Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections

Neisseria meningitidis is the most common cause of meningitis and causes epidemic outbreaks. One trait of N. meningitidis, which is associated with most of the currently recognized virulence determinants, is the presence of phase-variable genes that are suspected to enhance its ability to cause an invasive disease. To detect the immune responses to phase-variable expressed proteins, we applied protein microarray technology for the screening of meningitis patient sera. We amplified all 102 known phase-variable genes from N. meningitidis serogroup B strain MC58 by polymerase chain reaction and subcloned them for expression in Escherichia coli. With this approach, we were able to express and purify 67 recombinant proteins representing 66% of the annotated genes. These were spotted robotically onto coated glass slides to generate protein microarrays, which were screened using 20 sera of patients suffering from meningitis, as well as healthy controls. From these screening experiments, 47 proteins emerged as immunogenic, exhibiting a variable degree of seroreactivity with some of the patient sera. Nine proteins elicited an immune response in more than three patients, with one of them, the phase-variable opacity protein OpaV (NMB0442), showing responses in 11 patient sera. This is the first time that protein microarray technology has been applied for the investigation of genetic phase variation in pathogens. The identification of disease-specific proteins is a significant target in biomedical research, as such proteins may have medical, diagnostic, and commercial potential as disease markers.     

Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, Z(Taq) and Z(IgA), binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.     

Immobilization of a lipoxygenase in silica gels for application in aqueous media

The encapsulation of soybean lipoxygenase-1 (LOX-1) in silica gels and its application in an aqueous medium, were studied. The main silica precursor was tetramethoxysilane (TMOS) but the introduction of hydrophobic SiCH₃ groups brought with methyltrimethoxysilane (MTMS) was evaluated. Other sol–gel synthesis parameters investigated comprised partial or complete drying by evaporation and CO₂ supercritical drying. The influence on LOX-1 activity of the various chemicals with which the enzyme was in contact during encapsulation (acetone, methanol, polyvinyl alcohol), as well as the temperature and pH, were examined. The activity of free and encapsulated LOX-1 was assayed on the oxygenation reaction of linoleic acid by dioxygen from air dissolved in aqueous medium, in a UV–vis spectrophotometer. With free LOX-1, the reaction advancement could be followed in continuous in the spectrophotometer. With the gels, in a first approach, the conversion was simply determined after 15 min reaction after filtration of the liquid, to discriminate between active and inactive gels. For the most interesting gels, the kinetics were then assessed by continuous recording in the UV spectrophotometer, after placing a small piece of gel (≈15 mg) directly in the cell. The best gels had an activity ≈30% of free LOX. The present studies, supplemented by characterization of the gels texture and structure, respectively by nitrogen adsorption and ²⁹Si MAS NMR, showed that drying a gel before use in aqueous media was detrimental to the activity. This effect is due to a contraction of the gel network which occurs when a dry aerogel sample is dipped in water after drying. Hence gels containing LOX-1 enzyme must not be dried but kept in water impregnated state, for optimum use.     

Immobilization strategies for small molecule,peptide and protein microarrays

Protein, peptide and small molecule microarrays are valuable tools in biological research. In the last decade, substantial progress has been achieved to make these powerful technologies more reliable and available for researchers. This review describes chemical preparation methods for these microarrays with focus on site‐selective and bioorthogonal immobilization reactions, particularly the Staudinger ligation and the thiol‐ene reaction. In addition, the application of peptide microarrays, which were prepared by Staudinger ligation, to substrate specificity mapping is illustrated. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Immobilization and hybridization by single sub-millisecond electric field pulses, for pixel-addressed DNA microarrays

Single square voltage pulses applied to buried electrodes result in dramatic rate increases for (1) selective covalent bonding (immobilization) of single-stranded DNA (ssDNA) probes to a functionalized thin film SiO₂ surface on a plastic substrate and (2) hybridization of ssDNA to the immobilized probe. DNA immobilization and hybridization times are 100 ns and 10 μs, respectively, about 10⁹ times faster than the corresponding passive reactions without electric field. Surface coverage is comparable. Duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of ssDNA immobilization and hybridization. With rise times of 4.5 ns, pulses shorter than 1 ms and voltages below 1 V are effective. The ssDNA adsorbed on the surface is reoriented by the rapidly changing electric field. This reduces steric barriers and speeds the immobilization and hybridization reactions. These results open the way for pixel-addressed microarrays driven by silicon microelectronics circuits.     

Systematic comparison of surface coatings for protein microarrays

To process large numbers of samples in parallel is one potential of protein microarrays for research and diagnostics. However, the application of protein arrays is currently hampered by the lack of comprehensive technological knowledge about the suitability of 2-D and 3-D slide surface coatings. We have performed a systematic study to analyze how both surface types perform in combination with different fluorescent dyes to generate significant and reproducible data. In total, we analyzed more than 100 slides containing 1152 spots each. Slides were probed against different monoclonal antibodies (mAbs) and recombinant fusion proteins. We found two surface coatings to be most suitable for protein and antibody (Ab) immobilization. These were further subjected to quantitative analyses by evaluating intraslide and slide-to-slide reproducibilities, and the linear range of target detection. In summary, we demonstrate that only suitable combinations of surface and fluorescent dyes allow the generation of highly reproducible data.     

Infectivity of Anticarsia gemmatalis nucleopolyhedrovirus to different insect cell lines: Morphology, viral production, and protein synthesis

The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is currently used as an efficient biological pesticide for the control of the velvetbean caterpillar (A. gemmatalis), an important pest of soybean in Brazil. Until now, production of the virus has been achieved mainly by infection of larvae on local soybean farms. Studies for the development of in vitro systems and the optimization of mass production in insects reared on artificial diets is now important to help to meet the actual demand for the bioinsecticide. We therefore, investigated the infectivity of AgMNPV in cell culture, which might contribute to the selection of suitable cell lines that may be used for in vitro production of this virus. The cytopathic effects induced by the virus, the production of viral particles and the synthesis of viral polypeptides were examined and compared in the cell lines from A. gemmatalis (UFL-AG-286), Trichoplusia ni (BTI-Tn-5B1-4 and TN-368), Spodoptera frugiperda (IPLB-SF-21AE and Sf9), Lymantria dispar (IPLB-LD-652Y), and Bombyx mori (BM-5). Whereas, Tn-5B1-4 and AG-286 cells produced large numbers of occlusion bodies, no polyhedra were visualized in either Ld-652Y or BM-5 cells, although extensive cell lysis was observed in BM-5. Analysis of the kinetics of viral protein synthesis by SDS–PAGE after pulse labeling with [³⁵S]methionine, showed similar protein patterns in most of the cell lines tested. Exceptions were the LD-652Y and BM-5 cells, in which viral polypeptides, including polyhedrin, were not synthesized. In parallel, measurement of viral titers (budded virus) by the endpoint dilution method showed that Tn-5B1-4, AG-286, and SF-21AE cells were highly productive. Their TCID₅₀ values, at 48 h p.i., were about 10⁷ IU/ml. In addition to the lower formation of polyhedra, the viral titers determined in Sf9 and TN-368 cells were about 5 to10-fold lower. As expected, the viral titers obtained in LD-652Y and BM-5 cells were similar to basal levels.     

Advances in the development of human protein microarrays

Introduction: High-content protein microarrays in principle enable the functional interrogation of the human proteome in a broad range of applications, including biomarker discovery, profiling of immune responses, identification of enzyme substrates, and quantifying protein-small molecule, protein-protein and protein-DNA/RNA interactions. As with other microarrays, the underlying proteomic platforms are under active technological development and a range of different protein microarrays are now commercially available. However, deciphering the differences between these platforms to identify the most suitable protein microarray for the specific research question is not always straightforward.

Areas covered: This review provides an overview of the technological basis, applications and limitations of some of the most commonly used full-length, recombinant protein and protein fragment microarray platforms, including ProtoArray Human Protein Microarrays, HuProt Human Proteome Microarrays, Human Protein Atlas Protein Fragment Arrays, Nucleic Acid Programmable Arrays and Immunome Protein Arrays.

Expert commentary: The choice of appropriate protein microarray platform depends on the specific biological application in hand, with both more focused, lower density and higher density arrays having distinct advantages. Full-length protein arrays offer advantages in biomarker discovery profiling applications, although care is required in ensuring that the protein production and array fabrication methodology is compatible with the required downstream functionality.     

漆酶固定化及其在小型酶反应器中的应用

以尼龙网为载体,戊二醛为交联剂,固定化真菌漆酶。用固定化酶在自行设计的小反应器中降解低浓度造纸废水,试验结果如下：当废水COD的浓度控制在3000 mg/L左右,降解时间为26h,废水的COD去除率达到35%。     

Optimizing antibody immobilization strategies for the construction of protein microarrays

Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase.     

Method for collecting and immobilizing individual cumulus cells enabling quantitative immunofluorescence analysis of proteins

Most immunofluorescence methods rely on techniques dealing with a very large number of cells. However, when the number of cells in a sample is low (e.g., when cumulus cells must be analyzed from individual cumulus–oocyte complexes), specific techniques are required to conserve, fix, and analyze cells individually. We established and validated a simple and effective method for collecting and immobilizing low numbers of cumulus cells that enables easy and quick quantitative immunofluorescence analysis of proteins from individual cells. To illustrate this technique, we stained proprotein of a disintegrin and metalloproteinase with thrombospondin-like repeats-1 (proADAMTS-1) and analyzed its levels in individual porcine cumulus cells.     

Immobilization of thermostable exo-inulinase from mutant thermophilic Aspergillus tamarii-U4 using kaolin clay and its application in inulin hydrolysis

In this study, attempts were made to immobilize purified exo-inulinase from mutant thermophic Aspergillus tamarii-U4 onto Kaolinite clay by covalent bonding cross-linked with glutaraldehyde with an immobilization yield of 66% achieved. The free and immobilized inulinases were then characterized and characterization of the enzymes revealed that temperature and pH optima for the activity of the free and immobilized enzymes were both 65?°C and pH 4.5 respectively. The free inulinase completely lost its activity after incubation at 65?°C for 6 h while the immobilized inulinase retained 16.4% of its activity under the same condition of temperature and incubation time. The estimated kinetic parameters K_m and V_max for the free inulinase as estimated from Lineweaver-Burk plots were 0.39?mM and 4.21?µmol/min for the free inulinase and 0.37?mM and 4.01?µmol/min for the immobilized inulinase respectively. Inulin at 2.5% (w/v) and a flow rate of 0.1?mL was completely hydrolysed for 10?days at 60?°C in a continuous packed bed column and the operational stability of the system revealed that the half-life of the immobilized inulinase was 51?days. These properties make the immobilized exo-inulinase from Aspergillus tamarii-U4 a potential candidate for the production of fructose from inulin hydrolysis.     

Highly conserved structural properties of the C-terminal tail of HIV-1 gp41 protein despite substantial sequence variation among diverse clades: implications for functions in viral replication

Although the HIV-1 Env gp120 and gp41 ectodomain have been extensively characterized in terms of structure and function, similar characterizations of the C-terminal tail (CTT) of HIV gp41 remain relatively limited and contradictory. The current study was designed to examine in detail CTT sequence conservation relative to gp120 and the gp41 ectodomain and to examine the conservation of predicted physicochemical and structural properties across a number of divergent HIV clades and groups. Results demonstrate that CTT sequences display intermediate levels of sequence evolution and diversity in comparison to the more diverse gp120 and the more conserved gp41 ectodomain. Despite the relatively high level of CTT sequence variation, the physicochemical properties of the lentivirus lytic peptide domains (LLPs) within the CTT are evidently highly conserved across clades/groups. Additionally, predictions using PEP-FOLD indicate a high level of structural similarity in the LLP regions that was confirmed by circular dichroism measurements of secondary structure of LLP peptides from clades B, C, and group O. Results demonstrate that LLP peptides adopt helical structure in the presence of SDS or trifluoroethanol but are predominantly unstructured in aqueous buffer. Thus, these data for the first time demonstrate strong conservations of characteristic CTT physicochemical and structural properties despite substantial sequence diversity, apparently indicating a delicate balance between evolutionary pressures and the conservation of CTT structure and associated functional roles in virus replication.     

Immobilization of esterase SulE in cross-linked gelatin/chitosan and its application in remediating soils polluted with tribenuron-methyl and metsulfuron-methyl

The esterase SulE is known to be an important and primary enzyme for the degradation of many sulfonylurea herbicides such as tribenuron-methyl, metsulfuron-methyl, and chlorimuron-ethyl. The disadvantages of applying free enzyme include poor stability and rapid deactivation in the natural environment, but these problems can be mitigated by using immobilized enzymes. In this study, cross-linked gelatin/chitosan (GLT/CTS) was selected as the enzyme immobilization carrier because its SulE activity recovery was higher than those of γ-polyglutamate/gelatin (γ-PGA/GLT) and γ-polyglutamate/chitosan (γ-PGA/CTS). The immobilization conditions were further optimized, and the effects of pH and temperature on GLT/CTS-SulE and free SulE activities to degrade tribenuron-methyl and metsulfuron-methyl were investigated. A microcosm experiment was conducted to assess the effectiveness of GLT/CTS-SulE in remediating soils polluted with tribenuron-methyl and metsulfuron-methyl. The results showed that, compared with free SulE, GLT/CTS-SulE displayed better pH and temperature adaptabilities and higher degradation efficiencies for both herbicides in soil, especially tribenuron-methyl. The results indicate that this is an excellent method to immobilize SulE for enhanced remediation of soils polluted with tribenuron-methyl or metsulfuron-methyl in situ.     

A novel method for viral display of ER membrane proteins on budded baculovirus

The baculovirus expression system has been used to express large quantities of various proteins, including membrane receptors. Here, we reveal a novel property of this expression system to be that certain membrane proteins can be displayed on the budded virus itself. We introduced the genes encoding sterol regulatory element-binding protein-2 (SREBP-2) or SREBP cleavage-activating protein (SCAP), important integral membrane proteins of the endoplasmic reticulum (ER) and/or the Golgi apparatus related to cellular cholesterol regulation, into a baculovirus vector. When insect cells were infected with SREBP-2 or SCAP recombinant viruses, it was found that these ER membrane proteins appeared on the budded baculovirus in addition to the host cell membrane fraction. Compared to proteins expressed on the cell membrane, membrane proteins displayed on virus exhibited both less aggregation and less degradation upon immunoblotting. Using this viral displayed SCAP as the screening antigen, we then generated a new monoclonal antibody specific against SCAP, which was useful for immunological localization studies. This system, which takes advantage of the viral display of membrane proteins, should prove to be a powerful additional tool for postgenomic protein analysis.     

Current Status of Deltabaculoviruses, Cypoviruses and Chloriridoviruses Pathogenic for Mosquitoes

There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridoviruses (MIVs) (Iridoviridae: Chloriridovirus). Baculoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This review presents an overview of the current status and recent advancements in understanding the biology and molecular features of mosquito pathogenic viruses.     

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.     

Current Status of Deltabaculoviruses, Cypoviruses and Chloriridoviruses Pathogenic for Mosquitoes

There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridoviruses (MIVs) (Iridoviridae: Chloriridovirus). Baculoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This review presents an overview of the current status and recent advancements in understanding the biology and molecular features of mosquito pathogenic viruses.     

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.     

Thermus thermophilus-derived protein tags that aid in preparation of insoluble viral proteins

The expression and solubilization of insoluble proteins have been facilitated by the introduction of protein tags. In our analyses of viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1), however, several conventional tag proteins enhanced its expression but failed to solubilize it. Therefore, we decided to explore whether proteins derived from Thermus thermophilus HB8 (T. th.), a highly heat-stable bacterium, could be used as tag proteins to enhance the solubilization of Vpr. Based on the data accumulated during the recent structural genomics project of T. th., we selected 15 T. th. proteins with high expression levels and solubilities. From this group, we identified a T. th. tag protein that expressed Vpr in a soluble form. Furthermore, two T. th. tag proteins, including the identified one, were found to solubilize the extremely insoluble membrane-spanning domain of the envelope protein of HIV-1. When green fluorescent protein (GFP) was used as a passenger protein of T. th. tags, the brightness and stability of GFP were similar to those of untagged GFP, suggesting that the T. th. tags do not negatively affect the function of the passenger protein. Thus, data of structural genomics can be applied to generate a customized versatile protein tag for protein analyses.