Use Of Cross-linking Reagents Research Articles

Green preparation of covalently co-immobilized multienzymes on silica nanoparticles for clean production of reducing sugar from lignocellulosic biomass

Multienzyme co-immobilization has garnered increasing interest in biomass conversion due to the green catalytic properties. However, the conventional strategies for covalent co-immobilization were complicated (including carrier preparation, enzyme purification, and immobilization process) and not clean enough (such as harsh reaction conditions, high reagent and water consumption, and toxic chemical input). Herein, a simple and green strategy was proposed to address these issues by the integration of elastin-like polypeptide (ELP)-mediated green biomimetic silicification and the spontaneous covalent bond formation of SpyCatcher/SpyTag (SC/ST) protein pair. In this strategy, the SC was genetically fused to ELP and the resulting SC-ELP (SCE) was purified by inverse transition cycling (ITC) method. The nanocarrier (SCE@SiO2) was prepared rapidly via one-step biomimetic silicification at neutral pH and room temperature under ambient pressure. The obtained nanocarriers could be directly used for covalent co-immobilization without additional surface functionalization and activation. The ST-tagged recombinant bi-enzyme xylanase-ST-lichenase (XSTL) covalently self-conjugated onto SCE@SiO2 through the SC and ST reaction, which eliminated the use of cross-linking reagents. Notably, target-specific immobilization from cell crude extracts with excellent activity recovery (over 90%) eliminated reagent- and time-consuming purification process. Furthermore, the co-immobilized XSTL showed very good reusabilities, which retained 83.9% (lichenase) and 81.4% (xylanase) of the original activities in the seventh cycle, respectively. XSTL could accelerate the production of fermentable sugars from renewable biomass polysaccharide. Overall, this unique ELP-mediated silicification-based Spy-assisted enzyme self-immobilization (ESSESI) offered great potentials towards multienzyme co-immobilization for diverse applications in a cleaner manner.

Cross-linked nucleic acids: Formation, structure, and biological function

Published data on the main types of reagents capable of introducing covalent interstrand cross links into nucleic acids (NA) are summarized in the present review. The reactivity of cross-linking agents, their preferred binding sites, and methods of determining the cross-link localization in a duplex are discussed. Cell response to DNA cross linking, namely, the blocking of replication and transcription, the initiation of reparation processes, and apoptotic death of the cell, are analyzed, as well as the use of cross-linking reagents in therapy and molecular biology.

Covalent immobilization of urease on polysiloxane matrices containing 3-aminopropyl and 3-mercaptopropyl groups

A technique of covalent immobilization of urease on polysiloxane templates, involving the sol-gel method, based on the use of crosslinking reagents (such as glutaraldehyde and Ellman's reagent) has been proposed. Urease, covalently grafted onto the surface of a poly(3-mercaptopropyl)siloxane template, was shown to retain its activity (67-84%) and stability (a decrease of 10% was observed over a period of 300 days). Urease adsorbed onto the poly(3-mercaptopropyl)siloxane template exhibited a higher activity than the native enzyme. The 3-mercaptopropyl groups of the polysiloxane template could be brought into the vicinity of the active metal center of the adsorbed urease and start acting as proton donors, thereby increasing the rate of the reaction catalyzed by the enzyme. Covalent immobilization of urease onto a 3-aminopropyl-containing polysiloxane template was shown to be less efficient, because it resulted in considerable losses of the activity of the enzyme. Conversely, urease adsorbed onto this template exhibited a high activity (60-86%).

Intra-Molecular Cross-Linking of Acidic Residues for Protein Structure Studies

Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would help to develop structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine (lysine, the amino terminus) selective reagents. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution and solvent accessibility of the lysines in the protein sequence. To overcome these limitations, we have investigated the use of cross-linking reagents that can react with other reactive side chains in proteins. We used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E) and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO side chains can react to form "zero-length" cross-links with nearby primary amine containing residues, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO side chains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker arm of variable length. Using these reagents, we have found three new "zero-length" cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18 and K63-E64). Using the dihydrazide cross-linkers, we have identified two new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 A. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target will yield sufficient distance constraints to develop a structural model.

A New Method of Synthesizing Black Birnessite Nanoparticles: From Brown to Black Birnessite with Nanostructures

A new method for preparing black birnessite nanoparticles is introduced. The initial synthesis process resembles the classical McKenzie method of preparing brown birnessite except for slower cooling and closing the system from the ambient air. Subsequent process, including wet-aging at7∘Cfor 48 hours, overnight freezing, and lyophilization, is shown to convert the brown birnessite into black birnessite with complex nanomorphology with folded sheets and spirals. Characterization of the product is performed by X-ray diffraction (XRD), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), thermogravimetric analysis (TGA), andN2adsorption (BET) techniques. Wet-aging and lyophilization times are shown to affect the architecture of the product. XRD patterns show a single phase corresponding to a semicrystalline birnessite-based manganese oxide. TEM studies suggest its fibrous and petal-like structures. The HRTEM images at 5 and 10 nm length scales reveal the fibrils in folding sheets and also show filamentary breaks. The BET surface area of this nanomaterial was found to be 10.6 m2/g. The TGA measurement demonstrated that it possessed an excellent thermal stability up to400∘C. Layer-structured black birnessite nanomaterial containing sheets, spirals, and filamentary breaks can be produced at low temperature (−49∘C) from brown birnessite without the use of cross-linking reagents.

Detection of in vivo protein–DNA interactions using DamID in mammalian cells

Understanding gene regulatory networks in mammalian cells requires detailed knowledge of protein-DNA interactions. Commonly used methods for genome-wide mapping of these interactions are based on chromatin immunoprecipitation. However, these methods have some drawbacks, such as the use of crosslinking reagents, the need for highly specific antibodies and relatively large amounts of starting material. We present DamID, an alternative technique to map genome-wide occupancy of interaction sites in vivo, that bypasses these limitations. DamID is based on the expression of a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam). This leads to methylation of adenines near sites where the protein of interest interacts with the DNA. These methylated sequences are subsequently amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays. Using DamID, genome-wide maps of the binding of DNA-interacting proteins in mammalian cells can be constructed efficiently. Depending on the strategy used for expression of the Dam-fusion proteins, genome-wide binding maps can be obtained in as little as 2 weeks.

Nuclear localization signal binding proteins in higher plant nuclei.

The import of proteins into the nucleus is a vital process that is mediated by proteins which specifically recognize nuclear localization signals (NLSs). These factors have not been identified in plants. Previously, we demonstrated that higher plants possess a low-affinity binding site at the nuclear pore that specifically binds to several classes of functional NLSs. By the use of crosslinking reagents and a radiolabeled peptide to the bipartite NLS from the endogenous plant transcription factor Opaque2, two NLS binding proteins (NBPs) of 50-60 kDa and at least two NBPs of 30-40 kDa were identified. Competition studies indicated that labeling was specific for the functional NLS but not a mutant NLS impaired in vivo or a peptide unrelated to NLSs. Also, the apparent dissociation constant (100-300 microM) for labeling was similar to that of the binding site. Proteins of similar mass were labeled with two different crosslinking reagents, and concentration and time studies indicated that these NBPs were distinct proteins and not aggregates. Treatment with salt, detergent, or urea before or during NLS binding demonstrated that the properties of the binding site and the NBPs were identical. This tight correlation strongly indicates that some or all of the NBPs constitute the nuclear pore binding site. Overall, our results indicate that some components of NLS recognition are located at the nuclear pores in higher plants.

32] Use of cross-linking reagents for detection of subunit interactions of membrane-bound Na +,K +-ATPase

32] Use of cross-linking reagents for detection of subunit interactions of membrane-bound Na +,K +-ATPase

Isolation of cross-linked IgE-receptor complexes from rat macrophages.

Receptors for IgE on macrophages have been characterized by binding assays (1-3), but to date there has been only one report on the isolation of this receptor from macrophages, with use of the cell line U937 (4). In that report the receptor was isolated by using a heavily absorbed polyclonal antibody raised against lymphocytes bearing receptors for IgE (5). Monomeric IgE binds so weakly to macrophages that affinity chromatography of IgE-receptor complexes, such as has been used for isolation of the receptors for IgE on basophils (6) and for IgG on macrophages (7), cannot be readily accomplished. We have used oligomers of IgE to enhance the binding of IgE to macrophages (3), but this alone would not be sufficient because--depending on whether the receptors are multi- or univalent--once the cells are solubilized, multipoint attachment would again be reduced if not abrogated. In this report we describe the use of cross-linking reagents to stabilize further the interaction between IgE and its receptor on peritoneal macrophages. With this approach we have found that the receptor is likely to be composed of two chains whose gross properties are similar to the polypeptides constituting the receptor with high affinity for monomeric IgE on rat basophilic leukemia cells and mast cells.

Identification of neighboring protein pairs in rat liver 60S ribosomal subunits cross-linked with dimethyl suberimidate or dimethyl 3,3'-dithiobispropionimidate.

Protein-protein neighbors in rat liver 60S ribosomal subunits were investigated by using two bifunctional imidoesters; dimethyl suberimidate (DMS) and dimethyl 3,3'-dithiobispropionimidate (DTP). 1. Complexes cross-linked with DMS were separated by two-dimensional acrylamide/urea gel electrophoresis. Each complex in the gel was labeled with 125I (1) and was cleaved into the original monomeric protein constituents of ammonolysis. The products were analyzed by two-dimensional acrylamide/urea gel electrophoresis, followed by radioautography of the stained gel. Eight protein pairs are proposed according to our numbering system (2): L1-L3(L3-L5), L2-L21 (L4-L26), L5-L13 (L7/L7a-L15), L5-L34 L7/L7a-L36), L6-L34 (L8-L36), L6-L32 (L8-L35), L12-L32 (L13/L13a-L35), and L18-L27 (L18-L27/L27a). The designations according to the proposed uniform nomenclature (3) are given in parentheses. 2. Complexes cross-linked with DTP were analyzed by acrylamide/SDS diagonal gel electrophoresis (4), and the molecular weights of the complexes and their components were determined. The monomer components of cross-linked pairs were labeled with 125I in the gel, and identified by two-dimensional acrylamide/urea gel electrophoresis followed by radioautography. Six pairs are proposed; L1-L5 (L3-L7/L7a), L1-L6 (L3-L8). L5-L19 (L7-/L7a-L21/L23/L23a), L13-L15 (L15-L14), L13-L16 (L15-L19), and L23-L27 (L30-L27/L27a). 3. Two pairs which were formed by protein-protein interaction without the use of cross-linking reagents were identified as L2-L4 (L4-L6) and L4-L30 (L6-L29).

Purification and Properties of HeLa Cell Plasma Membranes

Abstract A simple method for the preparation of HeLa cell plasma membrane ghosts without the use of cross-linking reagents or fixatives has been developed. The membranes have been characterized by phase and electron microscopy and by the use of radioactive precursors to RNA, protein, glycoprotein, and lipid. The glycoprotein and polypeptide constituents have been analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis in conjunction with subfractionation by mild detergents. There appears to be a predominant species of polypeptide and [3H]-l-fucose-labeled glycoprotein having a molecular weight of 40,000 to 45,000. The number of plasma membrane ghosts in a preparation was determined by counting ghosts in a hemacytometer and the yield of ghosts was then found to be in the range of 10 to 20% of the number of cells used for the preparation. Purified plasma membranes labeled for 4 hours or more contained 70 to 100% of the total cell trichloroacetic acid insoluble [3H]-l-fucose. Thus, [3H]-l-fucose seems to be a specific label for HeLa cell plasma membranes.