Classification of cancer cell lines using an automated two-dimensional liquid mapping method with hierarchical clustering techniques

A two-dimensional liquid mapping method was used to map the protein expression of eight ovarian serous carcinoma cell lines and three immortalized ovarian surface epithelial cell lines. Maps were produced using pI as the separation parameter in the first dimension and hydrophobicity based upon reversed-phase HPLC separation in the second dimension. The method can be reproducibly used to produce protein expression maps over a pH range from 4.0 to 8.5. A dynamic programming method was used to correct for minor shifts in peaks during the HPLC gradient between sample runs. The resulting corrected maps can then be compared using hierarchical clustering to produce dendrograms indicating the relationship between different cell lines. It was found that several of the ovarian surface epithelial cell lines clustered together, whereas specific groups of serous carcinoma cell lines clustered with each other. Although there is limited information on the current biology of these cell lines, it was shown that the protein expression of certain cell lines is closely related to each other. Other cell lines, including one ovarian clear cell carcinoma cell line, two endometrioid carcinoma cell lines, and three breast epithelial cell lines, were also mapped for comparison to show that their protein profiles cluster differently than the serous samples and to study how they cluster relative to each other. In addition, comparisons can be made between proteins differentially expressed between cell lines that may serve as markers of ovarian serous carcinomas. The automation of the method allows reproducible comparison of many samples, and the use of differential analysis limits the number of proteins that might require further analysis by mass spectrometry techniques.     

Weak cell cycle dependency but strong distortive effects of transfection with Lipofectamine 2000 in near‐physiologically synchronized cell culture

Previously, we reported a method to generate and validate cell cycle‐synchronized cultures of multiple mammalian suspension cell lines under near‐physiological conditions. This method was applied to elucidate the putative interdependencies of the cell cycle and recombinant protein expression in the human producer cell line HEK293s using Lipofectamine 2000 and the reporter plasmid pcDNA3.3 enhanced green fluorescent protein, destabilized using PEST sequence. A population‐resolved modeling approach was applied to quantitatively assess putative variations of cell cycle dependent expression rates based on the obtained experimental data. We could not confirm results published earlier by other groups, based on nonphysiological synchronization attempts, reporting transfection efficiency being strongly dependent on the cell cycle phase at transfection time point. On the other hand, it is demonstrated that transfection and protein expression distort the progression of the cell cycle.     

The Bulk Isolation of Oligodendroglia from Whole Rat Forebrain: A New Procedure Using Physiologic Media

Abstract: A method for the isolation of oligodendroglia from undissected rat forebrain is described. The method has been applied to brains from 10-, 30- and 60-day-old rats. The procedure uses a balanced salt solution at pH 7.2 throughout. Tissue is briefly exposed to trypsin and DNase and dissociated and the cells are purified on a discontinuous sucrose gradient. The isolates were composed of 90% phase-bright rounded cells having diameters after fixation of 7-12 μm. The contamination was primarily by red blood cells and phase-dark nuclei. Neurons and astroglia were lysed by the procedure. The method is reproducible and should be applicable to other ages of rat or to other species. The cells have been examined by light and electron microscopy and analyzed for protein and nucleic acids. None of the cell parameters measured, including total protein (58 pg/cell), varied significantly with age. With this new method it should be possible to carry out studies on the development and metabolism of oligodendroglia in small laboratory animals.     

A refined method for the photoaffinity labelling of the nitrobenzylthioinosine-sensitive nucleoside transport protein: application to cell membranes of calf lung tissue

A refined method for the photoaffinity labelling of the NBI-sensitive nucleoside transport protein is described. It involves the use of low concentrations of the photolabile probe [3H]nitrobenzylthioinosine ([3H]NBI), whereas the usual inclusion of dithiothreitol in the protocol is omitted. The method was successfully applied to cell membranes of calf lung tissue, which was shown to be a rich source of this physiologically important protein with all the characteristics (both in membrane bound and solubilized form) known from similar proteins on other cell types. Specific covalent incorporation of radioactivity appeared to be pH independent. SDS-polyacrylamide gel electrophoresis revealed a specifically labelled protein with an apparent molecular weight of 55 kDa.     

Simultaneous phase transition of ELP tagged molecules and free ELP: an efficient and reversible capture system

In this paper, we demonstrate proof-of-principle for a method that allows selective recovery of molecules present at very low concentrations in complex mixtures. The method makes use of an elastin-like polypeptide (ELP) as a coaggregant for the capture of an ELP tagged recombinant protein present at concentrations as low as 10 pM, with a recovery higher than 90%. This coaggregation process was found to be independent of the concentration, at least up to 10 pM concentration of the ELP tagged protein. The coaggregation process is highly specific as was demonstrated by spiking crude cell lysate with the ELP tagged recombinant protein to a final concentration of 1 nM and recovering more than 80% of it to a high level of purity. The method should be particularly useful for high-throughput proteomic studies, where small amounts of poorly expressed proteins could be recovered for analysis by mass spectrometry. In a more general context, the concept presented in this paper provides a method that is highly efficient, specific, and fully reversible, which should render it useful in areas other than recombinant protein purification.     

Isolation of RNA and Protein from Guard Cells of Nicotiana glauca

Stomatal guard cells are critical for maintenance of plant homeostasis and represent an interesting cell type for studies of leaf cell differentiation and patterning. Here we describe techniques for the isolation of guard cell RNA and protein from blended epidermal peels of Nicotiana glauca. The RNA isolation procedure is a modification of the hot borate method, which is particularly well-suited for recalcitrant tissues. Protein was extracted by disrupting guard cell-enriched epidermis with a French® press. This system offers the following advantages: relatively high yield, low or no contamination by other cell types, fresh tissue as a source of RNA and protein rather than protoplasts, and a plant species that is readily transformable. These techniques will allow for cloning and analysis of genes expressed in guard cells, application of traditional biochemical techniques to guard cell proteins, as well as characterization of genetic manipulation of guard cell function in transgenic plants.     

Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression

This protocol details a method to identify CD4+ T cells that respond to antigens. The method relies on detection of CD154, a costimulatory cell surface protein that is expressed by CD4+ T cells upon activation, and can be used to purify live CD4+ T cells of diverse function. To detect CD154, fluorescently labeled antibodies are cultured with cell samples, peptides (or whole antigens) and monensin during a 6- to 24-h stimulation period. (Note that the assay is not compatible with brefeldin A.) After stimulation, cells are stained with any other antibodies of interest and then are analyzed by flow cytometry or purified by cell sorting. Unlike other assays, this method allows simultaneous assessment of other cell phenotypes or functions, is compatible with downstream RNA-based assays and preserves cell viability. This protocol can be completed in 9 h.     

Identification and isolation of Dictyostelium microtubule-associated protein interactors by tandem affinity purification

Tandem affinity purification (TAP) is a method originally established in yeast to isolate highly purified protein complexes in a very gentle and efficient way. In this work, we have modified TAP for Dictyostelium applications and have proved it as a useful method to specifically isolate and identify microtubule-associated protein (MAP) complexes. MAPs are known to interact with other proteins to fulfill their complex functions in balancing the dynamic instability of microtubules as well as anchoring microtubules at the cell cortex, controlling mitosis at the centrosome and guiding transport along them. DdEB1 and the Dictyostelium member of the XMAP215 protein family, DdCP224, are known to be part of complexes at the microtubule tips as well as at the centrosome. Employing TAP and mass spectrometry we were able to prove an interaction between EB1 and the DdCP224. Additionally, among other interactions that remain to be confirmed by other methods, an interaction between DdCP224 and a TACC-family protein could be shown for the first time in Dictyostelium and was confirmed by colocalization and co-immunoprecipitation analyses.     

Continuous cultivation of fission yeast: Analysis of single-cell protein synthesis kinetics

A fundamental problem in microbial reactor analysis is identification of the relationship between environment and individual cell metabolic activity. Population balance equations provide a link between experimental measurements of composition frequency functions in microbial populations on the one hand and macromolecular synthesis kinetics and cell division control parameters for single cells on the other. Flow microfluorometry measurements of frequency functions for single-cell protein content in Schizosaccharomyces pombe in balanced exponential growth have been analyzed by two different methods. One approach utilizes the integrated form of the population balance equation known as the Collins-Richmond equation, and the other method involves optimization of parameters in assumed kinetic and cell division functional forms in order to best fit measured frequency functions with corresponding model solutions. Both data interpretation techniques indicate that rates of protein synthesis increase most in small protein content cells as the population specific growth rate increases, leading to parabolic single-cell protein synthesis kinetics at large specific growth rates. Utilization of frequency function data for an asynchronous population is shown in this case to be a far more sensitive method for determination of single-cell kinetics than is monitoring the metabolic dynamics of a single cell or, equivalently, synchronous culture analyses.     

Protein diffusion in mammalian cell cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.     

Attachment to membranes of exogenous immunoglobulin conjugated to a hydrophobic anchor

A novel method has been developed for the attachment of exogenous protein to liposomal and other membranes. A new phospholipid-containing alkylating reagent, N-(N^α-idoacetyl, N^?-dansyl lysyl)-phosphatidylethanolamine, was synthesized for conjugation to sulfhydryl groups of proteins. Model experiments were carried out with a Bence-Jones dimer which was reduced to generate one sulfhydryl group per monomer. After alkylation with the reagent the modified protein spontaneously attached to preformed liposomes and red cell ghosts. The attachment and accessibility of the protein were demonstrated by the association of the protein with the liposomal fraction in gel filtration and the agglutination of treated vesicles and red cell ghosts with antisera to human λ chain and to the dansyl group.     

重组SARS冠状病毒M蛋白的表达、纯化及鉴定

SARS冠状病毒是人的严重急性呼吸综合征的病原体。根据对其他种类冠状病毒的研究结果 ,膜蛋白 (M蛋白 )是病毒主要的结构蛋白 ,重组M蛋白可被用来作为抗原检测对应冠状病毒的感染和制备疫苗。SARS病毒M蛋白基因克隆到原核表达载体pMAL cRI中 ,利用N端和C端分别融合麦芽糖结合蛋白 (maltosebindingprotein和MxeGyrAinteinCBD的策略 ,在大肠杆菌中初步表达了重组M蛋白 ,并通过Western印迹和质谱对蛋白质进行了鉴定。重组蛋白质经亲和层析得到了部分纯化 ,纯化后的蛋白质将用于功能研究与诊断试剂盒的研制。     

Single-cell mass cytometry adapted to measurements of the cell cycle

Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.     

Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain

Several protease negative mutant strains including HM114, HM126, and HM130 as well as their parent strain KS272 were compared for their growth and secretory production of a model fusion protein, protein A-beta-lactamase. HM114, a strain deficient in two cell envelope proteases, grew slightly faster and produced more fusion protein than the other strains deficient in more proteases. HM114 was grown to a cell dry weight of 47.86 g/L in 29 h using pH-stat, fed-batch cultivation. The beta-lactamase activity was 11.25 x 10(4) U/L, which was 30% higher than that obtained with its parent strain KS272. Up to 96% of protein A-beta-lactamase fusion protein could be recovered by a simple cold osmotic shock method. The specific beta-lactamase activity obtained with HM114 after fractionation was 4.5 times higher than that obtained with KS272.     

Spectrophotometric quantitation of anchorage-dependent cell numbers using the bicinchoninic acid protein assay reagent

A rapid and convenient method is described for the determination of the actual and relative number of adherent cells in tissue culture. The cell lines human melanoma C32, ATCC CRL 1585, mouse melanoma B16-F10, and pig epithelial LLC-PK1, suspended in Dulbecco's minimum essential medium containing no serum, were allowed to adhere to fibronectin adsorbed to wells of a 96-well microtiter plate. Nonadherent cells were removed by aspiration, wells were washed, and adherent cells were solubilized with 200 microliters of the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay reagent. Plates were heated to 60 degrees C for 30 min and absorbances read at 562 nm using a microtiter plate reader. A linear correlation was observed between the number of adherent cells in the range 2-8 X 10(5)/ml cells added and the protein content of the adherent cells as measured by the BCA protein reagent. The assay procedure gave absorbance values in the range of 0.100 to 1.30 making the method highly sensitive and reproducible. Blank wells containing only coupled protein and no cells gave little or no absorbance. Cell adhesion was fibronectin specific since little or no cell attachment was observed when microtiter plates were coupled with bovine serum albumin. Similar results were obtained with other cell types such as platelets. These results indicate that measurement of total cellular protein using the BCA protein reagent can be a rapid and sensitive assay for the detection and quantitation of adherent cells.     

The estimation of micro-algal protein content and its meaning to the evaluation of algal biomass I. Comparison of methods for extracting protein

The spectrophotometric evaluation of micro-algal protein needs a prior extraction from cells in order to liberate protein for measurement. The conditions of extraction (temperature, duration, normality of sodium hydroxide, pretreatment) which yield optimal protein content are tested with three algal cultures (Scenedesmus, Synechococcus, Asterionella). A standard method of extraction is presented. Comparison of this method with nine published methods reveals markedly lower protein yields for easy extractable (43–100%) and hard extractable (5–75%) algal species, relative to this method, depending on ease of cell wall breakage. The application of this standard method to field investigations is demonstrated and compared to other biochemical parameters. The advantages of this method over other protein extraction methods, with respect to field material, are discussed.     

Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin–proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high-resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes.     

Identification and isolation of Dictyostelium microtubule-associated protein interactors by tandem affinity purification

Tandem affinity purification (TAP) is a method originally established in yeast to isolate highly purified protein complexes in a very gentle and efficient way. In this work, we have modified TAP for Dictyostelium applications and have proved it as a useful method to specifically isolate and identify microtubule-associated protein (MAP) complexes. MAPs are known to interact with other proteins to fulfill their complex functions in balancing the dynamic instability of microtubules as well as anchoring microtubules at the cell cortex, controlling mitosis at the centrosome and guiding transport along them. DdEB1 and the Dictyostelium member of the XMAP215 protein family, DdCP224, are known to be part of complexes at the microtubule tips as well as at the centrosome. Employing TAP and mass spectrometry we were able to prove an interaction between EB1 and the DdCP224. Additionally, among other interactions that remain to be confirmed by other methods, an interaction between DdCP224 and a TACC-family protein could be shown for the first time in Dictyostelium and was confirmed by colocalization and co-immunoprecipitation analyses.