Atherosclerosis is a complex disease characterized by lipid accumulation in the vascular wall and influenced by multiple genetic and environmental factors. To understand the mechanisms of molecular regulation related to atherosclerosis better, a protein interaction network was constructed in the present study. Genes were collected in nucleotide database and interactions were downloaded from Biomolecular Object Network Database (BOND). The interactional data were imported into the software Cytoscape to construct the interaction network, and then the degree characteristics of the network were analyzed for Hub proteins. Statistical significance pathways and diseases were figured out by inputting Hub proteins to KOBAS2. 0. The complete pathway network related to atherosclerosis was constructed. The results identified a series of key genes related to atherosclerosis, which would be the potential promising drug targets for effective prevention.
Atherosclerosis ; genetics ; Databases, Factual ; Humans ; Protein Interaction Mapping ; methods ; Protein Interaction Maps ; Software

Animals ; Databases, Protein ; Humans ; Models, Molecular ; Protein Interaction Domains and Motifs ; Protein Interaction Mapping ; methods ; Protein Interaction Maps ; Proteins ; chemistry ; metabolism ; Proteome ; chemistry ; metabolism

Atomic force microscopy (AFM) has been applied in many biological investigations in recent years, and this review focuses on the application of AFM in DNA-protein interactions. AFM images of static DNA-protein complexes, in air and in liquid, can be used to obtain quantitative and qualitative information on the structure of different complexes. And dynamic AFM images of DNA-protein complexation in real time under liquid conditions will help to understand biological processes and mechanisms at single molecule level. In addition, the measurement of intermolecular forces between biomolecules also provides new opportunities for studying mechanical properties of biomolecules and the interactions in their native environment. AFM has revealed many mechanisms of gene regulation, and will play a more and more important role in life science research.
DNA ; chemistry ; ultrastructure ; Humans ; Microscopy, Atomic Force ; methods ; Protein Binding ; Protein Interaction Mapping ; Proteins ; chemistry ; ultrastructure

Bacterial two-hybrid system is a newly developed method for studying protein-protein interactions. However, in our studies of the interaction of regulatory proteins in Streptomyces, it was found that the bacterial two-hybrid system is not sensitive enough by the blue-and-white selection on X-gal plate. To overcome this drawback, the reason of false positive clone was firstly determined, which was the disturbance of other direct or indirect regulation on lacZ promoter. Then the disturbance was diluted by introducing multicopy lacZ promoter, which drive another reporter gene gfp. By such design, the sensitivity of the modified bacterial two-hybrid system was significantly inproved and the two different reporters also help to decrease the rate of the false positive clones. Further the evaluation of the modifiedd bacterial two-hybrid system indicated that the sensitivity was significantly improved.
Bacteria ; Genes, Reporter ; Promoter Regions, Genetic ; Protein Interaction Mapping ; methods ; Two-Hybrid System Techniques

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.
Endopeptidases ; genetics ; metabolism ; Escherichia coli ; genetics ; Plum Pox Virus ; enzymology ; genetics ; Protein Interaction Mapping ; methods ; Proteolysis

Membrane protein and its interaction network have become a novel research direction in bioinformatics. In this paper, a novel membrane protein interaction network simulator is proposed for system biology studies by integrated intelligence method including spectrum analysis, fuzzy K-Nearest Neighbor(KNN) algorithm and so on. We consider biological system as a set of active computational components interacting with each other and with the external environment. Then we can use the network simulator to construct membrane protein interaction networks. Based on the proposed approach, we found that the membrane protein interaction network almost has some dynamic and collective characteristics, such as small-world network, scale free distributing, and hierarchical module structure. These properties are similar to those of other extensively studied protein interaction networks. The present studies on the characteristics of the membrane protein interaction network will be valuable for its relatively biological and medical studies.
Algorithms ; Animals ; Artificial Intelligence ; Computational Biology ; methods ; Computer Simulation ; Humans ; Membrane Proteins ; chemistry ; Protein Binding ; Protein Interaction Mapping ; methods ; Protein Interaction Maps

The major bottle-neck in the way of constructing high density protein microarray is the availability and stability of proteins. The traditional methods of generating protein arrays require the in-vivo expression, purification and immobilization of hundreds or thousands of proteins. The cell-free protein array technology employs cell-free expression systems to produce proteins directly onto surface from co-distributed or pre-arrayed DNA or RNA, thus avoiding the laborious and often costly processes of protein preparation in the traditional approach. Here we provide an overview of recently developed novel technology in cell free based protein microarray and their applications in protein interaction analysis, in antibody specificity and vaccine screening, and in biomarker assay.
Cell-Free System ; DNA ; genetics ; Humans ; Protein Array Analysis ; methods ; Protein Biosynthesis ; Protein Interaction Mapping ; Proteins ; analysis ; genetics ; metabolism

OBJECTIVETo evaluate the effects of two sample preparation methods on two-dimensional polyacrylamide gel electrophoresis (2-DE)-based serum protein separation, and produce high-resolution and reproducible 2-DE images for identifying disease-related serum protein.

METHODSDirect solubilization and hot SDS methods were used separately to extract and handle the total proteins of serum samples from patients with colorectal carcinoma. Immobilized pH gradient 2-DE was used to separate the total proteins. After image analysis of silver-stained 2-D gels, 3 differential protein spots were identified by matrix-assisted laser desorption/time-of-flight mass spectrometry.

RESULTSThe total proteins treated with hot SDS method were used to perform 2-DE. 2-DE patterns with high resolution and reproducibility were obtained for human serum samples. 2-DE was performed 3 times for the samples treated by direct solubilization and hot SDS methods, respectively, resulting in the average number of spots of 675-/+46 and 702-/+49, respectively. The average matching protein spots were 573-/+42 and 623-/+52, with average matching rate of 85.3% and 89.6%, respectively. The average position deviation of matched spots in different gels was 0.85-/+0.30 mm and 0.81-/+0.28 mm in IEF direction, and 1.02-/+0.18 mm and 0.97-/+0.12 mm in SDS-PAGE direction. Mass spectrometry of the 2-D gels treated with hot SDS method generated high-quality mass spectra, and the sample preparation method allowed detection of relatively low abundance protein.

CONCLUSIONHot SDS method is more effective for human serum protein sample preparation and well-resolved, reproducible 2-DE profiles of human serum have been established in this study.

Blood Protein Electrophoresis ; Blood Proteins ; analysis ; isolation & purification ; Electrophoresis, Gel, Two-Dimensional ; methods ; Electrophoresis, Polyacrylamide Gel ; methods ; Humans ; Peptide Mapping ; Protein Interaction Mapping ; Reproducibility of Results

A new MS-titration method for the non-covalent binding of protein-ligand based on the research of berberine and alpha1-acid glycoprotein was established. The major presumption of new method is that the total concentration of protein-ligand complex is approximately the same as the total concentration of acting protein if a certain extent of affinity is existed between protein and ligand, in addition, the mole amount of acting ligand is more than that of acting protein. The non-covalent binding behaviours between berberine and alpha1-acid glycoprotein was studied by using electrospray ionization ion trap mass spectrometry (ESI-ITMS) , and the results were verified by fluorescence quenching method. The results showed that the binding behaviours between berberine and alpha1-acid glycoprotein, for example, stability constant, number of binding site, type of the main binding force, were almost the same by using the new MS-titration method and fluorescence quenching method. Comparing with the reported MS-titration method, the presented MS-titration method in this paper is more simple and applicable, does not demand much for the devices, and can lead to reliable results in same cases.
Berberine ; metabolism ; Ligands ; Orosomucoid ; metabolism ; Protein Binding ; Protein Interaction Mapping ; methods ; Proteins ; metabolism ; Spectrometry, Mass, Electrospray Ionization

The application of in vitro selection method to isolate nucleic acids, peptides and proteins according to their functions has been studied intensively in recent years. In vitro display technologies are not limited by cellular transformation efficiencies; thus, very large libraries of up to 10(13)-10(14) members can be built. The most popular in vitro display technologies are ribosome display and mRNA display; ribosome display achieves the mRNA-ribosome-nascent peptide complexes by stalling the translating ribosome in an in vitro translation reaction. In mRNA display, the mRNA-protein complex is achieved by binding the two macromolecules through a small adaptor molecule, typically puromycin; these mRNA-peptide fusions can then be purified and subjected to in vitro selection. In vitro display technologies provide a different approach to the in vitro selection and directed evolution of peptides and proteins. This review focuses on the principle and method of ribosome display and mRNA display technologies, and discusses their applications.
Animals ; Directed Molecular Evolution ; Gene Library ; Humans ; Peptide Library ; Protein Interaction Mapping ; methods ; RNA, Messenger ; chemistry ; genetics ; Ribosomes ; chemistry ; genetics