Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues

Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.     

DNA family shuffling within the chicken avidin protein family - A shortcut to more powerful protein tools

Avidins represent an interesting group of proteins showing high structural similarity and ligand-binding properties but low similarity in primary structure. In this study, we show that it is possible to create functional chimeric proteins from the avidin protein family when applying DNA family shuffling to the genes of the avidin protein family: avidin, avidin related gene 2 and biotin-binding protein A. The novel chimeric proteins were selected by phage display biopanning against biotin, and the selected enriched proteins were characterized, displaying diverse features distinct from the parental genes, including binding to cysteine.     

Vertical slab gel electrophoresis on a large number of samples: an apparatus with the capacity for central cooling

Following horizontal electroelution, or blotting, of proteins from polyacrylamide gels to immobilizing matrices, such as nitrocellulose or Zeta-bind paper, the transferred proteins can be derivatized in situ with pyridoxal 5'-phosphate and sodium borohydride. After a quenching step to eliminate nonspecific binding of antibody to the protein-binding matrix, the blot is incubated with a solution containing a mouse monoclonal antibody specific for the 5'-phosphopyridoxyl group. The transferred proteins can then be located on the blot with second antibody staining procedures employing either a peroxidase-linked goat anti-mouse F(ab')2 antibody or a peroxidase-linked avidin/biotin system. The solid-phase enzyme-linked immunosorbent assay method described in this report is a mild, general, and sensitive immunochemical method for the detection of proteins on protein-binding matrices.     

Efficient strategies for the conjugation of oligonucleotides to antibodies enabling highly sensitive protein detection

Three methods for the conjugation of oligonucleotides to antibodies and the subsequent application of these conjugates to protein detection at attomole levels in immunoassays are described. The methods are based on chemical modification of both antibody and oligonucleotide. Aldehydes were introduced onto antibodies by modification of primary amines or oxidation of carbohydrate residues. Aldehyde- or hydrazine-modified oligonucleotides were prepared either during phosphoramidite synthesis or by post-synthesis derivatization. Conjugation between the modified oligonucleotide and antibody resulted in the formation of a hydrazone bond that proved to be stable over long periods of time under physiological conditions. The binding activity of each antibody-oligonucleotide conjugate was determined to be comparable to the corresponding unmodified antibody using a standard sandwich ELISA. Each oligonucleotide contained a unique DNA sequence flanked by universal primers at both ends and was assigned to a specific antibody. Highly sensitive immunoassays were performed by immobilizing analyte for each conjugate onto a solid support with cognate capture antibodies. Binding of the antibody-oligonucleotide conjugate to the immobilized analyte allowed for amplification of the attached DNA. Products of amplification were visualized using gel electrophoresis, thus denoting the presence of bound analyte. The preferred conjugation method was used to generate a set of antibody-oligonucleotide conjugates suitable for high-sensitivity protein detection.     

A highly sensitive near‐infrared fluorescent detection method to analyze signalling pathways by reverse‐phase protein array

The comprehensive and quantitative analysis of the protein phosphorylation patterns in different cellular context is of considerable and general interest. The ability to quantify phosphorylation of discrete signalling proteins in large collections of biological samples would greatly favour the development of systems biology in the field of cell signalling. Reverse‐phase protein array (RPPA) potentially represents a very attractive approach to map signal transduction networks with high throughput. In the present report, we describe an improved detection method for RPPA combining near‐infrared with one or two rounds of tyramide‐based signal amplification. The LOQ was lowered from 6.84 attomoles with a direct detection protocol to 0.21 attomole with two amplification steps. We validated this method in the context of intracellular signal transduction triggered by follicle‐stimulating hormone in HEK293 cells. We consistently detected phosphorylated proteins in the sub‐attomole range from less than 1 ng of total cell extracts. Importantly, the method correlated with Western blot analysis of the same samples while displaying excellent intra‐ and inter‐slide reproducibility. We conclude that RPPA combined with amplified near‐infrared detection can be used to capture the subtle regulations intrinsic to signalling network dynamics at an unprecedented throughput, from minute amounts of biological samples.     

An accurate,sensitive, and scalable method to identify functional sites in protein structures

Functional sites determine the activity and interactions of proteins and as such constitute the targets of most drugs. However, the exponential growth of sequence and structure data far exceeds the ability of experimental techniques to identify their locations and key amino acids. To fill this gap we developed a computational Evolutionary Trace method that ranks the evolutionary importance of amino acids in protein sequences. Studies show that the best-ranked residues form fewer and larger structural clusters than expected by chance and overlap with functional sites, but until now the significance of this overlap has remained qualitative. Here, we use 86 diverse protein structures, including 20 determined by the structural genomics initiative, to show that this overlap is a recurrent and statistically significant feature. An automated ET correctly identifies seven of ten functional sites by the least favorable statistical measure, and nine of ten by the most favorable one. These results quantitatively demonstrate that a large fraction of functional sites in the proteome may be accurately identified from sequence and structure. This should help focus structure-function studies, rational drug design, protein engineering, and functional annotation to the relevant regions of a protein.     

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.     

A highly sensitive method for the determination of protein bound 3,4-dihydroxyphenylalanine as a marker for post-translational protein hydroxylation in human tissues ex vivo

A highly sensitive, specific and tissue-independent method is described to evaluate oxidative stress-mediated protein hydroxylation in red blood cells, frontal cortex, and liver by HPLC separation and electrochemical detection of protein-bound 3,4-dihydroxyphenylalanine (DOPA) following gas-phase amino acid hydrolysis of tissue protein extracts containing exclusively proteins larger than 3 kDa. Simultaneous measurement of protein tyrosine (Tyr) content using fluorescence detection results in a tissue specific DOPA/Tyr ratio that may reflect oxidative stress-mediated protein modifications in disease, or following the exposure to oxidative stress-inducing agents.     

Light-induced binding of 48-kDa protein to photoreceptor membranes is highly enhanced by phosphorylation of rhodopsin

The 48-kDa protein, a major protein of rod photoreceptor cells, is soluble in the dark but associates with the disk membranes when some (5-10%) of their rhodopsin has absorbed light and if this rhodopsin is additionally phosphorylated by ATP and rhodopsin kinase. If rhodopsin has been phosphorylated and regenerated prior to the protein binding experiment, the binding of 48-kDa protein depends on light but no longer on the presence of ATP. Another photoreceptor protein, GTP-binding protein, associates with both phosphorylated and unphosphorylated rhodopsin upon illumination. Excess GTP-binding protein thereby displaces 48-kDa protein from phosphorylated disks; this indicates competition between these two proteins for binding sites on illuminated phosphorylated rhodopsin molecules.     

Ligand supplementation as a method to increase soluble heterologous protein production

Ligand interactions are central to enzyme or receptor function, constituting a cornerstone in biochemistry and pharmacology. Here we discuss a ligand application that can be exploited to significantly increase the proportion of recombinant protein expressed in soluble form, by including ligands during the culture. Provided that a sufficiently soluble, cell-permeable and avid ligand is available, one can use it to stabilize nascently synthesized proteins, and in this manner promote solubility and prevent aggregation. To our knowledge, this concept has not been explored systematically and we provide here the first data on ligand supplementation in expression experiments across a whole human protein family: the short-chain dehydrogenases/reductases (SDR). We identified glycerrhitinic acid and its hemisuccinate ester, carbenoxolone (CBX), as ligands with variable affinities ranging from low nanomolar to micromolar binding constants against several SDRs. CBX was utilized as a culture additive in Escherichia coli expression systems against a total of approximately 500 constructs derived from 65 SDR targets, and significantly higher levels of soluble protein were obtained for more than four distinct targets. One of these, the glucocorticoid-activating enzyme type 1 11β-hydroxysteroid dehydrogenase (11β-HSD1), was solubly expressed only at a very low level (<10 µg/l culture) in the absence of ligand; however, soluble expression could be enhanced to mg/l levels by inclusion of CBX or other inhibitors. Other compounds with different chemical scaffolds were used against 11β-HSD1 in equivalent expression experiments yielding similar results. Taken together, if suitable ligands for a given protein are available, this approach could be tested quickly and might represent an easy and effective strategy to enhance soluble protein production, suitable for structural and functional characterization studies.     

Extension of a protein docking algorithm to membranes and applications to amyloid precursor protein dimerization

Novel adjustments are introduced to the docking algorithm, DOCK/PIERR, for the purpose of predicting structures of transmembrane protein complexes. Incorporating knowledge about the membrane environment is shown to significantly improve docking accuracy. The extended version of DOCK/PIERR is shown to perform comparably to other leading docking packages. This membrane version of DOCK/PIERR is applied to the prediction of coiled‐coil homodimer structures of the transmembrane region of the C‐terminal peptide of amyloid precursor protein (C99). Results from MD simulation of the C99 homodimer in POPC bilayer and docking are compared. Docking results are found to capture key aspects of the homodimer ensemble, including the existence of three topologically distinct conformers. Furthermore, the extended version of DOCK/PIERR is successful in capturing the effects of solvation in membrane and micelle. Specifically, DOCK/PIERR reproduces essential differences in the homodimer ensembles simulated in POPC bilayer and DPC micelle, where configurational entropy and surface curvature effects bias the handedness and topology of the homodimer ensemble. Proteins 2015; 83:2170–2185. © 2015 Wiley Periodicals, Inc.     

Investigation on bioactive protection of LEA protein to insulin by molecular simulation

Nowadays various protein medicines are increasingly playing a key role on treatment of many diseases, while the bioactivity of such kinds of protein medicines is unstable because of their heat sensitivity. In order to explore a protective method and to explain the protective mechanism of protein medicines, the bioactive protection of the late embryogenesis abundant (LEA) protein to insulin was researched by molecular dynamics simulation. The results suggest that LEA proteins preserve the native structure of the insulin well. Compared with the desiccated insulin without any protection, the structure of insulin protected by LEA protein have smaller values, more centralised configurational space, lower free energies and structural cluster more closer to the native structure. All the above results prove that the LEA protein does protect the bioactivity of insulin during desiccation. The LEA protein is a perfect bioactive protectant for heat-sensitive protein medicines. Such LEA proteins can match the shape of insulin and form multisite binding interaction with insulin.     

Dopamine receptors on photoreceptor membranes couple to a GTP-binding protein which is sensitive to both pertussis and cholera toxin

Dopamine receptors with a pharmacological profile similar to D2 receptors are coenriched with rhodopsin in preparations of bovine retinal membranes. A high density of these receptors are present on photoreceptor membranes. The affinity of the agonist apomorphine for these receptors is decreased by the guanine nucleotides GTP and GppNHp. Treatment of photoreceptor membranes with pertussis or cholera toxin also decreased the affinity of apomorphine and eliminated the effect of GTP.     

Stabilization of a proteolytically sensitive cytoplasmic recombinant protein during transition to downstream processing

The influence of aeration and glucose feeding on the stability of recombinant protein A in Escherichia coli during the transition period from a fed‐batch cultivation to downstream processing was studied. Neither interruption of the feeding under aerobic conditions nor anaerobic conditions in presence of glucose could stabilize protein A completely and the intracellular ATP pool did not decrease to less than 0.75–1 mM by this treatment. On the other hand, the absence of both oxygen and glucose resulted in a decrease of the ATP pool to less than 0.5 mM and almost complete stabilization of protein A. The decrease of ATP was more severe when sulfite was used instead of nitrogen gas to create anaerobic conditions in presence of glucose. This also resulted in nearly complete stabilization of protein A, which might be explained by an inhibiting effect of sodium sulfite on fermentation. Therefore, protein stabilization and decrease of the ATP pool were correlated in experiments in vivo. The concentrations of ADP and AMP increased during starvation and may also play a role in stabilization of the protein in vivo. ATP may be a limiting factor of proteolysis also during further steps of downstream processing. Its concentration decreases by 80–90% during harvesting and centrifugation of biomass and even further during disruption of cells. However, neither addition nor regeneration of ATP in cell disintegrate was enough to restore degradation of protein A, indicating that an additional factor limits proteolysis in vitro. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 730–738, 1999.     

Development of a conditional localization approach to control apicoplast protein trafficking in malaria parasites

Secretory proteins are of particular importance to apicomplexan parasites and comprise over 15% of the genomes of the human pathogens that cause diseases like malaria, toxoplasmosis and babesiosis as well as other diseases of agricultural significance. Here, we developed an approach that allows us to control the trafficking destination of secretory proteins in the human malaria parasite Plasmodium falciparum. Based on the unique structural requirements of apicoplast transit peptides, we designed three conditional localization domains (CLD1, 2 and 3) that can be used to control protein trafficking via the addition of a cell permeant ligand. Studies comparing the trafficking dynamics of each CLD show that CLD2 has the most optimal trafficking efficiency. To validate this system, we tested whether CLD2 could conditionally localize a biotin ligase called holocarboxylase synthetase 1 (HCS1) without interfering with the function of the enzyme. In a parasite line expressing CLD2‐HCS1, we were able to control protein biotinylation in the apicoplast in a ligand‐dependent manner, demonstrating the full functionality of the CLD tool. We have developed and validated a novel molecular tool that may be used in future studies to help elucidate the function of secretory proteins in malaria parasites.     

eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM)

The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H⁺ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter

The recombinant Ca²⁺-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of polymer methacrylate beads or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with the biotin residue on the 3′-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide was used as a DNA template. The probe in the hybridization complex was labeled by the elongation of the chain using a Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, provides a high sensitivity of the assay (no less than 10^?15 mol of DNA template), and ensures a quantitative determination of the amount of DNA template in the tested sample.     

A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

A Ca²⁺--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca²⁺ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.