A new method for detecting endocytosed proteins.

A new reagent, DPSgt, is described which has been designed to label cell surface proteins at 0 degree C. The reagent is easily made; it is water soluble and contains a reactive impermeant ester at one end, a tyrosine which can be radioiodinated at the other, and a disulphide in-between. The label can be removed from cells by cleaving the disulphide linkage in it with glutathione at 0 degree C. When cells are warmed to 37 degrees C between labelling and reduction, labelled proteins which are endocytosed acquire resistance to reduction. This provides a simple way of measuring the endocytosis of surface proteins. The intracellular pools of transferrin and LDL receptors in K562 cells and fibroblasts have been estimated. The results indicate that intracellular receptors are in non-reducing compartments, and that uptake of average cell surface (by non-coated pit processes) in K562 cells is small.     

Indirect tissue electrophoresis: a new method for analyzing solid tissue protein

1. The eye lens core (nucleus) has been a valuable source of molecular biologic information. 2. In these studies, lens nuclei are usually homogenized so that any protein information related to anatomical subdivisions, or layers, of the nucleus is lost. 3. The present report is of a new method, indirect tissue electrophoresis (ITE), which, when applied to fish lens nuclei, permitted (a) automatic correlation of protein information with anatomic layer, (b) production of large, clear electrophoretic patterns even from small tissue samples and (c) detection of more proteins than in liquid extracts of homogenized tissues. 4. ITE seems potentially applicable to a variety of solid tissues.     

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.     

Regulated readthrough: a new method for the alternative tagging and targeting of recombinant proteins

We report here a new method for the alternative peptide tagging of recombinant proteins from mammalian cell lines. This method, which we called regulated readthrough, exploits the property of aminoglycoside antibiotics to promote translational readthrough of nonsense codons. The basic expression cassette includes a translational fusion between a gene of interest and a membrane targeting peptide, which are separated by a nonsense codon. In the presence of an aminoglycoside antibiotic, translational readthrough is promoted and results in the targeting of the fusion protein to the cell membrane, thus allowing the efficient flow cytometry-based isolation of cells expressing very high levels of recombinant protein. For downstream applications requiring the production of soluble recombinant protein, the cells are cultured in the absence of aminoglycoside, leading to an efficient translational termination. By combining different translation termination signals that exhibit various susceptibilities to aminoglycoside-mediated translational readthrough with flow cytometry capabilities, it is possible to use this technology for other applications such as functional library screening or monitoring the stability of recombinant protein production.     

Human adenovirus serotype 3 fiber protein. Comparison of native and recombinant proteins

We were able to isolate viral fiber and penton from Ad3-infected KB cells using for their detection antibodies obtained against recombinant Ad3 fiber. The native material was examined by electron microscopy and the characteristic fiber shape of a shaft terminated by a globular head was observed. The native fiber was compared with two recombinant fibers synthesized in Escherichia coli cells. One, the Ad3 fiber protein expressed in E. coli with a 14-amino acid NH2-terminal fusion peptide, under the control of the T7 promoter has been described previously. The second is a recombinant Ad3 fiber without the fusion peptide (recAd3fib), expressed in the same system. As with the fusion protein recAd3fib was found to be insoluble upon expression. It was solubilized in 6 M urea and the gradual removal of urea during the purification cycle led to a soluble preparation. Biochemical and biophysical studies show that, similarly to fusion fiber, recAd3fib self-assembles as trimers in prokaryotic cells. Electron microscopy shows that, whereas the fusion fiber consists of a population of heterogeneous particles, recAd3fib has the characteristic morphology and size of the Ad3 trimeric native fiber. Small angle neutron scattering gives a molecular weight consistent with a trimeric fiber and a radius of gyration consistent with the dimensions derived from electron microscopy. These results suggest that the fusion peptide at the NH2 terminus prevents correct protein folding. They also indicate that after solubilization with urea and subsequent renaturation a correctly folded eukaryotic oligomeric protein can be produced in E. coli.     

Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.     

A general method of protein purification for recombinant unstructured non-acidic proteins

Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.     

Network-based diffusion analysis: a new method for detecting social learning

Social learning has been documented in a wide diversity of animals. In free-living animals, however, it has been difficult to discern whether animals learn socially by observing other group members or asocially by acquiring a new behaviour independently. We addressed this challenge by developing network-based diffusion analysis (NBDA), which analyses the spread of traits through animal groups and takes into account that social network structure directs social learning opportunities. NBDA fits agent-based models of social and asocial learning to the observed data using maximum-likelihood estimation. The underlying learning mechanism can then be identified using model selection based on the Akaike information criterion. We tested our method with artificially created learning data that are based on a real-world co-feeding network of macaques. NBDA is better able to discriminate between social and asocial learning in comparison with diffusion curve analysis, the main method that was previously applied in this context. NBDA thus offers a new, more reliable statistical test of learning mechanisms. In addition, it can be used to address a wide range of questions related to social learning, such as identifying behavioural strategies used by animals when deciding whom to copy.     

Plastoglobules: a new address for targeting recombinant proteins in the chloroplast

Background  

The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures) to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary.     

SVR_CAF: An integrated score function for detecting native protein structures among decoys

An accurate score function for detecting the most native‐like models among a huge number of decoy sets is essential to the protein structure prediction. In this work, we developed a novel integrated score function (SVR_CAF) to discriminate native structures from decoys, as well as to rank near‐native structures and select best decoys when native structures are absent. SVR_CAF is a machine learning score, which incorporates the contact energy based score ( C E_score), amino acid network based score ( A AN_score), and the fast Fourier transform based score ( F FT_score). The score function was evaluated with four decoy sets for its discriminative ability and it shows higher overall performance than the state‐of‐the‐art score functions. Proteins 2014; 82:556–564. © 2013 Wiley Periodicals, Inc.     

PCR method for detecting recombinant baculoviruses with the insertion of a large gene

A PCR strategy was developed using primers specific to an infectious bursal disease virus (IBDV) gene as well as primers flanking the polyhedrin region of baculovirus to verify the presence of IBDV gene in the recombinant baculovirus and confirm the absence of wild-type baculovirus contamination. This method can be applied to detect the presence of large genes in the recombinant baculovirus with greater sensitivity and avoid the need of modifying the typical PCR procedure provided by the manufacturer. © Rapid Science Ltd. 1998     

A new method for detecting neural interconnectivity

We propose a class of counting process models for analyzing firing times of an ensemble of neurons. We allow the counting process intensities to be unspecified, unknown functions of the times passed since the most recent firings. Under this assumption we derive a class of statistics with their respective thresholds as well as graphical methods for detecting neural connectivity. We introduce a model under which detection is shown to be certain for long series of observations. We suggest ways to classify interactions as inhibition or excitation and to estimate their strengths. The power of the proposed methods is compared by simulating observations from artificial networks. By analyzing empirically obtained series we obtain results which are consistent with those obtained from cross-correlation-based methods but in addition obtain new insights on further aspects of the interactions. Received: 7 February 1996 / Accepted in revised form: 5 March 1997     

A new method of detecting hormone-binding proteins electroblotted onto glass fiber filter: juvenile hormone-binding proteins from grasshopper hemolymph

We have developed a new method to identify juvenile hormone (JH)-binding proteins blotted onto glass fiber filter (GFF) after electrophoretic separation. Insect JH regulates reproduction in the two-striped grasshopper, Melanoplus bivittatus. A number of proteins are involved in the delivery of JH from its site of synthesis to the nuclei of fat body cells where it acts to induce vitellogenesis. To identify JH binding proteins, hemolymph was separated by PAGE, electroblotted onto GFF, and incubated in [10-3H]JH-III. The amount of hormone bound by blotted proteins increased with the amount of protein on the filter, was competitively displaced by excess non-labeled hormone, and was affiliated with individual bands on fluorograms of proteins blotted after electrophoretic separation. GFF etched with trifluoroacetic acid was better than nitrocellulose, Zeta Probe, cellulose acetate or unetched GFF. Phosphate (pH 6.0-7.3) or Tris buffers (pH 7.3-8.0) worked equally well for the procedure. Unbound hormone was easily removed by short washes in buffer, and adequate binding for detection was achieved in a 15 min incubation. Preliminary data suggest that this technique may be used to detect receptors, carriers, and binding proteins of steroid hormones.