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The invention provides peptides with high affinity for streptavidin. These peptides may be expressed as part of fusion proteins to facilitate the detection, quantitation, and purification of proteins of interest.
The invention provides peptides with high affinity for streptavidin. These peptides may be expressed as part of fusion proteins to facilitate the detection, quantitation, and purification of proteins of interest.
Biosensing devices comprised of carbon nanotubes and nanofibers have been developed for astronaut crew point-of-care. Their inherent nanometer scale, high conductivity, wide potential window, good biocompatibility and well-defined surface chemistry make them ideal candidates as biosensor electrodes. Here, we report two studies using carbon nanotube and carbon nanofiber electrodes for biomedical applications. First, a 3x3 electrode device, with each electrode containing 40,000 carbon nanofiber nanoelectrodes was fabricated on silicon using traditional microfabrication processing. The device was demonstrated as a multiplexed immunosensor for simultaneous, label-free detection of cardiac troponin-I, C-reactive protein and myoglobin. Antibodies specific to cardiac troponin-I, C-reactive protein and myoglobin were covalently bound to the CNF surface and were characterized using electrochemical impedance spectroscopy and differential pulse voltammetry. Each step of the modification process resulted in changes in resistance to charge transfer due to the changes at the electrode surface upon antibody immobilization and binding to the specific cardiac protein. The real-time label free detection of the three cardiac markers from pure components and mixtures was demonstrated with high sensitivity, down to 0.2 ng/mL, and good selectivity. Detection in human blood serum did not present false positives from non-specific protein adsorption. More recently, this detection scheme has been applied to inkjet printed carbon nanotube electrodes on Kapton and paper. Printed devices have several unique advantages including simple and inexpensive fabrication. The results demonstrate that these sensors can serve a miniaturized, low cost device for detection of proteins in complex mixtures making this platform a good candidate for early stage diagnosis of myocardial infarction. Future inkjet printed devices can be fabricated have the added advantage in their suitability to be manufactured in an in-space, microgravity environment.
Biosensing devices comprised of carbon nanotubes and nanofibers have been developed for astronaut crew point-of-care. Their inherent nanometer scale, high conductivity, wide potential window, good biocompatibility and well-defined surface chemistry make them ideal candidates as biosensor electrodes. Here, we report two studies using carbon nanotube and carbon nanofiber electrodes for biomedical applications. First, a 3x3 electrode device, with each electrode containing 40,000 carbon nanofiber nanoelectrodes was fabricated on silicon using traditional microfabrication processing. The device was demonstrated as a multiplexed immunosensor for simultaneous, label-free detection of cardiac troponin-I, C-reactive protein and myoglobin. Antibodies specific to cardiac troponin-I, C-reactive protein and myoglobin were covalently bound to the CNF surface and were characterized using electrochemical impedance spectroscopy and differential pulse voltammetry. Each step of the modification process resulted in changes in resistance to charge transfer due to the changes at the electrode surface upon antibody immobilization and binding to the specific cardiac protein. The real-time label free detection of the three cardiac markers from pure components and mixtures was demonstrated with high sensitivity, down to 0.2 ng/mL, and good selectivity. Detection in human blood serum did not present false positives from non-specific protein adsorption. More recently, this detection scheme has been applied to inkjet printed carbon nanotube electrodes on Kapton and paper. Printed devices have several unique advantages including simple and inexpensive fabrication. The results demonstrate that these sensors can serve a miniaturized, low cost device for detection of proteins in complex mixtures making this platform a good candidate for early stage diagnosis of myocardial infarction. Future inkjet printed devices can be fabricated have the added advantage in their suitability to be manufactured in an in-space, microgravity environment.
Tissue-specific dirigent protein gene expression and associated dirigent (site) localization were examined in various organs of Forsythia intermedia using tissue printing, in situ mRNA hybridization and immunolabeling techniques, respectively. Dirigent protein gene expression was primarily noted in the undifferentiated cambial regions of stem sections, whereas dirigent protein sites were detected mainly in the vascular cambium and ray parenchyma cell initials. Immunolocalization also revealed cross-reactivity with particular regions of the lignified cell walls, these being coincident with the known sites of initiation of lignin deposition. These latter regions are considered to harbor contiguous arrays of dirigent (monomer binding) sites for initiation of lignin biopolymer assembly. Dirigent protein mRNA expression was also localized in the vascular regions of roots and petioles, whereas in leaves the dirigent sites were primarily associated with the palisade layers and the vascular bundle. That is, dirigent protein mediated lignan biosynthesis was initiated primarily in the cambium and ray cell initial regions of stems as well as in the leaf palisade layers, this being in accordance with the occurrence of the lignans for defense purposes. Within lignified secondary xylem cell walls, however, dirigent sites were primarily localized in the S(1) sublayer and compound middle lamella, these being coincident with previously established sites for initiation of macromolecular lignin biosynthesis. Once initiation occurs, lignification is proposed to continue through template polymerization.
Derivatization with (omicron)-phthaldialdehyde (OPA) and the chiral thiol N-acetyl-L-cysteine (NAC) is a convenient and sensitive technique for the HPLC detection and resolution of protein amino acid enantiomers. The kinetics of the reaction of OPA-NAC with (alpha)-dialkylamino acids was investigated. The fluorescence yield of (alpha)-dialkylamino acids was only about 10% of that of protein amino acids when the derivatization was carried out at room temperature for 1-2 min, which is the procedure generally used for protein amino acid analyses. The fluorescence yield of (alpha)-dialkylamino acids can be enhanced by up to ten-fold when the derivatization reaction time is increased to 15 min at room temperature. The OPA-NAC technique was optimized for the detection and enantiomeric resolution of a-dialkylamino acids in geological samples which contain a large excess of protein amino acids. The estimated detection limit for a-dialkylamino acids is 1-2 pmol, comparable to that for protein amino acids.
The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.
Noting that protein synthesis declines in the gastrocnemius 6 hr after immobilization, the study sought to detect an increase of protein synthesis when the limb was freed, and to examine the effects of exercise on the rate of increase. Rats were used as subjects, with their hind legs in plaster of Paris in plantar flexion to eliminate strain on the gastrocnemius. Periods of immobilization were varied and samples of blood from the muscle were taken to track protein synthesis rates for different groups in immobilization and exercise regimens (running and weightlifting). Synthesis rates declined 3.6% during time in the cast, then increased 6.3%/day after the casts were removed. Both running and weightlifting were found to increase the fractional rate of protein formation in the gastrocnemius muscle when compared with contralateral muscles that were not exercised and were used as controls, suggesting that the mechanism controlling protein synthesis in skeletal muscles is rapidly responsive to changes in muscular contractile activity.
The possibility of using various optical techniques for detecting the onset of nucleation in protein crystal growth was investigated. Direct microscopy, general metrologic techniques, light scattering, ultraviolet absorption, and interferometry are addressed along with techniques for determining pH value. The necessity for collecting basic data on the optical properties of the growth solution as a prerequisite to the evaluation of monitoring techniques is pointed out.
In earlier sodium dodecylsulfate polyacylamide gel electrophoresis (SDS-PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026-0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS-PAGE analysis of OHEWL yielded two closely spaced dimer bands with M(sub r) = 28 000 and 27 500. In addition, larger HEWL oligomers with M, = 1.7 million and 320 000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 microgram(exp -1) mg ml(exp -1) OHEWL to 32 mg ml(exp -1) HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03-9,3 M Hydrogen peroxide. Repartioning of the dimer during crystallzation aat various temperatures between 277 and 293 K was analyzed by SDS-PAGE. The crystals contained <= 25 % weight by volume of the oligomers in the solution, with no apparent temperature dependence of the repartioning.
Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.
We present data describing a progression of continuing technology development - from expanding the detection capabilities of the current PTS unit to re-outfitting the instrument with a protein microarray increasing the number of detectable compounds. To illustrate the adaptability of the cartridge format, on-orbit operations data from the ISS demonstrate the detection of the fungal cell wall compound beta-glucan using applicable LOCAD-PTS cartridges. LOCAD-PTS is a handheld device consisting of a spectrophotometer, an onboard pumping mechanism, and data storage capabilities. A suite of interchangeable cartridges lined with four distinct capillaries allow a hydrated sample to mix with necessary reagents in the channels before being pumped to the optical well for spectrophotometric analysis. The reagents housed in one type of cartridge trigger a reaction based on the Limulus Amebocyte Lysate (LAL) assay, which results in the release of paranitroaniline dye. The dye is measured using a 395 nm filter. The LAL assay detects the Gram-negative bacterial cell wall molecule, endotoxin or lipopolysaccharide (LPS). The more dye released, the greater the concentration of endotoxin in the sample. Sampling, quantitative analysis, and data retrieval require less than 20 minutes. This is significantly faster than standard culture-based methods, which require at least a 24 hour incubation period.Using modified cartridges, we demonstrate the detection of Gram negative bacteria with protein microarray technology. Additionally, we provide data from multiple field tests where both standard and advanced PTS technologies were used. These tests investigate the transfer of target microbial molecules from one surface to another. Collectively, these data demonstrate that the new cartridges expand the number of compounds detected by LOCAD-PTS, while maintaining the rapid, in situ analysis characteristic of the instrument. The unit provides relevant data for verifying sterile sample collection protocols, which are critical for conducting accurate scientific experiments during future missions to the Moon and Mars.
The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth. Protein crystals grown in microgravity are often larger and have fewer defects than those grown on earth. The analysis of higher quality space-grown crystals will assist in structure-based drug design. We have successfully grown GCAT-infected Sf21 cells in both adhesion and suspension cultures. Expression levels of GCAT in cell lines such as Sf9 and High Five appear to be reduced. We intend to replicate GCAT expression in all three cell lines using the NASA rotating wall bioreactor which effectively duplicates a microgravity environment. The bioreactor itself could be launched to study the expression of the GFP and GCAT proteins in the actual microgravity environment achieved in orbit.
Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.
Upmass is the single most significant limitation of our current space mission capability. Although biomaterials and biocomposites have mass, strength, flexibility, and self-healing properties that could significantly reduce upmass, their use is limited by the following drawbacks: Expensive, specific production. Many biomaterials can only be produced as part of significant support ecosystem; Inaccessible functional customization. The grain of wood, the porosity of bone, and so on are an integral part of the materials' desired mechanical properties, but are not deterministic when the material is naturally grown; Limited compositions. Most biomaterials (unlike metal, plastic, etc.) cannot be easily combined or modified to produce new materials. This project builds on recent advances in: Synthetic biology. Libraries of standardized genetic parts which can be used for controlled cellular material production, delivery, and binding; 3D printing. Commercial off-the-shelf components which can be used to make of a pico- to nanoliter cell deposition system; Tissue engineering. Proven cell-compatible support hydrogels and scaffolds can be modified to bind the deposited biomaterials of interest. Objectives: Feasibility and benefit analysis. Two mission contexts span the concept's scope (see below); Proof-of-concept demonstration. A simple grid of two proteins, fluorescent for easy detection, to validate the core technology concept; Proposed implementations for follow-on work. Avenues for future work on each core component (host cell, production control, material delivery, material binding, etc.); Complementary studies exploration. A survey of other emerging areas (in situ resource utilization, protein engineering, etc.) with the potential to multiply our technology's impact. Potential Impacts: This application could dramatically expand manufacturing capabilities on Earth and in space: In situ resource utilization. A far greater range of materials and products will be available from the limited palette offered by in situ resource extraction techniques; Reduced equipment and material upmass for off-Earth habitats. Ready- to-use highly specialized construction materials (radiation hardened, compressive/tensile, light or dense) from an extremely low starting mass; Structured biomaterial production. New ready-to-use macro, micro, and molecular manufacturing techniques for traditional biomaterials such as wood, bone and shell; New and novel biocomposite creation. The ability to create completely novel material composites from any base material that cells can be engineered to produce. Suggested Mission Contexts: ISS part manufacturing. A 'minimal working example' making a finished biomaterial part aboard the International Space Station; A long-term Mars habitat. 'Cradle-to-grave' use at a hypothetical Mars habitat, covering everything from tools to construction materials. Alternate Abstract: Imagine being able to print anything from tools and composite building materials to food and human tissues. Imagine being on Mars with the ability to replace any broken part, whether it's a part of your spacesuit, your habitat, or your own body. We propose a technique that would allow just that. By printing 3D arrays of cells engineered to secrete the necessary materials, the abundant in situ resources of atmosphere and regolith become organic, inorganic, or organic-inorganic composite materials. Such materials include novel, biologically derived materials not previously possible to fabricate.
Upmass is the single most significant limitation of our current space mission capability. Although biomaterials and biocomposites have mass, strength, flexibility, and self-healing properties that could significantly reduce upmass, their use is limited by the following drawbacks: Expensive, specific production. Many biomaterials can only be produced as part of significant support ecosystem; Inaccessible functional customization. The grain of wood, the porosity of bone, and so on are an integral part of the materials' desired mechanical properties, but are not deterministic when the material is naturally grown; Limited compositions. Most biomaterials (unlike metal, plastic, etc.) cannot be easily combined or modified to produce new materials. This project builds on recent advances in: Synthetic biology. Libraries of standardized genetic parts which can be used for controlled cellular material production, delivery, and binding; 3D printing. Commercial off-the-shelf components which can be used to make of a pico- to nanoliter cell deposition system; Tissue engineering. Proven cell-compatible support hydrogels and scaffolds can be modified to bind the deposited biomaterials of interest. Objectives: Feasibility and benefit analysis. Two mission contexts span the concept's scope (see below); Proof-of-concept demonstration. A simple grid of two proteins, fluorescent for easy detection, to validate the core technology concept; Proposed implementations for follow-on work. Avenues for future work on each core component (host cell, production control, material delivery, material binding, etc.); Complementary studies exploration. A survey of other emerging areas (in situ resource utilization, protein engineering, etc.) with the potential to multiply our technology's impact. Potential Impacts: This application could dramatically expand manufacturing capabilities on Earth and in space: In situ resource utilization. A far greater range of materials and products will be available from the limited palette offered by in situ resource extraction techniques; Reduced equipment and material upmass for off-Earth habitats. Ready- to-use highly specialized construction materials (radiation hardened, compressive/tensile, light or dense) from an extremely low starting mass; Structured biomaterial production. New ready-to-use macro, micro, and molecular manufacturing techniques for traditional biomaterials such as wood, bone and shell; New and novel biocomposite creation. The ability to create completely novel material composites from any base material that cells can be engineered to produce. Suggested Mission Contexts: ISS part manufacturing. A 'minimal working example' making a finished biomaterial part aboard the International Space Station; A long-term Mars habitat. 'Cradle-to-grave' use at a hypothetical Mars habitat, covering everything from tools to construction materials. Alternate Abstract: Imagine being able to print anything from tools and composite building materials to food and human tissues. Imagine being on Mars with the ability to replace any broken part, whether it's a part of your spacesuit, your habitat, or your own body. We propose a technique that would allow just that. By printing 3D arrays of cells engineered to secrete the necessary materials, the abundant in situ resources of atmosphere and regolith become organic, inorganic, or organic-inorganic composite materials. Such materials include novel, biologically derived materials not previously possible to fabricate.
Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.
Recent studies indicate that hemodynamic forces such as cyclic strain and shear stress can increase prostacyclin (PGI2) secretion by endothelial cells (EC) but the effect of these forces on prostacyclin synthase (PGIS) gene expression remains unclear and is the focus of this study. Bovine aortic EC were seeded onto type I collagen coated flexible membranes and grown to confluence. The membranes and attached EC were subjected to 10% average strain at 60 cpm (0.5 sec deformation alternating with 0.5 sec relaxation) for up to 5 days. PGIS gene expression was determined by Northern blot analysis and protein level by Western blot analysis. The effect of cyclic strain on the PGIS promoter was determined by the transfection of a 1-kb human PGIS gene promoter construct coupled to a luciferase reporter gene into EC, followed by determination of luciferase activity. PGIS gene expression increased 1.7-fold in EC subjected to cyclic strain for 24 hr. Likewise, EC transfected with a pGL3B-PGIS (-1070/-10) construct showed an approximate 1.3-fold elevation in luciferase activity in EC subjected to cyclic strain for 3, 4, 8, and 12 hr. The weak stimulation of PGIS gene expression by cyclic strain was reflected in an inability to detect alterations in PGIS protein levels in EC subjected to cyclic strain for as long as 5 days. These data suggest that strain-induced stimulation of PGIS gene expression plays only a minor role in the ability of cyclic strain to stimulate PGI2 release in EC. These findings coupled with our earlier demonstration of a requisite addition of exogenous arachidonate in order to observe strain-induced PGI2 release, implicates a mechanism that more likely involves strain-induced stimulation of PGIS activity.