Identification of three continuous antigenic sites in horse muscle acylphosphatase

The main antibody-combining sites of horse skeletal muscle acylphosphatase were mapped by preparing and purifying CNBr, tryptic and peptic peptides from the pure enzyme, and looking for the immunoreactivity of each peptide by the dot-immunobinding assay using specific polyclonal antienzyme antibodies previously purified by immunoaffinity chromatography. The immunoreactive peptides were identified on the basis of either their elution times in the fingerprint analysis or amino acid composition, or both, by comparison with the known enzyme amino acid sequence. All the CNBr as well as two tryptic and two peptic peptides were immunopositive, leading to identification of three main continuous antigenic sites on the enzyme molecule. The strong inhibition (92%) of the antigen-antibody reaction carried out in the presence of antibodies previously incubated with the immunoreactive peptide mixture supports the possibility that, at our experimental condition, the three identified antigenic domains contain the main antigenic determinants of the enzyme. The relationship between structure and antigenicity of the immunoreactive peptides is discussed in detail.     

Methods of searching for antigenic determinants for proteins with known primary structure

Theoretical and experimental methods for locating antigenic determinants of proteins with known amino acid sequences are discussed. These methods are systematized on the basis of the theoretical approaches applied, and the efficiency of various predictive methods is compared. Some examples of experimental epitope determination for a number of proteins are given.     

Synthesis of multiple antigenic peptides (MAPs)—strategies and limitations

Dendrimeric platforms such as MAPs can be synthesized either entirely by solid‐phase methods (SPPS, direct approach) or by conjugation in solution of preformed, SPPS‐made building blocks (indirect approach). Although MAPs and MAP‐like constructs have been extensively and successfully used for various biological (mainly immunological) applications, experimental reports are most often lacking in chemical detail about their preparation and characterization. Here, we provide complete accounts of the synthesis and analytical documentation of MAPs and similar dendrimers by either all‐SPPS (direct) or chemoselective thioether ligation (indirect) methods. We have chosen as model epitopes a 24‐residue sequence of the ectodomain of protein M2 from influenza virus (M2e), which is found to be a rather challenging peptide epitope, and a far more manageable, shortened (12‐residue) version of the same peptide. The advantages and shortcomings of both direct and indirect methods are discussed. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Comparative antigenic proteins and proteomics of pathogenic Yersinia enterocolitica bio-serotypes 1B/O: 8 and 2/O: 9 cultured at 25°C and 37°C

Yersinia enterocolitica is a Gram-negative enteric pathogen responsible for a number of gastrointestinal disorders; the most pathogenic bio-serotype is 1B/O: 8. In this study, we compared the antigenicity of the outer membrane proteins and proteomics of the whole-cell proteins of a pathogenic bio-serotype 2/O: 9 isolated in China and a bio-serotype 1B/O: 8 strain isolated in Japan. Using two-dimensional gel electrophoresis, we showed that the outer membrane proteins A (OmpA), C (OmpC) and F (OmpF) were the major antigens for both strains, although proteins located on the bacterial cell membrane and enzymes involved in energy metabolism were also identified as antigenic. We compared the whole-cell proteins of the two strains cultured at 25°C and 37°C and found portions of the outer membrane proteins (OmpX, OmpF and OmpA) were downregulated when the bacteria were cultured at 37°C, whereas urease subunit gamma (UreA), urease subunit alpha (UreC) and urease accessory protein (UreE), which are involved in urease synthesis, were upregulated when the bacteria were grown at 37°C. These observations will lay a foundation to selection of diagnostic markers for pathogenic Yersinia enterocolitica, and maybe contribute to choose the vaccine targets.     

Eutherian phylogeny from a primate perspective

Determinant formulas for up to 22 serum proteins from nineteen species in seven Eutherian orders (Chiroptera, Carnivora, Artiodactyla, Perrissodactyl, Rodentia, Proboscidea, Xenarthra) and Metatheria were derived by comparative determinant analysis from a primate (human) perspective. Plesiomorphic character states for taxon-pairs were inferred, including epitope patterns of the putative last common ancestor of Primates and non-primate Eutheria. Our approach, comparative determinant analysis, is exceptional among immunological methods in being able to define molecular character states for cladistic systematics. Average degrees of phylogenetic conservation of protein determinants varied between loci by 6.5-fold. Against the background of possible lineage-specific rates of molecular substitution, the clear immunological differentiation of the Asiatic elephant from (other) ungulates is discussed as to its phylogenetic relevance, as are the well-marked distances between guinea-pig and myomorph rodents, fissipeds and harbour seal, Mega- and Microchiroptera, or pig and ruminants. It is concluded that genetic drift influenced the rate of molecular evolution of mammalian orders to a different extent, e.g. more in the Proboscidean lineage than in the clade leading to the horse. It is concluded that the substitution rate of antigenic determinants of serum proteins correlates positively with speciation frequency in a phyletic lineage.     

Accuracy of protein flexibility predictions

Protein structural flexibility is important for catalysis, binding, and allostery. Flexibility has been predicted from amino acid sequence with a sliding window averaging technique and applied primarily to epitope search. New prediction parameters were derived from 92 refined protein structures in an unbiased selection of the Protein Data Bank by developing further the method of Karplus and Schulz (Naturwissenschaften 72:212–213, 1985). The accuracy of four flexibility prediction techniques was studied by comparing atomic temperature factors of known three-dimensional protein structures to predictions by using correlation coefficients. The size of the prediction window was optimized for each method. Predictions made with our new parameters, using an optimized window size of 9 residues in the prediction window, were giving the best results. The difference from another previously used technique was small, whereas two other methods were much poorer. Applicability of the predictions was also tested by searching for known epitopes from amino acid sequences. The best techniques predicted correctly 20 of 31 continuous epitopes in seven proteins. Flexibility parameters have previously been used for calculating protein average flexibility indices which are inversely correlated to protein stability. Indices with the new parameters showed better correlation to protein stability than those used previously; furthermore they had relationship even when the old parameters failed. © 1994 Wiley-Liss, Inc.     

Improved Recovery of a Fusion Protein Containing the Antigenic Domain 1 of the Human Cytomegalovirus Glycoprotein B

Heterologous expression in Escherichia coli often leads to production of the expressed proteins as insoluble and inactive inclusion bodies. The general strategy for protein recovery includes isolation and washing of inclusion bodies, solubilization of aggregated protein and refolding of solubilized protein. The process of refolding, as well as the other steps involved in inclusion body recovery, must be optimized according to the characteristics of each protein. For the development of reliable and inexpensive serodiagnostic tests, the antigenic domain 1 (AD-1) of human cytomegalovirus glycoprotein B was expressed in E. coli and a process was developed to increase recovery of the fusion protein containing AD-1. A comparison of disruption methods and different conditions involved in recovery of this fusion protein from inclusion bodies is presented. The developed method gives a high yield of the fusion protein with a purity sufficient for use in diagnostic tests.     

Production of a monoclonal antibody specific for high molecular weight glutenin subunits (HMW-GS) in wheat and its antigenic determinant

Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to IgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T.aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye),Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.     

Integrative Structural Biology in the Era of Accurate Structure Prediction

Characterizing the three-dimensional structure of macromolecules is central to understanding their function. Traditionally, structures of proteins and their complexes have been determined using experimental techniques such as X-ray crystallography, NMR, or cryo-electron microscopy—applied individually or in an integrative manner. Meanwhile, however, computational methods for protein structure prediction have been improving their accuracy, gradually, then suddenly, with the breakthrough advance by AlphaFold2, whose models of monomeric proteins are often as accurate as experimental structures. This breakthrough foreshadows a new era of computational methods that can build accurate models for most monomeric proteins. Here, we envision how such accurate modeling methods can combine with experimental structural biology techniques, enhancing integrative structural biology. We highlight the challenges that arise when considering multiple structural conformations, protein complexes, and polymorphic assemblies. These challenges will motivate further developments, both in modeling programs and in methods to solve experimental structures, towards better and quicker investigation of structure–function relationships.     

Emerging computational approaches for the study of protein allostery

Allosteric regulation of protein function is key in controlling cellular processes so its underlying mechanisms are of primary concern to research in areas spanning protein engineering and drug design. However, due to the complex nature of allosteric mechanisms, a clear and predictive understanding of the relationship between protein structure and allosteric function remains elusive. Well established experimental approaches are available to offer a limited degree of characterization of mechanical properties within proteins, but the analytical capabilities of computational methods are evolving rapidly in their ability to accurately define the subtle and concerted structural dynamics that comprise allostery. This review includes a brief overview of allostery in proteins and an exploration of relevant experimental methods. An explanation of the transition from experimental toward computational methods for allostery is discussed, followed by a review of existing and emerging methods.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

The structure analysis and antigenicity study of the N protein of SARS-CoV

The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with o     

Structure- and sequence-based function prediction for non-homologous proteins

The structural genomics projects have been accumulating an increasing number of protein structures, many of which remain functionally unknown. In parallel effort to experimental methods, computational methods are expected to make a significant contribution for functional elucidation of such proteins. However, conventional computational methods that transfer functions from homologous proteins do not help much for these uncharacterized protein structures because they do not have apparent structural or sequence similarity with the known proteins. Here, we briefly review two avenues of computational function prediction methods, i.e. structure-based methods and sequence-based methods. The focus is on our recent developments of local structure-based and sequence-based methods, which can effectively extract function information from distantly related proteins. Two structure-based methods, Pocket-Surfer and Patch-Surfer, identify similar known ligand binding sites for pocket regions in a query protein without using global protein fold similarity information. Two sequence-based methods, protein function prediction and extended similarity group, make use of weakly similar sequences that are conventionally discarded in homology based function annotation. Combined together with experimental methods we hope that computational methods will make leading contribution in functional elucidation of the protein structures.     

Production of a monoclonal antibody specific for high molecular weight glutenin subunits (HMW-GS) in wheat and its antigenic determinant

Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to lgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T. aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye), Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.     

Trypanosoma congolense: Surface glycoproteins of two early bloodstream variants: III. Immunochemical characterization

A radioimmunoassay (RIA) for the variant-specific glycoproteins (VSG-1 and VSG-2) of two sequentially appearing variants of Trypanosoma congolense has been devised. When the isoelectrically focused VSG-1 components (VSG-1a, VSG-1b, and VSG-1c) are used as inhibitors of the VSG-1-anti-VSG-1 interaction, the RIA inhibition curves resemble each other, although minor differences in the high-affinity region of the curves can be detected. The heterologous antigen (VSG-2) does not inhibit the VSG-1-anti-VSG-1 interaction except at very high concentrations, indicating there is little cross-reactivity between highly purified VSG-1 and VSG-2. Nevertheless, heterologous antiserum, directed against VSG-2, will inhibit the VSG-1 -anti-VSG-1 interaction, and this property is shared to a significant degree by rabbit antiserum directed against an unrelated antigen. We have interpreted these findings as suggesting that: (1) there may be a constant region common to both VSG proteins, and (2) the constant region of the immunoglobulin molecule may also bind VSG proteins. Preliminary experiments show that the VSG-1 molecule augments binding of the Clq component of complement to the Fc region of immunoglobulin G.     

汉滩病毒S基因的分段表达及其线性和构象型抗原表位分析

构建汉滩病毒76—118N蛋白及其分别从N-端和C-端缺失的共6个突变体,在大肠杆菌BL-21中进行表达,并对其中一些蛋白进行了纯化。通过Western blot、酶联免疫吸附试验(ELISA)进行汉滩病毒N蛋白的抗原表位分析,N蛋白及6个缺失突变体都与组特异性抗体L13F3呈阳性反应,而缺失突变体与型特异性抗体AH30呈阴性反应。构建汉滩病毒76—118N蛋白及其6个缺失突变体的真核表达载体,并在COS-7细胞中进行表达。通过间接免疫荧光试验(IFA)进行汉滩病毒N蛋白的抗原表位分析,病人血清与真核表达的N蛋白及6个缺失突变体呈阳性反应。而仅有N蛋白及缺失N端1～30位氨基酸序列的NPN30与型特异性抗体AH30呈阳性反应。证实组特异性抗体L13F3结合的抗原表位位于N端1～30位氨基酸;而C端抗原表位对于型特异性抗体AH30与N蛋白的识别和结合具有重要意义,缺失N端100位氨基酸序列可能破坏羧基端构象型表位,也可以影响N蛋白与AH30的结合。     

Evaluation of transmembrane helix predictions in 2014

Experimental structure determination continues to be challenging for membrane proteins. Computational prediction methods are therefore needed and widely used to supplement experimental data. Here, we re‐examined the state of the art in transmembrane helix prediction based on a nonredundant dataset with 190 high‐resolution structures. Analyzing 12 widely‐used and well‐known methods using a stringent performance measure, we largely confirmed the expected high level of performance. On the other hand, all methods performed worse for proteins that could not have been used for development. A few results stood out: First, all methods predicted proteins in eukaryotes better than those in bacteria. Second, methods worked less well for proteins with many transmembrane helices. Third, most methods correctly discriminated between soluble and transmembrane proteins. However, several older methods often mistook signal peptides for transmembrane helices. Some newer methods have overcome this shortcoming. In our hands, PolyPhobius and MEMSAT‐SVM outperformed other methods. Proteins 2015; 83:473–484. © 2014 Wiley Periodicals, Inc.     

SARS-CoV单克隆抗体的制备及抗原表位的初步鉴定

参照已发表的SARS冠状病毒BJ01株基因序列 ,利用计算机软件预测并选取该病毒S、M、N三种主要结构蛋白部分抗原性优势区域 ,以编码Gly-Pro-Gly序列相连接合成两段嵌合基因A和B。并分别克隆于pGEX -6p- 1载体上用IPTG进行诱导表达 ,以纯化的嵌合蛋白A和B为抗原 ,分别免疫BALB c小鼠制备单克隆抗体。利用单克隆抗体亚型检测试剂盒和SARS CoV商品化ELISA检测试剂盒对其进行亚型和特异性鉴定。结果表明融合表达两段嵌合基因产物 ,其大小分别为 34kD和35kD ,Westernblot分析证实两种表达产物都能被SARS病人康复期血清所识别。获得了 6株能稳定分泌特异性抗体的阳性细胞克隆株。亚型鉴定结果除D3C5为IgG2a外其他单抗均为IgG1,而且所有单抗的轻链均为κ链。特异性鉴定发现除D3D1外 ,其余的 5株单抗均能与SARS CoV商品化ELISA检测试剂盒发生特异性反应。将D3D1与灭活后经超声波裂解的SARS CoV进行Westernblot分析 ,发现它能特异性识别 180kD的蛋白带。分别融合表达了 6个S蛋白的寡肽 (S1- S6 ) ,并对筛选出的单克隆…     

Computational methods for generating models of denatured and partially folded proteins

Partially folded and denatured proteins can give important insights into protein folding, misfolding, and aggregation. Such non-native states of proteins are however very difficult to characterise in detail as they are dynamic, heterogeneous systems comprising of ensembles of interconverting conformers. This article describes methods that produce models for non-native proteins in atomic detail. A variety of molecular dynamics based protocols are discussed together with some recent procedures that include restraints from experimental data. These models provide an important framework for interpreting experimental data from studies of non-native states using nuclear magnetic resonance spectroscopy, fluorescence, circular dichroism, and small angle scattering techniques.     

Data variance and statistical significance in 2D-gel electrophoresis and DIGE experiments: comparison of the effects of normalization methods

Identifying changes in the relative abundance of proteins between different biological samples is often confounded by technical noise. In this work, we compared eight normalization methods commonly used in two-dimensional gel electrophoresis and difference gel electrophoresis (DIGE) experiments for their ability to reduce noise and for their influence on the list of proteins whose difference in abundance between two samples is determined to be statistically significant. With respect to reducing noise we find that, while all methods improve upon unnormalized data, cyclic linear normalization is the least well suited to gel-based proteomics and the performances of the other methods are similar. We also find in DIGE data that the choice of normalization method has less of an impact on the noise than does the decision to use an internal reference in the experimental design and that both normalization and standardization using the internal reference are required to maximally reduce variance. Despite the similar noise reduction achieved by most normalization methods, the list of proteins whose abundance was determined to differ significantly between biological groups differed depending on the choice of normalization method. This work provides a direct comparison of the impact of normalization methods in the context of common experimental designs.