentration from clinical samples to verify the performance of the developed microfluidic system. The results indicate that the measured CRP concentrations from human serums are consistent with those using a benchtop system. The developed system can also detect CRP concentrations from human whole blood without any external sample pretreatment process. This microfluidic system may be promising for point-of-care applications for CRP detection in the future.     

Exploiting knowledge ontology and software agents for PPI network analysis

One major goal of functional genomics has been to identify and analyze molecular interactions in a cellular context to better understand the underlying design principles and mechanisms. To investigate into a PPI network from both topological and functional points of view, this work proposes a methodology that exploits ontology-based biological knowledge for network analysis. To speed up the procedure, an agent-based framework is also presented for supporting distributed computing. The preliminary results show that through the knowledge obtained from gene ontology, our work in analyzing building blocks of PPI networks can give a higher resolution than that of previous ones. Also our agent-based framework can successfully speed up the task of network analysis in an adaptive manner.     

基于模式驱动和模糊查询的模式发现方法

在蛋白质序列的比对研究中,拥有相似模式的蛋白质常常具有相似的功能.通过已知的蛋白质序列模式可以很方便地对新蛋白质序列的功能结构进行研究和确认.蛋白质序列的发现已成为一个很有意义的题目.对基于模式驱动Pratt算法进行改进以提高其效率,在原来基础上引入模糊查询方法,能够更为快捷地从互不相关的蛋白质序列集合中找出最具代表性的蛋白质模式.