Solid-state protein kinase. A tool for post-synthetically modifying and radioactively labeling proteins in vitro.

The capacity of partially purified rat muscle protein kinase coupled to cyanogen-bromide-activated Sepharose 4B to (radio-)phosphorylate proteins in vitro was evaluated using histones from calf thymus and rat liver and certain proteins as substrates. Data are presented which point to a low substrate specificity of this enzyme. It is demonstrated that even within a short time period histones are efficiently phosphorylated without the introduction of contaminating (phospho-)proteins. Therebye phosphoserine residues are formed. The phosphorylation reaction usually performed at 30 degrees C is shown to function quite efficiently also at 4 degrees C. It proceeds even at 30 degrees C for several hours at pH values close to the physiological range without the release of proteins from the solid matrix. The phosphorus transfer can be largely increased with the use of high ATP concentrations. The stability of the substrates is sufficient to suggest a wide applicability of this solid-state protein kinase in the phosphorylation of proteins either for labeling or as a tool to modify proteins post-synthetically under gentle conditions. The solid enzyme seems to be suitable for radioactively labeling proteins of more complex biological structures, such as membrane surfaces.     

Experimental design principles for isotopically instationary 13C labeling experiments

13C metabolic flux analysis (MFA) is a well-established tool in Metabolic Engineering that found numerous applications in recent years. However, one strong limitation of the current method is the requirement of an-at least approximate-isotopic stationary state at sampling time. This requirement leads to a principle lower limit for the duration of a 13C labeling experiment. A new methodological development is based on repeated sampling during the instationary transient of the 13C labeling dynamics. The statistical and computational treatment of such instationary experiments is a completely new terrain. The computational effort is very high because large differential equations have to be solved and, moreover, the intracellular pool sizes play a significant role. For this reason, the present contribution works out principles and strategies for the experimental design of instationary experiments based on a simple example network. Hereby, the potential of isotopically instationary experiments is investigated in detail. Various statistical results on instationary flux identifiability are presented and possible pitfalls of experimental design are discussed. Finally, a framework for almost optimal experimental design of isotopically instationary experiments is proposed which provides a practical guideline for the analysis of large-scale networks.     

Fluorescence labeling method for estimation of soluble polymers in the living material

The highly fluorescent derivatives of fluorescein, bearing the aliphatic primary amino groups, N-(2-aminoethylcarbonyl)-5(6)-aminofluorescein and 5-[N′-(2-aminoethyl)thioureido]fluorescein, were prepared for labeling of soluble polymers. The absorption and emission properties of these labels and polymers labeled with them were compared with properties of fluorescein and fluorescein isothiocyanate (FITC)-bovine serum albumin conjugate. Effects of the chemical structure of the polymer on the relative fluorescence quantum yield of a covalently attached label were evaluated using ionogenic, olefinic, or phenolic groups in side chains. The fluorescence of labeled polymers was adequate for their tracing in all the cases studied. The most pronounced quenching of fluorescence in the presence of phenolic groups is comparable with the quenching of fluorescence of FITC observed in FITC-protein conjugates. The long-term stability of the polymer-fluorochrome bond was checked in solutions of pH 2.10, 7.46, and 11.84; a higher stability of simple amide over amide plus the thiourea bond was found. The quantitative method of measurement of the concentration of labeled polymers in the biological material in a range of about 10 ng was developed; factors affecting the reproducibility are discussed.     

Computational tools for isotopically instationary 13C labeling experiments under metabolic steady state conditions

(13)C metabolic flux analysis (MFA) has become an important and powerful tool for the quantitative analysis of metabolic networks in the framework of metabolic engineering. Isotopically instationary (13)C MFA under metabolic stationary conditions is a promising refinement of classical stationary MFA. It accounts for the experimental requirements of non-steady-state cultures as well as for the shortening of the experimental duration. This contribution extends all computational methods developed for classical stationary (13)C MFA to the instationary situation by using high-performance computing methods. The developed tools allow for the simulation of instationary carbon labeling experiments (CLEs), sensitivity calculation with respect to unknown parameters, fitting of the model to the measured data, statistical identifiability analysis and an optimal experimental design facility. To explore the potential of the new approach all these tools are applied to the central metabolism of Escherichia coli. The achieved results are compared to the outcome of the stationary counterpart, especially focusing on statistical properties. This demonstrates the specific strengths of the instationary method. A new ranking method is proposed making both an a priori and an a posteriori design of the sampling times available. It will be shown that although still not all fluxes are identifiable, the quality of flux estimates can be strongly improved in the instationary case. Moreover, statements about the size of some immeasurable pool sizes can be made.     

A new method for protein labeling with 99mTc

A method is described for labeling proteins with ^99mTc, the radionuclide of choice in diagnostic nuclear medicine. Labeling efficiency, stability of label attachment and retained biological behaviour, e.g. immunoreactivity of monoclonal antibodies after radiolabeling are demonstrated. An application of a ^99mTc-labeled anti-fibrin monoclonal antibody in radioimmunoimaging of thrombi is presented.     

A method for efficient isotopic labeling of recombinant proteins

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for ¹³C labeling of the C-terminal domain of angiopoietin-2, ¹⁵N labeling of ubiquitin and for ²H/¹³C/¹⁵N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.     

A rapid and inexpensive labeling method for microarray gene expression analysis

Background  

Global gene expression profiling by DNA microarrays is an invaluable tool in biological research. However, existing labeling methods are time consuming and costly and therefore often limit the scale of microarray experiments and sample throughput. Here we introduce a new, fast, inexpensive method for direct random-primed fluorescent labeling of eukaryotic cDNA for gene expression analysis and compare the results obtained on the NimbleGen microarray platform with two other widely-used labeling methods, namely the NimbleGen-recommended double-stranded cDNA protocol and the indirect (aminoallyl) method.     

A new method for labeling and autoradiographic localization of androgen receptors

We have used a novel receptor labeling and autoradiographic technique to localize androgen receptors in the intact rat ventral prostate at the morphological level. Frozen slide-mounted prostate tissue sections (10 micron thick) were incubated with increasing concentrations of [3H]-R1881 in the absence and presence of excess unlabeled R1881. Tissue sections labeled in this way were subjected to concurrent biochemical and autoradiographic analysis. After incubation and washing to remove free [3H]-steroid, some of the sections were wiped from the slides for scintillation counting in order to characterize and quantitate [3H]-R1881 binding. Androgen receptors could indeed be labeled in slide-mounted tissue sections, and specific [3H]-R1881 binding to these receptors was high-affinity (Kd = 1 nM), saturable, and androgen-specific. All cellular androgen receptors appear to be retained, because receptor content in sections was comparable to the sum of receptors in subcellular fractions of homogenized tissue. Replicate labeled slide-mounted tissue sections were dried rapidly, apposed to dry emulsion-coated coverslips, and exposed in the dark for autoradiography. Silver grains were counted over nuclei or cytoplasm of epithelium or stroma to evaluate specific androgen receptor location. Autoradiographic analysis demonstrated androgen receptor localization almost exclusively in the epithelial nuclei, with little or none in the stroma. We discuss here the unique features and advantages of labeling androgen receptors in slide-mounted frozen tissue sections for autoradiographic localization.     

A rapid and inexpensive labeling method for microarray gene expression analysis

Background

Polyethyleneimine (PEI), a cationic polymer, is one of the successful and widely used vectors for non-viral gene transfection in vitro. However, its in vivo application was greatly limited due to its high cytotoxicity and short duration of gene expression. To improve its biocompatibility and transfection efficiency, PEI has been modified with PEG, folic acid, and chloroquine in order to improve biocompatibility and enhance targeting.

Results

Poly(ε-caprolactone)-Pluronic-Poly(ε-caprolactone) (PCFC) was synthesized by ring-opening polymerization, and PCFC-g-PEI was obtained by Michael addition reaction with GMA-PCFC-GMA and polyethyleneimine (PEI, 25 kD). The prepared PCFC-g-PEI was characterized by ¹H-NMR, SEC-MALLS. Meanwhile, DNA condensation, DNase I protection, the particle size and zeta potential of PCFC-g-PEI/DNA complexes were also determined. According to the results of flow cytometry and MTT assay, the synthesized PCFC-g-PEI, with considerable transfection efficiency, had obviously lower cytotoxicity against 293 T and A549 cell lines compared with that of PEI 25 kD.

Conclusion

The cytotoxicity and in vitro transfection study indicated that PCFC-g-PEI copolymer prepared in this paper was a novel gene delivery system with lower cytotoxicity and considerable transfection efficiency compared with commercial PEI (25 kD).     

A new two-color Fab labeling method for autoantigen protein microarrays

Antigen microarrays hold great promise for profiling the humoral immune response in the settings of autoimmunity, allergy and cancer. This approach involves immobilizing antigens on a slide surface and then exposing the array to biological fluids containing immunoglobulins. Although these arrays have proven extremely useful as research tools, they suffer from several sources of variability. To address these issues, we have developed a new two-color Fab labeling method that allows two samples to be applied simultaneously to the same array. This straightforward labeling approach improves reproducibility and reliably detects changes in autoantibody concentrations. Using this technique we profiled serum from a mouse model of systemic lupus erythematosus (SLE) and detected both expected and previously unrecognized reactivities. The improved labeling and detection method described here overcomes several problems that have hindered antigen microarrays and should facilitate translation to the clinical setting.     

A new reagent for preparing radioactively labeled nucleoside derivatives.

The identification of benzimidazole incorporated into RNA of Escherichia coli as benzimidazole nucleoside by means of mass spectrometry is reported. Trimethylsilylation of an enzymatic digest of bacterial RNA allowed the separation of the different nucleosides by gas chromatography. The coupled mass spectrometer was used as a mass specific detector and allowed the sensitive detection of single components of the complex mixture. Thus, benzimidazole ribonucleoside could be detected in hydrolysates of RNA from E. coli fed benzimidazole in the culture broth, although this nucleoside could not be completely separated from uridine by the gas chromatographic systems explored. Quantitation of the benzimidazole nucleoside content revealed that benzimidazole is incorporated into RNA amounting to 16% relative to adenosine.     

An efficient method for the production of isotopically enriched cholesterol for NMR

(13)C-Cholesterol was produced with high efficiency by a genetically engineered yeast strain. The method produces ～ 1 mg of cholesterol per gram of glucose using 100 ml of culture medium. Uniform 94% enrichment where the most abundant product is the fully enriched isotopomer (u-(13)C(27)) is obtained using (u-(13)C(6), 99%) glucose medium. High enrichment is very important for relaxation experiments, but for NMR applications where carbon-carbon couplings are measured, this is problematic. A good compromise between sensitivity and cost consists in diluting (u-(13)C(6), 25%) with natural-abundance glucose. With a 2:3 ratio, the maximal amount of singlets can be obtained in 1 dimensional (D) carbon and 2D heteronuclear single-quantum correlation (HSQC) spectra with 6× intensity increase relative to natural-abundance samples. The use of (1-(13)C(1)-glucose, 99%) or (2-(13)C(1)-glucose, 99%) as isotope sources allows the labeling of the cholesterol in multiple mostly nonvicinal positions and reach 45× intensity increase. As an alternative, the dilution of (u-(13)C(6), 99%) glucose can be used to simultaneously enrich eleven pairs of (13)C up to ～ 1,000× natural-abundance probability, which should be very beneficial to double-quantum NMR experiments including the INADEQUATE and related pulse sequences. The flexibility of the method and the potential to adapt the culture protocol to specific needs should find many applications in chemistry and biology and in different fields of NMR and MS.     

A simple and novel method for tritium labeling of gangliosides and other sphingolipids

A very simple method for introducing tritium specifically into the ceramide portion of gangliosides, neutral glycosphingolipids and sphingomyelin has been developed using potassium boro [3H]hydride and palladium as catalyst. In this way six different gangliosides, five different neutral glycosphingolipids and sphinomyelin with high specific radioactivity were prepared for the first time. This simple procedure which does not require sophisticated apparatuses is suitable for large scale preparation of tritium-labeled sphingolipids as well as for nanogram quantities of individual sphingolipids so as to serve for analytical purposes. During the course of the labeling procedure no degradation of even the most labile trisialosyl-gangliotetraosylceramide has been observed. The yield of tritiated compounds is almost quantitative. The specific radioactivity depends on the unsaturation of the ceramide moiety and the specific activity of the boro[3H]hydride employed.     

A novel method for selective isotope labeling of bacterially expressed proteins

Summary A novel method for isotope labeling in selected amino acids is presented for use with the T7 RNA polymerase system. The protocol is illustrated with the DNA-binding domain from the E2 protein of bovine papillomavirus, BPV-1. On addition of rifampicin, protein expression occurs exclusively from the gene controlled by the T7 promoter. Since the bacteria are now dedicated to the production of E2 protein, labeling with specific amino acids is efficiently performed. For example, 10 mg/l of ¹⁵N-labeled phenylalanine is shown to be sufficient for incorporation of the label, without scrambling, and without the use of an auxotrophic strain.     

Immunofluorescent labeling using covalently linked anti-phycoerythrin antibodies and phycoerythrin polymers

We have developed methods for covalently attaching anti-phycoerythrin (PE) MAbs to other antibodies, and for using PE or polymers of PE in conjunction with these conjugates to rapidly produce specific, high intensity fluorescent labeling of antigens. The performance of these systems was examined on the surface of microspheres and on the cell surface. The noncovalent means by which PE is incorporated into the label complex in this method makes it possible to use crude algal homogenate successfully as a source of PE in immunofluorescence assays. The intensity of labeling achieved using this method is comparable to, or in some cases better than, that obtained using a direct PE conjugate. With the aim of amplifying the intensity of fluorescence obtained, we produced two types of covalently linked complexes. These were an anti-PE MAb, designated PE6, linked to itself (i.e., PE6-PE6), and PE linked to itself (i.e., PE-PE). When used in a two-stage procedure in place of monomeric PE alone, these complexes increased the intensity of fluorescence obtained on the surface of microspheres by more than fourfold. On the cell surface, the performance of this system varied from one antigen to the next but in most cases was restricted to, at best, a 60% increase in the intensity of fluorescence and in many cases only about a 30% increase.     

New miRNA labeling method for bead-based quantification

Background  

microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies.     

A non-perturbation method for the optical properties of helical polymers

The optical absorption and circular dichroism (CD) of poly-L -alanine in an α-helical conformation are computed in terms of monomer properties by use of Bogoliubov exciton theory (a non-perturbation method). It was shown earlier that a proper formulation of the theory of CD for large molecular systems permits the use of periodic boundary conditions, a feature which greatly simplifies calculations as well as conceptual understanding. If is found that the shape of the observed CD spectrum can be explained in terms of the two rotational strength components and an electric dipole strength component referred to as the helix term. Comparisons of the results of perturbation theory and Bogoliubov exciton theory show that perturbation theory is valid for absorption but is inadequate for CD if the coupling among monomers is sufficiently large lo predict the observed hypochromism.     

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.Subject terms: Environmental microbiology, Sequencing, Microbiology